CN112569223B

CN112569223B - Plant-derived flavone antibacterial compound and application thereof

Info

Publication number: CN112569223B
Application number: CN202110001827.4A
Authority: CN
Inventors: 沈建忠; 朱奎; 丁双阳
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-09-24
Filing date: 2021-01-04
Publication date: 2022-05-24
Anticipated expiration: 2041-01-04
Also published as: CN114796188A; CN112569223A

Abstract

The invention relates to a flavone antibacterial compound from plant and application thereof. The application provides 37 flavonoids compounds with gram-positive drug-resistant bacteria and 31 gram-negative drug-resistant bacteria with combined Colistin resistance to mcr positive. The compound has no hemolytic property and cytotoxicity and has better safety; can effectively inhibit infection or pollution caused by MDR gram-positive bacteria, including treating wound skin infection caused by MRSA, eliminating intestinal tract colonization of VRE and having good anti-putrefaction and disinfection effects. In addition, the flavone compound AMG or IBC is derived from plants, has small molecular weight and simple structure, and can be obtained and prepared in large quantity.

Description

Plant-derived flavone antibacterial compound and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a flavone antibacterial compound derived from plants and application thereof.

Background

The rapid spread of multiple drug-resistant bacteria poses a serious threat to global public health safety, and a novel antibacterial drug or antibacterial synergist is urgently needed. Historically, antibiotics have been discovered primarily by screening soil microorganisms for secondary metabolites. Preventing the growth of pathogenic bacteria. However, this method has high repetition rate and low yield, and the development of new sources of antibacterial compounds is urgently needed. Small molecule compounds have been previously discovered from plants, and great progress has been made in the development of drugs for different purposes. The chemical diversity of the herbal medicine demonstrates the excellent efficacy of natural compounds for the treatment of infection, particularly malaria with quinine and artemisinin. Due to the lack of the advanced immune system of mammals, plants have evolved to optimize specific drug-like molecules against bacterial diseases. However, the chemical diversity not yet developed in traditional herbal medicine has been neglected since the golden age of antibiotics.

We conclude that plants produce active compounds against bacterial pathogens. Plants are rich in a variety of polyphenolic flavonoids, including 15 carbon skeleton structures (C6-C3-C6). Flavonoids are widely found in herbs and have a variety of uses in medicine, nutrition, and agrochemical fields. We screened a number of prenyl-bearing and antibacterial flavonoid compounds, such as alpha-mangostin (AMG) and Isobavachalcone (IBC), that not only have antibacterial effects against MDR gram-positive pathogens, but also have outer membrane penetration effectiveness against MDR gram-negative pathogens or different gram-negative pathogens. In addition, further mechanism research shows that the flavonoid compound has a unique action mode in the sterilization process. Therefore, plant-derived antibacterial molecules are an undeveloped treasure house to discover new antibacterial drugs to avoid the increasing prevalence of antibiotic resistance. In conclusion, the flavone antibacterial compound and the antibacterial agent synergist can provide favorable guarantee for controlling the development of drug-resistant bacteria, and have higher development and research values.

Disclosure of Invention

The object of the present invention is to provide antibacterial preparations of plant origin.

The invention firstly provides 37 flavonoids compounds with gram-positive resistant bacteria resistance, and in addition, the research also provides 31 gram-negative resistant bacteria with combined Colistin resistance to mcr positive resistance. a) The structure of the mother ring of the flavonoid compound is as follows: the structural formula of the following figure shows a mother ring structure of the flavonoid compound: namely a series of compounds with C6-C3-C6 as basic skeletons. In the flavone mother ring structure, C3, C6, C8, C3 'and C5' are more easily substituted by isopentenyl;

The structure-activity relationship of the flavone antibacterial compound belongs to the protection scope of the invention; mainly comprises the influence of the existence, quantity and position of isopentenyl on the antibacterial activity of gram-positive bacteria, and also comprises other substituents and modifications comprising cyclization, wherein hydroxyl and methoxyl groups can influence the antibacterial activity of the isopentenyl flavonoid compound.

