Application of combination of phenolic acid compound and antibiotic in preparation of medicine for treating infection of drug-resistant pathogenic bacteria

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of a phenolic acid compound and an antibiotic in preparation of a medicine for treating drug-resistant pathogenic bacterium infection. The phenolic acid compound provided by the invention is combined with antibiotics to completely kill multi-drug-resistant escherichia coli in an in-vitro experiment; under the same antibacterial effect, compared with the single use of the antibiotics, the use concentration of the antibiotics can be reduced by 8-32 times by the combined medication method; the compound can be used for improving the bioavailability of antibiotics and exerting a good effect in the treatment of in-vivo infection; the method of combined use of phenolic acid and antibiotics can also effectively reduce the problem of rapid evolution of bacterial drug resistance caused by independent use of antibiotics. The method is simple, efficient, stable, reliable and high in application value, and a brand new and feasible technical scheme is provided for effectively restraining clinical antibiotic multi-drug-resistant pathogenic bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of phenolic acid compounds combined with antibiotics in the preparation of drugs for treating drug-resistant pathogenic bacteria infections. Background Art

[0002] In recent years, the problem of bacterial resistance has become increasingly serious due to the extensive use and even abuse of antibiotics. The highly persistent outbreak of resistant pathogens has brought unprecedented challenges to global public health. The development of new antibiotics or the restoration of the effectiveness of existing antibiotics is a strategy to solve the increasingly serious problem of bacterial resistance. Phenolic acid compounds are plant aromatic secondary metabolites, which are widely distributed throughout the plant kingdom. Due to their important biological and pharmacological properties, they have received increasing attention in terms of anti-inflammatory, antioxidant, anti-cancer and anti-mutagenic properties. At present, some studies have reported that the combined use of antibiotics and traditional Chinese medicine can cope with infection, avoid antibiotic abuse and bacterial resistance, effectively reduce the generation of resistant strains, and promote wound healing. However, it cannot be ignored that traditional Chinese medicine has poor antibacterial effect, few clinical trials, and great limitations in drug development. Therefore, in order to effectively deal with the increasingly severe problem of clinical antibiotic resistance, it is urgent to explore new methods to deal with the problem of antibiotic resistance. Summary of the invention

[0003] The purpose of the present invention is to provide an application of a phenolic acid compound combined with an antibiotic in the preparation of a drug for treating infection by drug-resistant pathogenic bacteria. The phenolic acid compound combined with an antibiotic is suitable for treating infection in vivo, and can stably and long-term curb multiple clinical Escherichia coli and inhibit the generation of antibiotic-resistant bacteria.

[0004] The present invention provides the use of phenolic acid compounds combined with antibiotics in the preparation of medicines for treating drug-resistant pathogenic bacteria infection.

[0005] As a preferred embodiment, the phenolic acid compound includes an organic acid compound containing a phenol ring.

[0006] As a preferred embodiment, the antibiotics include tetracycline antibiotics or aminoglycoside antibiotics.

[0007] As a preferred embodiment, when the antibiotic is tetracycline, the phenolic acid compound includes any one of the following: salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, pyrocatechuic acid, β-resorcinic acid, gentisic acid, 2,6-dihydroxybenzoic acid, protocatechuic acid, α-resorcinic acid, 2,3,4-trihydroxybenzoic acid, gallic acid, vanillic acid, caffeic acid, sinapinic acid and chlorogenic acid.

[0008] As a preferred embodiment, when the antibiotic is tetracycline, the phenolic acid compound also includes a combination of the following two compounds: gallic acid and chlorogenic acid.

[0009] As a preferred embodiment, when the antibiotic is kanamycin, the phenolic acid compound includes any one of the following: salicylic acid, α-resorcinol, gallic acid, vanillic acid and caffeic acid.

[0010] The present invention also provides a pharmaceutical composition, which comprises: an antibiotic and a phenolic acid compound, wherein the mass volume concentration ratio of the antibiotic to the phenolic acid compound is 1:0.2-1.6.

[0011] As a preferred embodiment, the phenolic acid compound includes an organic acid compound containing a phenol ring.

[0012] As a preferred embodiment, the antibiotics include tetracycline antibiotics or aminoglycoside antibiotics.

[0013] As a preferred embodiment, when the antibiotic is tetracycline, the phenolic acid compound includes any one of the following: salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, pyrocatechuic acid, β-resorcinic acid, gentisic acid, 2,6-dihydroxybenzoic acid, protocatechuic acid, α-resorcinic acid, 2,3,4-trihydroxybenzoic acid, gallic acid, vanillic acid, caffeic acid, sinapinic acid and chlorogenic acid.

[0014] As a preferred embodiment, when the antibiotic is tetracycline, the phenolic acid compound also includes a combination of the following two compounds: gallic acid and chlorogenic acid.

[0015] As a preferred embodiment, when the antibiotic is tetracycline, the mass volume concentration ratio of tetracycline to phenolic acid compound is 1:0.2-0.8.

[0016] As a preferred embodiment, when the antibiotic is kanamycin, the phenolic acid compound includes any one of the following: salicylic acid, α-resorcinol, gallic acid, vanillic acid and caffeic acid.

[0017] As a preferred embodiment, when the antibiotic is kanamycin, the mass volume concentration ratio of kanamycin to phenolic acid compound is 1:0.4-1.6.

[0018] As a preferred embodiment, the pharmaceutical composition is used at a pH of 6.0 to 7.0.

