Application of Pinocembrin combined with polymyxin in preparation of antibacterial drugs

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of pinocembrin combined with polymyxin in preparation of antibacterial drugs, wherein the bacteria targeted by the antibacterial drugs are one or more of escherichia coli, salmonella and klebsiella pneumoniae. The bacteria targeted by the antibacterial drugs are polymyxin-resistant bacteria. The bacteria targeted by the antibacterial drugs are multiple drug-resistant gram-negative bacteria. The application proves that pinocembrin effectively restores the sensitivity of bacteria to polymyxin through checkerboard minimum inhibitory concentration test and in-vitro time-kill curve, and simultaneously proves that pinocembrin can effectively enhance the antibacterial activity of polymyxin on bacteria by destroying the integrity of bacterial cell membranes and inhibiting the transcription and expression of polymyxin-resistant genes through staining experiment and fluorescence quantitative PCR. mcr-1 The application provides a new use of pinocembrin in synergizing the antibacterial activity of polymyxin antibiotics, and can solve the technical problems of polymyxin clinical drug resistance and low therapeutic index.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of pinocembrin combined with polymyxin in the preparation of antibacterial drugs. Background Technology

[0002] Gram-negative bacteria are common pathogens in clinical practice, seriously affecting human health and livestock breeding. Polymyxins are one of the important antibacterial drugs for treating multidrug-resistant Gram-negative bacterial infections. However, in recent years, due to non-standard drug use in clinical treatment, drug-resistant bacteria have emerged, severely weakening the clinical efficacy of polymyxins. In recent years, the development of new antibacterial drugs has stagnated; utilizing existing drugs to restore the efficacy of existing antibiotics is an economical and effective approach. Pinosylvin is a plant secondary metabolite antitoxin, mainly found in pine, eucalyptus, and spruce trees. Its structure is very similar to resveratrol, and it possesses biological activities such as preventing cardiovascular disease, antioxidation, anticancer, and treating arthritis. Furthermore, studies have shown that pinosylvin has antibacterial activity against *Escherichia coli* and *Staphylococcus aureus*, with a minimum inhibitory concentration (MIC) of 250 μg / mL for both. However, to date, the antibacterial activity of pinosylvin as an adjuvant to enhance the efficacy of antibacterial drugs has not been reported. Summary of the Invention

[0003] The purpose of this invention is to provide the application of pinocembrin combined with polymyxin in the preparation of antibacterial drugs, in order to solve the problems mentioned in the background above.

[0004] One aspect of the present invention provides the application of pinocembrin combined with polymyxin in the preparation of antibacterial drugs.

[0005] Furthermore, the antibacterial drug targets one or more of the following bacteria: Escherichia coli, Salmonella, and Klebsiella pneumoniae.

[0006] Furthermore, the antibacterial drug targets bacteria that are resistant to polymyxins.

[0007] Furthermore, the antibacterial drug targets multidrug-resistant Gram-negative bacteria.

[0008] Another aspect of the present invention provides an antibacterial drug comprising pinocembrin and polymyxin.

[0009] Furthermore, the polymyxin is polymyxin E sulfate or polymyxin B.

[0010] Furthermore, when the polymyxin is polymyxin E sulfate, the mass ratio of the red pinocembrin to polymyxin E sulfate is 5~40:0.25~16.

[0011] Furthermore, the dosage form of the drug is one of the following: premix, tablet, oral liquid, capsule, sustained-release tablet, injection, cream, or lyophilized powder for injection.

[0012] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0013] The application of pinocembrin combined with polymyxin in the preparation of antibacterial drugs provided in this invention embodiment is not a simple addition of functions, but rather achieves a synergistic antibacterial effect. This invention embodiment also discloses the antibacterial combination ratio of pinocembrin and polymyxin E sulfate, providing a new treatment strategy for the clinical treatment of bacterial infections, especially multidrug-resistant Gram-negative bacterial infections, specifically bacterial infections with polymyxin resistance genes (MCR). Attached Figure Description

[0014] Figure 1 The results of the checkerboard method for the synergistic antibacterial effect of pinocembrin combined with polymyxin E sulfate against multiple Escherichia coli provided in the embodiments of the present invention.