The application of the flavone antibacterial compound also belongs to the protection scope of the invention; the compounds may be applied b1) or b2) or b3) or b4) or b5) or b6) or b7) or b 8): b1) inhibiting bacteria; b2) preparing an antibacterial preparation; b3) preventing diseases caused by bacterial infection; b4) preparing a product for preventing diseases caused by bacterial infection; b5) treating diseases caused by bacterial infection; b6) preparing a product for treating diseases caused by bacterial infection; b7) preventing food spoilage; b8) preparing a product for preventing food spoilage.

The application of the flavone compound combined with polymyxin also belongs to the protection scope of the invention; the application of the flavone compound combined with polymyxin can be c1), c2), c3), c4), c5) or c 6): c1) inhibiting bacteria; c2) preparing an antibacterial preparation; c3) preventing diseases caused by bacterial infection; c4) preparing a product for preventing diseases caused by bacterial infection; c5) treating diseases caused by bacterial infection; c6) preparing the product for treating diseases caused by bacterial infection.

The diseases caused by any of the above bacterial infections may be in particular MRSA-induced skin tissue infections and VRE intestinal colonization.

Any of the above products may be a medicament or a vaccine.

The invention also protects the use of said flavonoid compounds for restoring the sensitivity of polymyxin-resistant gram-negative bacteria to polymyxins.

In the above application, the gram-negative bacterium is a plurality of gram-negative bacteria. The polymyxin-resistant gram-negative bacterium can be a gram-negative bacterium containing mcr drug-resistant genes. The gram-negative bacteria containing the mcr drug-resistant gene can be escherichia coli containing the mcr-1 drug-resistant gene, klebsiella pneumoniae containing the drug-resistant gene, acinetobacter baumannii containing the mcr-1 drug-resistant gene, citrobacter freundii containing the mcr-1 drug-resistant gene, uracillus ornithii containing the mcr-1 drug-resistant gene, serratia marcescens containing the mcr-1 drug-resistant gene, salmonella containing the mcr-1 drug-resistant gene, aeromonas veronii containing the mcr-3 drug-resistant gene, providencia alcaligenes containing the mcr-1 drug-resistant gene, uracilus planticola containing the mcr-1 drug-resistant gene or enterobacter cloacae containing the mcr-1 drug-resistant gene. The Escherichia coli containing the mcr-1 drug-resistant gene can be specifically Escherichia coli (Escherichia coli)12120478(mcr-1), Escherichia coli (Escherichia coli)1794(mcr-1), Escherichia coli (Escherichia coli) WZ3909(mcr-1), Escherichia coli (Escherichia coli)878(mcr-1), Escherichia coli (Escherichia coli) C3(mcr-1) or Escherichia coli (Escherichia coli) B2(NDM-5+ mcr-1). The Klebsiella pneumoniae containing drug resistance gene can be Klebsiella pneumoniae (Klebsiella pneumoniae)38 or Klebsiella pneumoniae (Klebsiella pneumoniae) 40. The Acinetobacter baumannii containing mcr-1 drug-resistant gene can be specifically Acinetobacter baumannii (Acinetobacter baumannii)17QD (mcr-1). The Citrobacter freundii containing mcr-1 drug-resistant gene can be Citrobacter freundii (Citrobacter freumdii)2AP4RC (mcr-1). The Uralensis ornithinolyticus containing mcr-1 drug-resistant gene can be specifically Uralensis ornithinolyticus (Raoultella ornithinolytica)68BC (mcr-1). The Serratia marcescens containing mcr-1 drug-resistant gene can be Serratia marcescens (Serratiamarcescens)99RC (mcr-1). The salmonella containing mcr-1 drug resistance gene can be salmonella (Salmonella enterica) SH30(mcr-1) or salmonella (Salmonella enterica) SH170 (mcr-1). The Aeromonas veronii containing mcr-3 drug-resistant gene can be specifically Aeromonas veronii 172 (mcr-3). The alkali-producing providencia strain containing mcr-1 drug-resistant gene can be specifically alkali-producing providencia strain (providencia calixafaciens) SLRD1WC (mcr-1). The Uralensis planticola containing mcr-1 drug-resistant gene can be specifically Uralensis planticola (Raoultella) DRRF15BC (mcr-1). The Enterobacter cloacae containing mcr-1 drug-resistant gene can be Enterobacter cloacae ZWRF15BC (mcr-1).