[0019] Beneficial effects: The present invention provides the use of phenolic acid compounds combined with antibiotics in the preparation of drugs for drug-resistant pathogenic bacteria infection. Based on the principle of increasing the permeability of the cell outer membrane and inhibiting the expression of efflux pump proteins provided by phenolic acid compounds, the present invention exerts a synergistic antibacterial effect by combining phenolic acid compounds with antibiotics, and completely kills multidrug-resistant Escherichia coli in in vitro experiments. Phenolic acid at a concentration of 1 / 4MIC and antibiotics at a concentration of 1 / 4MIC acted on the model strain E.coli ATCC 25922 sensitive to tetracycline for 30 consecutive days. Its MIC value was the same as the initial MIC value, and no drug resistance was evolved. However, the MIC of the tetracycline treatment group increased by 8 times and evolved drug resistance. It shows that the combined use of phenolic acid and tetracycline can effectively inhibit the development of antibiotic resistance in sensitive Escherichia coli. The method of the present invention can significantly curb the generation of clinical multi-antibiotic resistance and maintain long-term stability, which has a guiding role in the clinical treatment of drug-resistant pathogens and is of great significance for restoring the effectiveness of existing antibiotics.

[0020] The present invention selects hydroxybenzoic acid-type phenolic acid, hydroxycinnamic acid-type phenolic acid or desphenolic acid for combined use with antibiotics, and designs and determines the optimal ratio of phenolic acid compounds and antibiotics by constructing in vivo and in vitro combined antibacterial experiments and in vitro long-term evolution experiments. Within the optimal ratio range designed by the present invention, the combined antibacterial degree of antibiotics and phenolic acid compounds on Escherichia coli can reach more than 95%; through the in vivo infection treatment using phenolic acid compounds and antibiotics in combination, it is found that the survival rate of the combined medication group is higher than that of the single medication group, and the survival rate is increased by about 20%; at the same time, under the same antibacterial effect, compared with the use of antibiotics alone, the combined medication method can reduce the use concentration of antibiotics by 8 to 32 times; further, the method can improve the bioavailability of antibiotics and play a good role in the treatment of in vivo infections. While exerting the synergistic antibacterial effect of phenolic acid compounds and antibiotics, the method of the present invention can improve the bioavailability of antibiotics and is efficiently applied to the in vivo infection treatment of multidrug-resistant pathogens. The method of the invention is simple, efficient, stable, reliable, and has high application value, and provides a new and feasible technical method for effectively curbing clinical antibiotic multidrug-resistant pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1The results of the micro-checkerboard dilution method for the combined use of phenolic acid compounds and tetracycline in Example 1 are shown in Figure 1, wherein (A) is a micro-checkerboard dilution method result diagram for the combined use of salicylic acid and tetracycline, (B) is a micro-checkerboard dilution method result diagram for the combined use of m-hydroxybenzoic acid and tetracycline, (C) is a micro-checkerboard dilution method result diagram for the combined use of p-hydroxybenzoic acid and tetracycline, (D) is a micro-checkerboard dilution method result diagram for the combined use of pyrocatechinic acid and tetracycline, (E) is a micro-checkerboard dilution method result diagram for the combined use of β-resorcinol and tetracycline, (F) is a micro-checkerboard dilution method result diagram for the combined use of gentisic acid and tetracycline, and (G) is a micro-checkerboard dilution method result diagram for the combined use of 2,6-dihydroxybenzoic acid and tetracycline. The result graph of the method is shown in Figure 1, (H) is the result graph of the micro-checkerboard dilution method for the combined use of protocatechuic acid and tetracycline, (I) is the result graph of the micro-checkerboard dilution method for the combined use of α-resorcinic acid and tetracycline, (J) is the result graph of the micro-checkerboard dilution method for the combined use of 2,3,4-trihydroxybenzoic acid and tetracycline, (K) is the result graph of the micro-checkerboard dilution method for the combined use of gallic acid and tetracycline, (L) is the result graph of the micro-checkerboard dilution method for the combined use of vanillic acid and tetracycline, (M) is the result graph of the micro-checkerboard dilution method for the combined use of caffeic acid and tetracycline, (N) is the result graph of the micro-checkerboard dilution method for the combined use of sinapinic acid and tetracycline, and (O) is the result graph of the micro-checkerboard dilution method for the combined use of chlorogenic acid and tetracycline;

[0023] Figure 2 The results of the micro-checkerboard dilution method for the combined use of the phenolic acid compounds and kanamycin in Example 1 are shown in Figure 1, wherein (A) is a micro-checkerboard dilution method result diagram for the combined use of salicylic acid and kanamycin, (B) is a micro-checkerboard dilution method result diagram for the combined use of α-resorcinic acid and kanamycin, (C) is a micro-checkerboard dilution method result diagram for the combined use of gallic acid and kanamycin, (D) is a micro-checkerboard dilution method result diagram for the combined use of vanillic acid and kanamycin, and (E) is a micro-checkerboard dilution method result diagram for the combined use of caffeic acid and kanamycin;

[0024] Figure 3 The results of the micro-chessboard dilution method of the phenolic acid compounds in Example 1 at pH = 6.0 are shown, wherein (A) is the combined use of pyrocatechinic acid and tetracycline, (B) is the combined use of α-resorcinic acid and tetracycline, (C) is the combined use of caffeic acid and tetracycline, and (D) is the combined use of gallic acid and chlorogenic acid and tetracycline;