[0015] Figure 2 The results of the checkerboard method for the synergistic antibacterial effect of pinocembrin combined with polymyxin E sulfate against multiple Salmonella bacteria provided in the embodiments of the present invention.

[0016] Figure 3 The results of the checkerboard method for the synergistic antibacterial effect of red pinocembrin combined with polymyxin E sulfate against multiple Klebsiella pneumoniae provided in the embodiments of the present invention.

[0017] Figure 4 The synergistic bactericidal results of red pinocembrin combined with polymyxin E sulfate against Escherichia coli B2 provided in the embodiments of the present invention.

[0018] Figure 5 The results of dead cell staining tests for the control group, polymyxin E sulfate group, pinocembrin group, and polymyxin E sulfate combined with pinocembrin group provided in the embodiments of the present invention.

[0019] Figure 6 The red pinocembrin provided in this embodiment of the invention inhibits polymyxin resistance genes. mcr-1 The results of RT-qPCR expression. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0022] The pinosylvin used in the embodiments of this invention has CAS number 22139-77-1 and molecular formula C. 14 H 12 O2, molecular weight: 212.24, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity of ≥95%; a certain amount of pinocembrin was weighed and prepared into a pinocembrin stock solution with a concentration of 20 µg / mL using DMSO, and stored at -20℃; Polymyxin E sulfate (Colistin), CAS No.: 1264-72-8, Molecular Formula: C 52 H 100 N 16 O 17 S, molecular weight: 1253.53, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity ≥19,000U / mg; a certain amount of polymyxin E sulfate was weighed and prepared into a polymyxin E sulfate stock solution with a concentration of 2 µg / mL, and stored at -20℃; Pathogens in the embodiments of this invention: Escherichia coli B2, Escherichia coli EYAP10-258, Escherichia coli GZP08-8, Escherichia coli GZP11-9, Escherichia coli GZP11-6, Escherichia coli 13h1, Salmonella-white 6, Salmonella-white 8, Salmonella-white 2, Salmonella 106, Salmonella 107, Salmonella 155, Klebsiella pneumoniae 1202+45-5, Klebsiella pneumoniae K57-KP3 (CM), and Klebsiella pneumoniae K57-KP6 (CM) were all preserved by the National Veterinary Drug Safety Evaluation Center of China Agricultural University; among them, Escherichia coli EYAP10-258 and Escherichia coli GZP1 ... were preserved by the National Veterinary Drug Safety Evaluation Center of China Agricultural University. 08-8, Escherichia coli GZP11-9, Escherichia coli GZP11-6, Escherichia coli 13h1, Salmonella-white 6, Salmonella-white 8, Salmonella-white 2, and Klebsiella pneumoniae 1202+45-5 are polymyxin-resistant strains containing MCR-1; Escherichia coli B2 is a multidrug-resistant strain containing MCR-1 and blaNDM-5; Klebsiella pneumoniae K57-KP3 (CM) and Klebsiella pneumoniae K57-KP6 (CM) are polymyxin-resistant mutant strains without MCR-1; Salmonella 106, Salmonella 107, and Salmonella 155 are polymyxin-sensitive strains.

[0023] The bacterial culture medium used in the embodiments of this invention: CAMHB broth culture medium was purchased from BD Medical Devices (Shanghai) Co., Ltd., and the preparation method is as follows: weigh 22.0g into 1 L of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15 min, and set aside for later use; MH agar medium was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: weigh 38.0 g into 1 L of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15 min, cool to 50℃ and pour into plates for later use. Brain-Heart Infusion Culture Medium (BHI Broth Culture Medium) was purchased from Beijing Luqiao Technology Co., Ltd., and the preparation method is as follows: Weigh 24.5 g of this product, heat and stir to dissolve in 1 L of distilled water, autoclave at 121℃ for 15 min, and set aside.