The invention also protects two antibacterial preparations with good antibacterial effect, which comprise flavone antibacterial compounds AMG and IBC.

Experiments prove that the plant-derived antibacterial molecule is an undeveloped treasury to find compounds aiming at MDR pathogenic bacteria, the research firstly provides 37 flavonoid compounds with gram-positive-resistant bacteria, and in addition, the research also provides 31 gram-negative-resistant bacteria with combined Colistin resisting mcr positive. Structurally, the position and number of isopentenyl groups plays a crucial role for antibacterial activity. In addition, other substituents and modifications including cyclization, hydroxyl and methoxy groups may also have a significant effect on the antibacterial activity of prenylated flavonoids. The research takes the compound AMG or IBC as a representative of the prenylated flavonoid compound, and finds that the compound has no hemolysis and cytotoxicity and has better safety; in animal experiments, the flavonoid AMG or IBC can effectively inhibit infection or pollution caused by MDR gram-positive bacteria, including treatment of wound skin infection caused by MRSA, elimination of intestinal colonization of VRE and better anti-putrefaction and disinfection effects. In addition, the flavone compound AMG or IBC is derived from plants, has small molecular weight and simple structure, and can be obtained and prepared in large quantity. Therefore, the invention has great application value.

Drawings

FIG. 1 shows the synergistic effect of flavonoid antibacterial compounds AMG and IBC with different classes of antibiotics.

FIG. 2 shows hemolytic tests of flavonoid antibacterial compounds AMG and IBC.

FIG. 3 shows the skin infection of the wound of mice treated with the flavone antibacterial compounds AMG and IBC; wherein PBS is PBS group, IBC is IBC treatment group, AMG is AMG treatment group, and antibiotic group is vancomycin treatment group.

FIG. 4 shows that flavonoid antibacterial compounds AMG or IBC are effective in clearing VRE intestinal colonization; wherein PBS is PBS group, IBC is IBC treatment group, AMG is AMG treatment group, and antibiotic group is tiamulin treatment group.

FIG. 5 is a model of the antiseptic decay of the flavonoid antibacterial compound AMG or IBC;

FIG. 6 shows disinfection models of flavonoid antibacterial compounds AMG or IBC.

Examples

The invention is further illustrated by the following examples. It should be understood that the method described in the examples is only for illustrating the present invention and not for limiting the present invention, and that simple modifications of the preparation method of the present invention based on the concept of the present invention are within the scope of the claimed invention.

The BALB/c female mouse is a product of experimental animal technology Limited of Beijing Wittingerli.

MHB broth medium is an aqueous solution containing 1.5g/L of beef powder, 1.5g/L of soluble starch and 17.5g/L of acid hydrolyzed casein.

Example 1 screening of plant-derived flavone antibacterial Compounds

It is estimated that there are approximately 450000 species existing in the plant kingdom, and that 301 families 3408 and 31142 vascular plants in China are described in "journal of plants in China". Flavonoids are widely distributed in plants and perform many functions. Currently, over 5000 natural flavonoids have been identified from various plants. The invention firstly searches 85 flavonoids compounds from the literature, including 23 flavones, 20 isoflavones, 13 flavanones, 10 chalcones and 19 chalcones

A ketone. Structurally, the compound mainly contains isopentene group. Statistically, these compounds were derived from 39 families, 271 different plants. Antibacterial activity against bacterial pathogens was determined according to CLSI 2019 standard guidelines (antibacterial susceptibility test execution standards), and the antibacterial effects of the flavone compounds and their synergistic antibacterial effects with various antibacterial agents (such as β -lactams, macrolides, aminoglycosides, tetracyclines, glycopeptides, quinolones, chloramphenicol, and rifamycins) were evaluated.