[0025] Figure 4 The phenolic acid in Example 2 enhances the bioavailability of antibiotics to bacteria, wherein (A) is the linear relationship between the fluorescence intensity of E. coli MC4100 / pTGM and the concentration of tetracycline, and (B) is the average fluorescence intensity of E. coli MC4100 / pTGM at 32 μg / L tetracycline and different phenolic acid concentrations;

[0026] Figure 5 The results of the in vivo infection treatment using the phenolic acid compounds and antibiotics in combination in Example 3 are shown in Figure 3, wherein (A) is the survival curve of the wax moth larvae inoculated with different concentrations of E. coli C4313 bacterial liquid, (B) is the survival curve of the wax moth larvae inoculated with different types of phenolic acid compounds (4096 μg / mL), (C) is the survival curve of the wax moth larvae after being infected with E. coli C4313 and treated with tetracycline and gentisic acid, wherein the four treatments are 0.9% NaCl, 1 / 2MIC tetracycline (40 μg / mL), 1 / 2MIC gentisic acid (128 μg / mL) and 1 / 2MIC tetracycline + 1 / 2MIC gentisic acid, and (D) is the survival curve of the wax moth larvae infected with E. coli C4313 and treated with tetracycline and gentisic acid. After C4313 was infected with G. mellonella larvae, the survival curve of G. mellonella larvae treated with tetracycline and α-resorcinol. The four treatments were 0.9% NaCl, 1 / 2MIC of tetracycline (40μg / mL), 1 / 2MIC of α-resorcinol (128μg / mL), and 1 / 2MIC of tetracycline + 1 / 2MIC of α-resorcinol.

[0027] Figure 6 This is an evolution experiment of the phenolic acid compound of Example 4 combined with tetracycline at a concentration of 1 / 4 MIC on E.coli ATCC25922;

[0028] Figure 7 The figure shows the effect of the combined use of phenolic acid compounds and tetracycline on the bacterial outer membrane in Example 5, wherein light purple is the blank control group, dark purple is the group with only tetracycline, light gray is the phenolic acid alone, and dark gray is the combination of phenolic acid and tetracycline. DETAILED DESCRIPTION

[0029] The present invention provides the use of phenolic acid compounds combined with antibiotics in the preparation of medicines for treating drug-resistant pathogenic bacteria infection.

[0030] The phenolic acid compounds of the present invention include organic acid compounds containing phenol rings.

[0031] The antibiotics described in the present invention include tetracycline antibiotics or aminoglycoside antibiotics.

[0032] In the present invention, when the antibiotic is tetracycline, the phenolic acid compound may include any one of the following: salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, pyrocatechuic acid, β-resorcinic acid, gentisic acid, 2,6-dihydroxybenzoic acid, protocatechuic acid, α-resorcinic acid, 2,3,4-trihydroxybenzoic acid, gallic acid, vanillic acid, caffeic acid, sinapinic acid and chlorogenic acid. The phenolic acid compounds of the present invention have the ability to increase the permeability of the outer membrane. The phenolic acid compounds used in combination with tetracycline increase the permeability of the outer membrane, so that other ions or compounds can enter the bacteria more easily, thereby affecting the physiological activity of the bacteria and obtaining a synergistic antibacterial effect. As a specific embodiment, the phenolic acid compounds of the present invention have a synergistic antibacterial effect on E.coli MG1655 / RP4 and E.coli C4313 when used in combination with tetracycline.

[0033] In the present invention, when the antibiotic is tetracycline, the phenolic acid compound can also include the following two compositions for use together: gallic acid and chlorogenic acid. As a specific embodiment, the combination of chlorogenic acid and gallic acid with tetracycline can inhibit Escherichia coli E.coliMG1655 / RP4 in vitro, and the graded inhibitory concentration index FIC is 0.313, which is less than 0.5, indicating that the combination of chlorogenic acid and gallic acid with tetracycline has a synergistic antibacterial effect and an antibacterial sensitization effect.

[0034] In the present invention, when the antibiotic is kanamycin, the phenolic acid compound may include any one of the following: salicylic acid, α-resorcinol, gallic acid, vanillic acid and caffeic acid. As a specific embodiment, after adding different concentrations of CCCP, a known bacterial multidrug resistance efflux pump inhibitor, it was found that the fluorescence intensity of bacteria stimulated by some phenolic acid compounds on E.coliMC4100 / pTGM was higher than that of CCCP, a known bacterial multidrug resistance efflux pump inhibitor. This indicates that phenolic acid is a potential bacterial multidrug resistance efflux pump inhibitor.

[0035] The present invention also provides a pharmaceutical composition, which may include: antibiotics and phenolic acid compounds, wherein the mass volume concentration ratio of the antibiotics to the phenolic acid compounds may be 1:0.2 to 1.6, such as 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 and 1:1.6. Within the mass volume concentration ratio range of the antibiotics to the phenolic acid compounds of the present invention, the combined antibacterial degree of the antibiotics and the phenolic acid compounds against Escherichia coli can reach more than 95%.

[0036] In the present invention, when the antibiotic is tetracycline, the phenolic acid compound may include any one of the following: salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, pyrocatechuic acid, β-resorcinic acid, gentisic acid, 2,6-dihydroxybenzoic acid, protocatechuic acid, α-resorcinic acid, 2,3,4-trihydroxybenzoic acid, gallic acid, vanillic acid, caffeic acid, sinapinic acid and chlorogenic acid. As a specific embodiment, the phenolic acid compound and the antibiotic can be used in combination to treat the infection in vivo, and the effect of the combined medication can be compared, so that it is found that the survival rate of the combined medication group is higher than that of the single medication group, and the survival rate is increased by about 20%.