[0024] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0025] Example 1: Evaluation of the antibacterial effect of the combined use of pinocembrin and polymyxin E sulfate: Combined Antimicrobial Index (FICI) of Pinocene and Polymyxin E Sulfate Against Pathogens: The checkerboard method was used to determine the combined antibacterial effects of Pinocene and Polymyxin E Sulfate against Escherichia coli B2, Escherichia coli EYAP10-258, Escherichia coli GZP08-8, Escherichia coli GZP11-9, Escherichia coli GZP11-6, Escherichia coli 13h1, Salmonella-white 6, Salmonella-white 8, Salmonella-white 2, Salmonella 106, Salmonella 107, Salmonella 155, Klebsiella pneumoniae 1202+45-5, Klebsiella pneumoniae K57-KP3 (CM), and Klebsiella pneumoniae K57-KP6 (CM). Specifically, *E. coli* B2, *E. coli* EYAP10-258, *E. coli* GZP08-8, *E. coli* GZP11-9, *E. coli* GZP11-6, *E. coli* 13h1, *Salmonella*-white 6, *Salmonella*-white 8, *Salmonella*-white 2, *Klebsiella pneumoniae* 1202+45-5, *Klebsiella pneumoniae* K57-KP3 (CM), and *Klebsiella pneumoniae* K57-KP6 (CM) were cultured in CAMHB broth medium under normal conditions. The checkerboard method was performed as follows: 100 μL of CAMHB broth medium was added to a 96-well plate. Pinocene (as drug A) and polymyxin E sulfate (as drug B) were serially diluted with CAMHB broth medium at maximum concentrations of 40 μg / mL and 16 μg / mL, respectively. 100 μL of CAMHB broth medium containing different concentrations of the two drugs was added along the horizontal and vertical axes of the 96-well plate. μL, then add 100 μL of bacterial suspension for each pathogen to make the final bacterial count per well 2×10. 5CFU was incubated at 37℃ for 16–18 h, and the results were observed. The minimum inhibitory concentrations (MICs) of the two drugs were recorded when used alone and in combination, and the FICI value (combination drug index) was calculated according to the following formula. Specifically, Salmonella 106, Salmonella 107, and Salmonella 155 were cultured normally using CAMHB broth. The checkerboard method was performed as follows: 100 μL of CAMHB broth was added to a 96-well plate. Pinocene (as drug A) and polymyxin E sulfate (as drug B) were serially diluted with CAMHB broth at maximum concentrations of 40 μg / mL and 1 μg / mL, respectively. 100 μL of CAMHB broth containing different concentrations of the two drugs was added along the horizontal and vertical axes of the 96-well plate, respectively. Then, 100 μL of each pathogenic bacterial suspension was added to each well, resulting in a final bacterial count of 2 × 10⁶ bacteria per well. 5 CFU was incubated at 37℃ for 16-18 hours, and the results were observed. The minimum inhibitory concentrations (MICs) of the two drugs were recorded when used alone and in combination. The FICI value (combination drug index) was calculated according to the following formula. FICI = MIC (Drug A combined) / MIC (Drug A alone) + MIC (Drug B combined) / MIC (Drug B alone). Judgment criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, no effect; FICI > 2, antagonistic effect. That is, the FICI of pinocembrin and polymyxin E sulfate = MIC (pinocembrin combined) / MIC (pinocembrin alone) + MIC (polymyxin E sulfate combined) / MIC (polymyxin E sulfate alone).