The method comprises the following steps: the antimicrobial activity of 85 flavonoids was tested by broth dilution (Clinical and Laboratory Standards Institute, CLSI, 2019) and tested strains were staphylococcus aureus ATCC 29213 and methicillin-resistant staphylococcus aureus (MRSA). The steps for detecting the minimum inhibitory concentration of 85 flavone compounds to the MRSA strain are as follows:

1. Suspending the test strain in MHB broth to obtain a concentration of 1X 10⁸CFU/mL of bacterial suspension.

2. One of 85 flavone compounds was dissolved in DMSO and diluted with MHB broth to give a solution of 512. mu.g/mL.

3. Adding MHB broth culture medium (100 mu L per well) into a 96-well plate, adding the compound solution prepared in the step 2 (100 mu L per well) into each well of the first row, diluting the mixture in a multiple proportion to a tenth well, discarding 100 mu L of liquid from the tenth well, then adding 100 mu L of the bacterial suspension prepared in the step 1 into each well, wherein the concentration of the final compound solution in each well is 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 mu g/mL, 64.0 mu g/mL or 128.0 mu g/mL, standing and culturing at 37 ℃ for 16-20 h, and the lowest concentration for inhibiting the bacterial growth in each well is observed to be MIC. Negative control wells and positive control wells were set in a 96-well plate. 200 μ LMHB broth was added to each negative control well. Each positive control well was added 100. mu.L of LMHB broth and 100. mu.L of the bacterial suspension prepared in step 1.

The method 2 comprises the following steps: a chessboard analysis method is adopted to detect the synergistic effect of 85 flavone compounds and different antibacterial drugs, the test strains are Escherichia coli ATCC 25922 and Escherichia coli (Escherichia coli) B2(NDM-5+ mcr-1) with multiple drug resistance, and the chessboard analysis method comprises the following specific steps:

1) Test strains were suspended in MHB broth to a concentration of 1X 10⁸CFU/mL of bacterial suspension.

2) One of 85 flavonoids was dissolved in DMSO and diluted with MHB broth to give a solution of 256. mu.g/mL of the flavonoid.

3) Taking antibacterial agent (ampicillin; ceftriaxone; erythromycin; florfenicol; gentamicin; meropenem; ofloxacin; polymyxin; rifampin; a tetracycline; florfenicol) dissolved in DMSO and diluted with MHB broth to give an antibacterial solution at a concentration of 128 μ g/mL.

4) Taking a 96-well plate, adding 100 mu of LMHB broth culture medium into each well, adding 100 mu of the flavone compound solution prepared in the step 2) into each well in the last row (the concentration of the first well is 512 mu g/mL), and diluting to the second row from the eighth row in a multiple ratio; adding 100 mu L of the antibacterial agent solution prepared in the step 3) into each well of the first row, diluting to the tenth row in a multiple ratio, adding 100 mu L of the bacterial suspension prepared in the step 1) into each well, standing and culturing at 37 ℃ for 16-20 h, and observing the lowest concentration combination of the flavone compound and the different antibacterial agents when the flavone compound and the different antibacterial agents are used in combination to inhibit the growth of bacteria. Positive control wells were set, and 100. mu.L of LMHB broth and 100. mu.L of the bacterial suspension prepared in step 1) were added to each positive control well.

The fractional bacteriostatic concentration FIC index is calculated according to the following formula:

FIC-MIC (combined application of A)/MIC (used alone A) + MIC (combined application of B)/MIC (used alone B)

The experimental results are shown in Table 1, and the results show that almost half of the flavonoids (37/85) have strong antibacterial activity against drug-sensitive Staphylococcus aureus ATCC 29213 and drug-resistant MRSA T144(1-8 μ g/mL). It is emphasized that although all flavonoids have no antibacterial effect on the gram-negative bacterium escherichia coli, one third of the flavonoids (31/85) restore the sensitivity of the E.coli B2 gene with mcr drug resistance to the antibiotic Colistin.

TABLE 1

a represents the minimum concentration of colistin required to inhibit the growth of E.coli B2 (μ g/mL) under the action of 8 μ g/mL of flavone compound; b shows FICI when colistin and a flavonoid compound (8 μ g/mL) inhibit growth of e.