[0037] In the present invention, when the antibiotic is tetracycline, the phenolic acid compound can also be used together with a composition of the following two: gallic acid and chlorogenic acid. The phenolic acid compound of the present invention used in combination with tetracycline can increase the average fluorescence intensity of a single bacterium of E. coli MC4100 / pTGM by about 2.83 times at most, and the corresponding bioavailable tetracycline concentration is increased by about 1.66 times. As a specific embodiment, the combination of tetracycline at 1 / 4 minimum inhibitory concentration and phenolic acid compound at 1 / 4 minimum inhibitory concentration can still effectively inhibit the development of antibiotic resistance in sensitive Escherichia coli during 30 days of continuous treatment.

[0038] In the present invention, when the antibiotic is tetracycline, the mass volume concentration ratio of tetracycline to phenolic acid compounds can be 1:0.2 to 0.8, for example 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7 and 1:0.8.

[0039] In the present invention, when the antibiotic is kanamycin, the phenolic acid compound may include any one of the following: salicylic acid, α-resorcinic acid, gallic acid, vanillic acid and caffeic acid. The phenolic acid compound of the present invention is used in combination with an antibiotic, and the combined use can reduce the concentration of tetracycline or kanamycin by 8 to 32 times compared with the single use of antibiotics under the same antibacterial effect.

[0040] In the present invention, when the antibiotic is kanamycin, the mass volume concentration ratio of kanamycin to phenolic acid compounds can be 1:0.4-1.6, for example, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 and 1:1.6.

[0041] The use conditions of the pharmaceutical composition of the present invention can be pH=6.0-7.0, such as 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0. As a specific embodiment, pyrocatechinic acid, α-resorcinic acid and caffeic acid are used in combination with tetracycline at pH=6.0, and have a synergistic antibacterial effect compared with the previous neutral condition (pH=7.0), indicating that phenolic acid has an antibacterial sensitization effect on tetracycline under neutral or acidic conditions. As a specific embodiment, the pH range in which antibiotics and phenolic acid compounds exert synergistic antibacterial effects is shown in Table 1.

[0042] Table 1 pH range in which antibiotics and phenolic acid compounds exert synergistic antibacterial effects

[0043]

[0044] To further illustrate the present invention, the application of the phenolic acid compounds provided by the present invention in combination with antibiotics in the preparation of drugs for treating drug-resistant pathogenic bacteria infections is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] The antibiotics used in the examples are tetracycline and kanamycin; there are 15 phenolic acid compounds used, and their molecular names are shown in Table 2. The four strains of Escherichia coli used are E.coli MG1655 / RP4, E.coli MC4100 / pTGM, E.coli C4313 and the model strain E.coli ATCC 25922, wherein the RP4 plasmid of E.coli MG1655 / RP4 carries tetA, bla and neo genes, which are resistance genes for tetracycline, ampicillin and kanamycin respectively. The pTGM plasmid of E.coli MC4100 / pTGM carries tetA, tetM and tetR tetracycline resistance genes. E.coli C4313 has the tetracycline resistance gene tetA and the multidrug resistance efflux pump gene mdfA on its genome.

[0046] The bacterial strains involved in this study include E. coli MG1655 / RP4 (this study used the plasmid RP4 carrying the lacIq-Plpp-mCherry marker that can bind to a wide range of hosts, which was provided by Dr. Barth F. Smets of the Technical University of Denmark) (Reference: Klümper U, Riber L, Dechesne A, et al. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community [J]. The ISME journal, 2015, 9 (4): 934-945.);

[0047] E.coli MC4100 / pTGM (References: Bahl MI, Hansen LH, SJ. Construction of an extended range whole-cell tetracycline biosensor by use of the tet(M)resistance gene[J]. FEMS Microbiology Letters, 2005, 253(2):201-205.);

[0048] E.coli C4313 and E.coliATCC 25922 (Reference: DuanY, Gao H, Zheng L, etal. Antibiotic resistance and virulence of extraintestinal pathogenic Escherichia coli (ExPEC) vary according to molecular types [J]. Frontiers inmicrobiology, 2020, 11: 598305.).

[0049] The reagents used in the present invention are shown in Table 2. The LB medium used in the present invention: sodium chloride: 5 g / L, yeast powder: 5 g / L, beef extract: 3 g / L, peptone: 10 g / L, these components are dissolved in distilled water, fixed to volume, and sterilized by high-pressure steam to obtain the LB medium.

[0050] Table 2 Reagents required for the experiment

[0051]

[0052]

[0053] Example 1 Synergistic antibacterial effect of phenolic acid compounds and antibiotics when used in combination