[0026] The combined anti-Escherichia coli results of pinocembrin and polymyxin E sulfate are as follows: Figure 1 As shown: like Figure 1 As shown in Figure A, it targets Escherichia coli. E. coli For strain B2, the MICs for both pinene and polymyxin E sulfate alone are >40 µg / mL and 8 µg / mL, respectively. When pinene is in the concentration range of 5-40 µg / mL and polymyxin E sulfate is in the concentration range of 0.25-4 µg / mL, the two exhibit significant synergistic or additive effects. Preferably, when pinene is at 20 µg / mL and polymyxin E sulfate is at 0.25 µg / mL, the two exhibit significant synergistic effects, with a synergistic index FICI of 0.28125. like Figure 1As shown in Figure B, for Escherichia coli strain EYAP10-258, the MICs for using pinocembrin and polymyxin E alone are >40 µg / mL and 8 µg / mL, respectively. When pinocembrin is in the concentration range of 10-40 µg / mL and polymyxin E is in the concentration range of 0.25-4 µg / mL, the two have a significant synergistic or additive effect. Preferably, when pinocembrin is at 20 µg / mL and polymyxin E is at 2 µg / mL, the two have a significant synergistic effect, with a synergistic index FICI of 0.5. like Figure 1 As shown in Figure C, for Escherichia coli GZP08-8 strain, the MICs of pinene and polymyxin E sulfate used alone are >40 µg / mL and 16 µg / mL, respectively. When pinene is in the concentration range of 5-40 µg / mL and polymyxin E sulfate is in the concentration range of 0.25-8 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinene is at 20 µg / mL and polymyxin E sulfate is at 2 µg / mL, the two have obvious synergistic effects, and the synergistic index FICI is 0.375. like Figure 1 As shown in Figure D, for Escherichia coli GZP11-9 strain, the MICs of pinene and polymyxin E sulfate used alone are >40 µg / mL and 8 µg / mL, respectively. When pinene is in the concentration range of 10-40 µg / mL and polymyxin E sulfate is in the concentration range of 0.25-4 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinene is at 20 µg / mL and polymyxin E sulfate is at 2 µg / mL, the synergistic index FICI is 0.5. like Figure 1 As shown in Figure E, for Escherichia coli GZP11-6 strain, the MICs of pinene and polymyxin E sulfate used alone are >40 µg / mL and 8 µg / mL, respectively. When pinene is in the concentration range of 10-40 µg / mL and polymyxin E sulfate is in the concentration range of 0.25-4 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinene is at 20 µg / mL and polymyxin E sulfate is at 1 µg / mL, the synergistic index FICI is 0.375. like Figure 1 As shown in Figure F, for Escherichia coli strain 13h1, the MICs of pinene and polymyxin E sulfate used alone are >40 µg / mL and 8 µg / mL, respectively. When pinene is in the concentration range of 5-40 µg / mL and polymyxin E sulfate is in the concentration range of 0.25-4 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinene is at 20 µg / mL and polymyxin E sulfate is at 2 µg / mL, the synergistic index FICI is 0.5.

[0027] The combined anti-Salmonella results of pinocembrin and polymyxin E sulfate are as follows: Figure 2 As shown: like Figure 2 As shown in Figure A, for Salmonella White 6 strain, the MICs for using pinene and polymyxin E alone are >40 µg / mL and 8 µg / mL, respectively. When pinene is in the concentration range of 5-40 µg / mL and polymyxin E is in the concentration range of 0.25-4 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinene is at 20 µg / mL and polymyxin E is at 1 µg / mL, the synergistic index FICI is 0.375. like Figure 2 As shown in Figure B, for Salmonella strain B-8, the MICs for using pinocembrin and polymyxin E alone are >40 µg / mL and 8 µg / mL, respectively. When pinocembrin is in the concentration range of 10-40 µg / mL and polymyxin E is in the concentration range of 0.25-4 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinocembrin is at 20 µg / mL and polymyxin E is at 1 µg / mL, the synergistic index FICI is 0.375. like Figure 2 As shown in Figure C, for Salmonella strain White 2, the MICs for using pinocembrin and polymyxin E alone are >40 µg / mL and 8 µg / mL, respectively. When pinocembrin is in the concentration range of 10-40 µg / mL and polymyxin E is in the concentration range of 0.25-4 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinocembrin is at 20 µg / mL and polymyxin E is at 1 µg / mL, the synergistic index FICI is 0.375. like Figure 2 As shown in Figure D, for Salmonella strain 106, the MICs for using pinocembrin and polymyxin E alone are >40 µg / mL and 0.5 µg / mL, respectively. Preferably, when pinocembrin is at 40 µg / mL and polymyxin E is at 0.25 µg / mL, the two have a significant additive effect, with a synergistic index FICI of 1. like Figure 2 As shown in Figure E, for Salmonella strain 107, the MICs for using pinene and polymyxin E alone are >40 µg / mL and 0.5 µg / mL, respectively. When pinene is in the concentration range of 5-40 µg / mL and polymyxin E is in the concentration range of 0.25 µg / mL, the two have a significant additive effect. Preferably, when pinene is at 5 µg / mL and polymyxin E is at 0.25 µg / mL, the synergistic index FICI is 0.5625. like Figure 2 As shown in Figure F, for Salmonella strain 155, the MICs for using pinene and polymyxin E alone are >40 µg / mL and 0.5 µg / mL, respectively. When pinene is in the concentration range of 5-40 µg / mL and polymyxin E is in the concentration range of 0.25 µg / mL, the two have a significant additive effect. Preferably, when pinene is at 5 µg / mL and polymyxin E is at 0.25 µg / mL, the synergistic index FICI is 0.5625.