[0054] Determine the MIC and MBC of antibiotics and phenolic acid against bacteria. The specific steps are as follows: First, add 200 μL of high-concentration antibiotics (20.48 mg / mL) or phenolic acid stock solution (16.384 mg / mL) dissolved in methanol to the first column of a sterile 96-well plate, then draw 100 μL from the first column of wells and mix with 100 μL of sterile LB solution in the second column of wells, then draw 100 μL to the next well, and so on to the last column (antibiotic concentration (μg / mL): 20480, 1 0240, 5120, 2560, 1280, 640, 320, 160, 80, 40, 20, 10; 5.12, 2.56, 1.28, 0.64, 0.32, 0.16; phenolic acid concentration (μg / mL): 16384, 8192, 4096, 2048, 1024, 512, 256, 128, 64, 32, 16, 8; 4, 2, 1, 0.5, 0.25, 0.125). Finally, 100 μL of the diluted bacterial solution was added to a 96-well plate (the final bacterial concentration was 5×10 5 CFU / mL), OD of the diluted bacterial solution 600 The strain to be tested was diluted 50 times with sterile LB solution at a concentration of 0.5. The positive control was to add only 100 μL of sterile LB solution and 100 μL of diluted bacterial solution, and the negative control was 200 μL of sterile LB solution. After the addition of samples, the 96-well plate was placed in a 37°C incubator in the dark for 16 hours, and the absorbance value of each well was read at λ=600nm using an enzyme reader. The lowest concentration that completely inhibited bacterial growth in the well was taken as MIC. After the MIC was determined, 100 μL was taken from each well where the lowest concentration to the highest concentration completely inhibited bacterial growth into 1 mL of sterile LB solution. After culturing at 37°C, 180 rpm in the dark for 24 hours, the absorbance value was measured at λ=600nm using an enzyme reader, and the lowest concentration equal to the value of the blank sterile LB control group was taken as MBC.

[0055] Next, the micro-checkerboard dilution method was used to conduct an in vitro combined antibacterial test of phenolic acid and antibiotics. The specific steps are as follows: First, in row A and column 1 of a sterile 96-well plate, phenolic acid and antibiotics were diluted in multiples using 2 times the MIC as the initial concentration, and 100 μL of the diluted phenolic acid and antibiotic solutions were added to each well. Then, from row B, column 2 to row H, column 8, 50 μL of 2 times the concentration of phenolic acid and antibiotics in the row and column were added in sequence, so that the final concentration of the well was equal to the corresponding antibiotic concentration in row A and the phenolic acid concentration in column 1. Finally, the OD 600The test strain with a value of 0.5 was diluted 50 - fold with sterile LB solution, and then 100 μL was taken and added to a 96 - well plate (final concentration: 5×10 5 CFU / mL). The positive control was adding only 100 μL of sterile LB solution and 100 μL of the diluted bacterial solution, and the negative control was 200 μL of sterile LB solution. All experiments had at least 3 biological replicates. After adding the samples, the 96 - well plate was placed in an incubator at 37 °C in the dark for 16 hours, and then the absorbance values of each well were read using an enzyme - linked immunosorbent assay (ELISA) reader at λ = 600 nm.

[0056] The calculation formula for the inhibition rate (%) is as follows: { (OD positive control - OD negative control) - (OD experimental group - OD negative control)} / (OD positive control - OD negative control) × 100%. An inhibition rate greater than or equal to 50% is defined as having a synergistic effect. Then, the fractional inhibitory concentration (FIC) index is calculated. FIC = MIC (phenolic acid and antibiotic combination) / MIC (phenolic acid alone) + MIC (phenolic acid and antibiotic combination) / MIC (antibiotic alone). The judgment criteria for the interaction between phenolic acid and antibiotic: FIC ≤ 0.5 is judged as a synergistic effect, 0.5 < FIC ≤ 1 is judged as an additive effect, 1 < FIC ≤ 2 is judged as an irrelevant effect, and FIC > 2 is judged as an antagonistic effect.

[0057] The MIC and MBC values of phenolic acid compounds and antibiotics against Escherichia coli when used alone are shown in Tables 3 and 7. The results of the in vitro combined antibacterial test of 15 phenolic acid compounds and antibiotics against E. coli MG1655 / RP4 using the micro - checkerboard dilution method are as Figure 1 and Figure 2 shown, where the abscissa represents different tetracycline concentrations, the ordinate represents different phenolic acid concentrations, and the darker the color of the color block, the lower the antibacterial degree, and white represents 100% antibacterial. The fractional inhibitory index was calculated using the experimental results to evaluate the synergistic antibacterial effect. When different phenolic acid compounds were used in combination with tetracycline, they all had a synergistic antibacterial effect on E. coli MG1655 / RP4 and E. coli C4313, as Figure 1As shown in Table 4, Table 6 and Table 7, when the concentration of tetracycline is 0.64 mg / mL and the concentration of phenolic acid compounds is 0.128-0.512 mg / mL (i.e., the combined mass volume concentration ratio of phenolic acid compounds to tetracycline concentration is 0.2-0.8:1), the combined inhibition of Escherichia coli can reach more than 95%; when the combined mass volume concentration ratio of phenolic acid compounds to tetracycline concentration (0.64 mg / mL) is 0.2-0.8:1, and the combined mass volume concentration ratio of phenolic acid compounds to kanamycin concentration (0.64 mg / mL) is 0.4-1.6:1, the combined inhibition of Escherichia coli can reach more than 95%. It can be seen from Tables 4, 6 and 7 that the combined use of pyrocatechuic acid, protocatechuic acid, etc. with tetracycline can reduce the concentration of tetracycline by more than 8 times, and the combined use of salicylic acid, gallic acid, etc. with kanamycin can reduce the concentration of kanamycin by 32 times. It can be seen that compared with the use of a single antibiotic, the combined use of tetracycline or kanamycin can reduce the concentration of tetracycline or kanamycin by 8 to 32 times to achieve the same antibacterial effect.

[0058] Table 5 and Figure 3 (A), (B), and (C) show the in vitro combined antibacterial effects of pyrocatechin, α-resorcin and caffeic acid on E. coli MG1655 / RP4 when used in combination with tetracycline at pH = 6.0. The horizontal axis represents different concentrations of tetracycline, and the vertical axis represents different concentrations of phenolic acid. The darker the color block, the lower the degree of inhibition, and white represents 100% inhibition. The results showed that the FIC (0.188, 0.323, and 0.375) of these three phenolic acids with tetracycline were all less than 0.5, and they were judged to have a synergistic antibacterial effect. Compared with the previous neutral conditions (pH = 7.0), they all have a synergistic antibacterial effect, indicating that phenolic acids have an antibacterial sensitization effect on tetracycline under neutral or acidic conditions. Figure 3 Middle (D) shows the in vitro combined antibacterial effect of chlorogenic acid and gallic acid in combination with tetracycline on E. coli MG1655 / RP4. The FIC value (0.313) was less than 0.5, which was judged to have a synergistic antibacterial effect. This shows that the use of two phenolic acids together can also have an antibacterial sensitization effect on tetracycline.

[0059] Table 3 MIC and MBC values ​​of phenolic acid compounds and tetracycline against Escherichia coli when used alone (μg / mL)

[0060]

[0061]

[0062] Table 4 FIC values ​​of phenolic acid compounds and tetracycline against Escherichia coli when used in combination

[0063]

[0064]

[0065] Note: In the column of 'Results' in Table 2, 'S' means synergy; 'PA / TC' means the ratio of phenolic acid to tetracycline (0.64 mg / mL); 'TC' means tetracycline.

[0066] Table 5 Combinations of phenolic acid compounds and tetracycline with synergistic antibacterial effect at pH = 6.0 and their FIC values ​​against E.coli MG1655 / RP4

[0067]

[0068] Note: In the column of 'Results' in Table 2, 'S' means synergy; 'PA / TC' means the ratio of phenolic acid to tetracycline (0.64 mg / mL); 'TC' means tetracycline.

[0069] Table 6 FIC values ​​of phenolic acid compounds and tetracycline against E.coli MC4100 / pTGM

[0070]

[0071] Note: In the column of 'Results', 'S' means synergy; 'TC' means tetracycline.

[0072] Table 7 MIC, MBC and FIC values ​​of phenolic acid compounds and kanamycin against Escherichia coli

[0073]

[0074] Note: In the column of 'Results', 'S' means synergy; 'PA / TC' means the ratio of phenolic acid to tetracycline (0.64 mg / mL); 'Kan' means kanamycin.

[0075] Example 2 The combined use of phenolic acid compounds and antibiotics can improve the bioavailability of antibiotics Experiment on the bioavailability of phenolic acid and antibiotics to bacteria: Take 10 μL of E. coli MC4100 / pTGM (OD 6001) to 990 μL of a solution of tetracycline and phenolic acid compounds or a solution of tetracycline and CCCP, the concentration of tetracycline is 32 μg / L, the concentration of phenolic acid compounds is 64 μg / L, 128 μg / L, 256 μg / L, 512 μg / L and 1024 μg / L, and the concentration of CCCP solution is 64 μg / L, 128 μg / L, 256 μg / L, 512 μg / L and 1024 μg / L. Tetracycline, phenolic acid compounds and CCCP solutions are all solutions filtered by a 0.22 μm filter membrane. After the addition, the solution makes the bacterial concentration in a 2 mL sterile centrifuge tube be 10 6 CFU / mL. Place in a 37°C constant temperature incubator away from light, culture at 180rpm for 6 hours, wash 3 times with sterile PBS, take 100μL to a 96-well plate, measure its absorbance at λ=600nm with an enzyme marker, and place the remaining solution in a fluorescent quartz cuvette, and measure its fluorescence intensity at an excitation wavelength of 488nm and an emission wavelength of 511nm with a fluorescence spectrophotometer. Finally, the total fluorescence intensity was divided by the total number of bacteria to calculate the average fluorescence intensity of a single bacterium.

[0076] E.coli MC4100 / pTGM acts as a tetracycline sensor. When it comes into contact with tetracycline in the solution, it will emit fluorescence. Its fluorescence intensity will show a linear correlation with the concentration of tetracycline in the solution. The higher the tetracycline concentration, the stronger the fluorescence brightness emitted by a single bacterium. Figure 4 As shown in (A), when the tetracycline concentrations were 16 μg / L, 32 μg / L, 64 μg / L, 128 μg / L, and 256 μg / L, the corresponding average fluorescence intensity of a single bacterium was 1.08×10 -6 , 1.87×10 -6 , 6.21×10 -6 , 2.37×10 -5 and 5.46×10 -5 When the concentration of tetracycline in the fixed system was 32 μg / L, the addition of phenolic acid compounds at different concentrations (64 μg / L, 128 μg / L, 256 μg / L and 512 μg / L) could increase the average fluorescence intensity of E. coli MC4100 / pTGM single bacteria to varying degrees. Among them, the most effective agent for increasing the fluorescence intensity of E. coli MC4100 / pTGM single bacteria was 512 μg / L 2,6-dihydroxybenzoic acid, followed by 1024 μg / L pyrocatechuic acid and 256 μg / L β-resorcylic acid. The corresponding fluorescence intensities of E. coli MC4100 / pTGM single bacteria in these three cases were 5.1×10-6 4.1×10 -6 and 3.9×10 -6 ; When the concentration of tetracycline was 32 μg / L (the fluorescence intensity of a single bacterium in E. coli MC4100 / pTGM was 1.8×10 -6 ), the fluorescence intensity can be increased by about 2.83, 2.28 and 2.17 times, respectively, and the corresponding bioavailable tetracycline concentrations are increased by about 1.66, 1.31 and 1.18 times, respectively.