[0028] The combined anti-Klebsiella pneumoniae treatment results of pinocembrin and polymyxin E sulfate are as follows: Figure 3 As shown: like Figure 3 As shown in Figure A, for Klebsiella pneumoniae strain 1202+45-5, the MICs of pinene and polymyxin E used alone are >40 µg / mL and 16 µg / mL, respectively. When pinene is in the concentration range of 5-40 µg / mL and polymyxin E is in the concentration range of 0.25-8 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinene is at 10 µg / mL and polymyxin E is at 1 µg / mL, the synergistic index FICI is 0.1875. like Figure 3 As shown in Figure B, for Klebsiella pneumoniae K57-KP3 (CM) strain, the MICs of pinene and polymyxin E used alone are >40 µg / mL and >16 µg / mL, respectively. When pinene is in the concentration range of 10-40 µg / mL and polymyxin E is in the concentration range of 0.25-16 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinene is at 20 µg / mL and polymyxin E is at 1 µg / mL, the synergistic index FICI is 0.28125. like Figure 3 As shown in Figure C, for Klebsiella pneumoniae K57-KP6 (CM) strain, the MICs of pinene and polymyxin E sulfate used alone are >40 µg / mL and >16 µg / mL, respectively. When pinene is in the concentration range of 10-40 µg / mL and polymyxin E sulfate is in the concentration range of 0.25-16 µg / mL, the two have obvious synergistic or additive effects. Preferably, when pinene is at 10 g / mL and polymyxin E sulfate is at 4 µg / mL, the synergistic index FICI is 0.25.

[0029] Example 2: Synergistic bactericidal analysis of pinocembrin combined with polymyxin E sulfate: 2.1 Time-based sterilization curve test: Single colonies of *E. coli* B2 were picked and cultured in BHI broth until the logarithmic growth phase, then used for later use. A mixture of pinocembrin, polymyxin E sulfate, and pinocembrin E sulfate was added to CAMHB broth, and bacterial culture was added to bring the initial bacterial count to 1 × 10⁻⁶. 6 CFU / mL.

[0030] The grouping and dosage are as follows: Control group: Contains only bacterial culture, with a final concentration of 0 for pinocembrin and polymyxin E sulfate; Colistin sulfate group: The final concentrations of polymyxin E sulfate were 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively; Pinosylvin group: The final concentrations of pinosylvin were 40 µg / mL, 80 µg / mL, and 160 µg / mL, respectively; The combined groups of polymyxin E sulfate and pinocembrin were: 0.5 μg / mL polymyxin E sulfate + 40 µg / mL pinocembrin, 1 μg / mL polymyxin E sulfate + 80 µg / mL pinocembrin, and 2 μg / mL polymyxin E sulfate + 160 µg / mL pinocembrin. After thoroughly mixing the culture medium, place it in a constant temperature incubator and incubate at 37°C. At 1 h, 3 h, 6 h, 12 h and 24 h, respectively, take 100 μL of bacterial solution and spread it on MH agar plates with a diameter of 10 cm. After incubating overnight, count the colonies.

[0031] 2.2 Test Results: like Figure 4 As shown, for Escherichia coli B2 strain, compared with the control group, polymyxin E sulfate treatment alone had no significant effect on bacterial colony count. Pinocene alone reduced the number of viable bacteria, but the reduction was weaker compared to the control group. When pinocene and polymyxin E sulfate were treated together, the number of viable bacterial colonies was significantly lower than that of the control group treated with pinocene or polymyxin E sulfate alone. Preferably, when pinocene was treated at a concentration of 160 µg / mL and polymyxin E sulfate at a concentration of 2 µg / mL for 12 h, the number of viable bacterial colonies could be reduced to 0 Log. 10 CFU / mL, meaning that this combined concentration can quickly and effectively kill bacteria; These data indicate that the combined treatment with pinocembrin and polymyxin E sulfate has a significant synergistic bactericidal effect on Escherichia coli.