[0077] Example 3 Treatment of infection in vivo using phenolic acid compounds and antibiotics in combination

[0078] The specific steps of the in vivo infection treatment experiment are as follows:

[0079] The wax moth larvae used in the in vivo infection treatment experiment: healthy wax moth larvae of about 20 mm in length were selected.

[0080] E.coil C4313 strain virulence test: 1.0×10 8 CFU / mL of the bacterial solution were diluted to 1.0×10 7 CFU / mL, 1.0×10 6 CFU / mL and 1.0×10 5 CFU / mL, and 2 mL of each dilution was reserved for later use. This experiment was divided into 4 groups, with 8 larvae in each group. After grouping, the larvae were placed in the culture box according to the group and marked accordingly. Each larva in Group 1 was injected with a concentration of 1.0×10 8 CFU / mL of bacterial solution, 10 μL each; the injection concentration of group 2 was 1.0×10 7 CFU / mL of bacterial solution, 10 μL each; the injection concentration of group 3 was 1.0×10 6 CFU / mL of bacterial solution, 10 μL each; the injection concentration of group 4 was 1.0×10 5 CFU / mL of bacterial solution were injected into each group; 10 μL of sterile saline was injected into the fifth group as a control. All larvae were returned to the culture box and incubated in a 37°C incubator for 120 h, and the number of larval deaths was recorded every 24 h; the survival data of the greater wax moth was recorded for 5 consecutive days. Figure 5 As shown in (A), when the concentration of bacterial solution is 1.0×10 7 CFU / mL, the mortality of the wax moth larvae reached 100% on the second day, and the concentration of the bacterial solution that reached 80% mortality was about 1.0×10 6 CFU / mL, therefore, 1.0×10 6 The bacterial concentration in CFU / mL was used as the inoculation concentration.

[0081] Phenolic acid toxicity test: Use sterile saline to inject the prepared 15 phenolic acid solutions with a concentration of 4096 μg / mL into the larvae of each group, and divide them into 16 groups. The 16th group is a sterile saline blank control group. The rest of the test steps are the same as above. Figure 5 As shown in (B), the injection of 15 phenolic acid compounds (at a concentration of 4096 μg / mL) into the larvae of G. mellonella had little effect on the survival rate of G. mellonella larvae.

[0082] In vivo antimicrobial drug combination experiment: The bacterial suspension of E.coil C4313 was prepared at a concentration of 80% lethal dose. The wax moth larvae in groups 1 to 4 were injected with 10 μL of 80% lethal E.coil C4313 bacterial suspension, and group 5 was given sterile saline as a control. All infected larvae were returned to the 37°C culture box and incubated in a 37°C incubator for 2 hours. After 2 hours of incubation, the culture box was taken out, and the larvae in group 1 were given tetracycline 40 μg / mL; group 2 was given phenolic acid 128 μg / mL; group 3 was given tetracycline 40 μg / mL + phenolic acid 128 μg / mL; groups 4 and 5 were given sterile saline, and group 5 was used as a control group to evaluate the effect of mechanical damage on larval mortality. After the administration, all larvae were returned to the culture dish, and the culture dish was placed in a constant temperature incubator at 37°C. The number of dead wax moth larvae in all groups was observed every 24 hours until 120 hours, and the data of each group was recorded in detail.

[0083] pass Figure 5 (C) and (D) show that the 120h survival rate of the wax moth larvae infected with E. coli C4313 was higher than that of the larvae in the saline control group after receiving combined treatment with phenolic acid (1 / 2MIC) or antibiotic (1 / 2MIC), and the survival rate of the combined drug group was higher than that of the single drug group, with a survival rate increase of about 20%. When tetracycline was used alone, the survival rate of the wax moth larvae infected with E. coli C4313 was about 60% at 100h, while the survival rate of the combined drug group was about 80% at 100h;

[0084] Example 4: Suppressing the development of antibiotic resistance and maintaining long-term stability

[0085] Evolution experiment of phenolic acid compounds combined with antibiotics on Escherichia coli: OD 6002 μL of E. coli ATCC25922 bacterial solution with a concentration of 1 was taken into a 198 μL 96-well plate. The 198 μL contained 0.25×MIC phenolic acid and 0.25×MIC tetracycline dissolved in sterile LB liquid culture medium. The concentration of phenolic acid was 64 μg / mL, 32 μg / mL or 16 μg / mL, and the concentration of tetracycline was 0.16 μg / mL. The 198 μL containing only 0.25×MIC tetracycline concentration (0.16 μg / mL) was used as a control group. After culturing at 37°C in the dark for 24 hours, the OD was measured using a Nanodrop micro-spectrophotometer. 600 The OD value was adjusted to 0.05 with sterile LB liquid medium. 600 1, and then take 2 μL to 198 μL of 96-well plate containing phenolic acid and tetracycline. After culturing at 37°C for 24 hours, repeat the above operation for 30 days and test the MIC of tetracycline on the remaining bacterial liquid.

[0086] like Figure 6 As shown in the figure, at a concentration of 1 / 4 MIC, phenolic acid and antibiotics acted on the model strain E. coli ATCC 25922 sensitive to tetracycline for 30 consecutive days, and its MIC value was the same as the initial MIC value, and no resistance was evolved. However, the MIC of the group treated with tetracycline alone increased by 8 times, and resistance was evolved. These results show that the combination of phenolic acid and tetracycline can effectively inhibit the development of antibiotic resistance in sensitive Escherichia coli.