[0032] Example 3: Live / dead cell staining using pinocembrin combined with polymyxin E sulfate: 3.1 Dead cell staining test using pinocembrin combined with polymyxin E sulfate: The bacterial culture was the same as in Experiment 2. After culturing at 37°C for 2 h, 1 μg / mL PI dye was added to label dead cells. The cells were then incubated at 37°C in the dark for 30 min. The bacterial activity was measured at a wavelength of 561 nm using a laser confocal microscope.

[0033] 3.2 Test Results: like Figure 5 As shown, for Escherichia coli B2 strain, compared with the control group, treatment with polymyxin E sulfate alone did not change the red fluorescence, while treatment with pinene alone significantly increased the red fluorescence. When pinene and polymyxin E sulfate were treated together, the increase in red fluorescence was even more significant, indicating that the combined use of pinene and polymyxin E sulfate increased the number of dead bacteria.

[0034] Example 4: Pinus julibrissin inhibits polymyxin resistance genes mcr-1 Express: 4.1. Escherichia coli B2 polymyxin resistance gene mcr-1 Expression level measurement: Single colonies of *E. coli* B2 were picked and inoculated into BHI broth medium and cultured with shaking at 37°C to induce the bacteria into the logarithmic growth phase. The culture was then prepared for use. A mixture of pinocembrin, polymyxin E sulfate, and pinocembrin E sulfate was added to fresh BHI broth medium, and bacterial culture was added to bring the initial bacterial count to 5 × 10⁻⁶. 8 CFU / mL.

[0035] The grouping and dosage are as follows: Control group: Contains only bacterial culture, with a final concentration of 0 for pinocembrin and polymyxin E sulfate; Colistin sulfate group: The final concentration of polymyxin E sulfate was 1 μg / mL; Pinosylvin group: Pinosylvin final concentration was 80 µg / mL; The combined group of polymyxin E sulfate and pinocembrin: the final concentration was 1 μg / mL polymyxin E sulfate + 80 µg / mL pinocembrin; After thoroughly mixing the culture medium, place it in a constant temperature incubator and incubate at 37°C for 3 hours. Centrifuge the culture medium at 8000 rpm for 10 minutes to collect the bacterial cells. Wash twice with pre-cooled PBS solution, centrifuge at 8000 rpm for 5 minutes, discard the supernatant and collect the precipitated bacterial cells. Add 1 mL of TRIzol reagent to each sample, vortex for 15 minutes, centrifuge at 12000 rpm for 8 minutes at 4°C, transfer the supernatant to a 1.5 mL centrifuge tube, add 200 μL of chloroform, vortex vigorously for 15 seconds, let stand for 2 minutes, pre-cool on ice for 4 minutes, centrifuge at 12000 rpm for 15 minutes at 4°C, transfer the supernatant to a 1.5 mL centrifuge tube, add an equal volume of isopropanol, incubate at 4°C for 3 hours, centrifuge at 12000 rpm for 15 minutes at 4°C, discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 12000 rpm for 5 minutes at 4°C. After 2 min, discard the supernatant, allow to dry completely, and add 30 μL of RNase-free water; after RNA extraction, reverse transcription is performed using the Evo M-MLV reverse transcription premix kit, and quantitative real-time PCR is performed using the SYBR Green I dye method. -ΔΔCt Methods targeting drug resistance genes mcr-1 The transcriptional level was relatively quantified.

[0036] 4.2 Test Results: like Figure 6 As shown, quantitative real-time PCR results for Escherichia coli B2 strain indicate that pinocembrin significantly inhibits polymyxin resistance genes. mcr-1 The expression.

[0037] In summary, the embodiments of the present invention achieve synergistic antibacterial effects by combining pinocembrin with polymyxin, providing a new approach for the preparation of antibacterial drugs. Antibacterial drugs can be prepared into different dosage forms by adding different excipients, carriers, etc. as needed.

[0038] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of pinocembrin combined with polymyxin in the preparation of antibacterial drugs, characterized in that, The polymyxin is polymyxin E sulfate, and the antibacterial drug targets polymyxin-resistant Escherichia coli, Salmonella, and Klebsiella pneumoniae carrying the MCR-1 gene.

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