[0087] Example 5 Inhibition of efflux pump expression and increase in cell outer membrane permeability

[0088] Cell outer membrane permeability assay: Take 10 μL of E. coli MG1655 / RP4 (OD 600 1) to 990 μL of 320 μg / mL tetracycline and 0, 128 and 256 μg / mL phenolic acid solution after passing through a 0.22 μm filter membrane, and then add the fluorescent probe 1-Nphenylnaphthylamine (NPN) to a final concentration of 10 μmol / L, which is used to evaluate the outer membrane integrity of Escherichia coli after treatment with phenolic acid and tetracycline. Place in a 37°C constant temperature incubator away from light, react at 180 rpm for 20 minutes, wash with PBS three times, transfer the solution to a fluorescent quartz cuvette, and measure the fluorescence intensity with an excitation wavelength of 350 nm and an emission wavelength of 420 nm using a fluorescence spectrophotometer.

[0089] like Figure 4As shown in (B), after adding different concentrations of CCCP, a known bacterial multidrug resistance efflux pump inhibitor, the fluorescence intensity of E. coli MC4100 / pTGM single bacteria also increased, indicating that the increase in the bioavailability of tetracycline by bacteria is related to bacterial multidrug efflux pumps, and the fluorescence intensity of bacteria stimulated by some phenolic acid compounds on E. coli MC4100 / pTGM is higher than that of CCCP, a known bacterial multidrug resistance efflux pump inhibitor. This indicates that phenolic acid may be a potential bacterial multidrug resistance efflux pump inhibitor.

[0090] from Figure 7 It can be seen that the use of tetracycline alone will increase the outer membrane permeability of bacteria compared to those without tetracycline, while the outer membrane permeability of phenolic acid alone is significantly higher than that of tetracycline alone. The most prominent one is that when gentisic acid is used alone (the fluorescence intensity corresponding to the outer membrane permeability of bacteria treated with 256μg / mL gentisic acid is 6350.55), it is more than 800 times higher than when tetracycline is used alone (the fluorescence intensity corresponding to the outer membrane permeability of bacteria treated with 320μg / mL tetracycline is 7.15). In general, most phenolic acids have the ability to increase the permeability of the outer membrane, thereby increasing the increase in permeability when used in combination with tetracycline, allowing other ions or compounds to enter the bacteria more easily, thereby affecting the physiological activity of the bacteria and obtaining a synergistic antibacterial effect.

[0091] It can be seen that the method of using phenolic acid compounds in combination with antibiotics designed by the present invention can significantly reduce the usage of antibiotics and improve the bioavailability of antibiotics while exerting a synergistic antibacterial effect, and is suitable for the treatment of in vivo infections. It can stably curb the development of clinical Escherichia coli antibiotic resistance in the long term, which is of great significance for achieving the inhibition of bacterial development of resistance.

[0092] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of phenolic acid compounds combined with antibiotics in the preparation of drugs for treating drug-resistant pathogenic bacteria infections; The phenolic acid compounds include organic acid compounds containing phenol rings; The antibiotics include tetracycline antibiotics or aminoglycoside antibiotics.

2. The use according to claim 1, characterized in that: When the antibiotic is tetracycline, the phenolic acid compound includes any one of the following: salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, pyrocatechuic acid, β-resorcinic acid, gentisic acid, 2,6-dihydroxybenzoic acid, protocatechuic acid, α-resorcinic acid, 2,3,4-trihydroxybenzoic acid, gallic acid, vanillic acid, caffeic acid, sinapinic acid and chlorogenic acid.

3. The use according to claim 1, characterized in that: When the antibiotic is tetracycline, the phenolic acid compound also includes a combination of the following two: gallic acid and chlorogenic acid.

4. The use according to claim 1, characterized in that: When the antibiotic is kanamycin, the phenolic acid compound includes any one of the following: salicylic acid, α-resorcinol, gallic acid, vanillic acid and caffeic acid.

5. A pharmaceutical composition, characterized in that The composition comprises: an antibiotic and a phenolic acid compound, wherein the mass volume concentration ratio of the antibiotic to the phenolic acid compound is 1:0.2-1.6; The phenolic acid compounds include organic acid compounds containing phenol rings; The antibiotics include tetracycline antibiotics or aminoglycoside antibiotics.

6. The composition according to claim 5, characterized in that When the antibiotic is tetracycline, the phenolic acid compound includes any one of the following: salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, pyrocatechuic acid, β-resorcinic acid, gentisic acid, 2,6-dihydroxybenzoic acid, protocatechuic acid, α-resorcinic acid, 2,3,4-trihydroxybenzoic acid, gallic acid, vanillic acid, caffeic acid, sinapinic acid and chlorogenic acid.

7. The composition according to claim 5, characterized in that When the antibiotic is tetracycline, the phenolic acid compound further comprises a combination of the following two: gallic acid and chlorogenic acid.

8. The composition according to claim 5, characterized in that When the antibiotic is tetracycline, the mass volume concentration ratio of tetracycline to phenolic acid compound is 1:0.2-0.

8.

9. The composition according to claim 5, characterized in that When the antibiotic is kanamycin, the phenolic acid compound includes any one of the following: salicylic acid, α-resorcinic acid, gallic acid, vanillic acid and caffeic acid; When the antibiotic is kanamycin, the mass volume concentration ratio of kanamycin to phenolic acid compound is 1:0.4-1.

6.

10. The composition according to claim 5, characterized in that The use condition of the pharmaceutical composition is pH=6.0-7.0.

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