Example 2 screening of plant-derived flavone antibacterial Compounds

In most flavonoids prenylation can be detected. The literature reports that the flavonoid compounds exert antibacterial activity and are related to isopentenyl. In the present study, by comparing the structure-activity relationship, we found that isopentenyl is an essential group for flavonoid compounds to exert antibacterial activity, but not all prenylated flavonoids have antibacterial activity. Of the 85 compounds, 79 compounds had isopentenyl groups, 46 of which had one isopentenyl group, 25 had two isopentenyl groups, and 8 had three isopentenyl groups. However, of the 85 compounds, 37 had antibacterial activity against MRSA, of which 36 were prenylated and 1 was not prenylated. Wherein two prenylated (69.2%, 18/26) flavone antibacterial compounds predominate compared to one or three prenylated compounds. Furthermore, C8 (44.4%, 16/36), C6 (30.6%, 11/36), C3 (22.2%, 8/36) and C3' (16.7%, 6/36) with isopentenyl groups located on the backbone of the flavonoid compound are prerequisites for their antibacterial activity, consistent with previous findings. The presence of isopentenyl groups increases the hydrophobicity of the flavonoid compounds, which results in higher affinity for bacterial biofilms and better interaction with target proteins, a currently well-established explanation for antibacterial action. In addition, the position and amount of isopentenyl groups in a particular backbone also play an important role in antibacterial activity. Of all the flavonoid compounds tested in this study, the better active candidate had two isopentenyl groups. In addition, other substituents and modifications including cyclization, hydroxyl and methoxy groups may also have a significant effect on the antibacterial activity of prenylated flavonoids.

Example 3 measurement of antibacterial Activity of flavonoid Compound AMG, IBC

In order to better find lead compounds with the best effect, the study respectively screens the morin, 5,7,4 '-trihydroxy-6, 3' -diisopentenyl isoflavone, glabrene alcohol, Isobavachalcone (IBC) and alpha-mangostin (AMG) from 5 major flavonoids. As AMG and IBC are found to have better activity against gram-positive bacteria and synergistic Colistin activity against gram-negative bacteria, and lower hemolytic property and cytotoxicity, further antibacterial activity determination is performed on AMG and IBC.

This section mainly measured the antibacterial activity of AMG and IBC against the MDR bacteria MRSA (23 strains) and VRE (54 strains). Also included are quality control strains ATCC 29213, ATCC25922, and control strain e.

The strains to be tested are detailed in Table 2.

TABLE 2

Note: a. staphylococcus aureus (Staphylococcus aureus) ATCC No.29213 and Escherichia coli (Escherichia coli) ATCC No.25922 are both deposited in the American Type Culture Collection (ATCC, address: American Type Culture Collection (ATCC)10801University Boulevard Manassas, VA 20110USA), and publicly available from the American Type Culture Collection.

b. Enterococcus faecalis (Enterococcus faecalis) VRE a4 is a drug-resistant strain of vancomycin. Staphylococcus aureus (Staphylococcus aureus) MRSA T144 is methicillin-resistant Staphylococcus aureus. The bacterial strains containing 'NDM' are all bacterial strains which produce new Delhi metallo beta-lactamase mediated beta-lactam antibiotics resistance. The strains containing the 'mcr' in the name are all strains carrying a polymyxin drug resistance gene mcr.

c. Reference 1 is Liu Y, Ding S, Dietrich R,

E，Zhu K.A biosurfactant inspired heptapeptide with improved specificity to kill MRSA[J].Angewandte Chemie international Edition, 2017, 56(6), 1486-.

d. Literature 3 is link Z, Yin W, Li H, et al, chromosome-mediated mcr-3variants in Aeromonas veronii from chip mean [ J ]. Antimicrobial agents and chemitherapy, 2017, 61 (11): e01272-17 Aeromonas veronii (Aeromonas veronii)172(mcr-3) is named Aeromonas veronii 172 in reference 3.

e. The isolation of the strains from which they were self-isolated was as follows: (1) collecting pig nose swabs in slaughterhouses and breeding farms with long distance in Shandong, Henan, Sichuan provinces, and the like, retail chicken, pork, and the like in supermarkets, soaking in BHI liquid culture medium for 5min, filtering, and storing the filtrate in ESwab tubes for later use; (2) different filtrates are dipped by an inoculating loop and spread on a Kemajia urethra positioning and developing culture medium containing polymyxin of 2 mu g/mL, the mixture is cultured for 18h at 37 ℃, monoclonal strains with red (escherichia coli), blue (Klebsiella pneumoniae, Citrobacter and Enterobacter) or semitransparent (Pseudomonas) cream color are selected and transferred to a BHI plate, and the mixture is cultured for 18h at 37 ℃. Scraping appropriate bacteria from the flat plate by using an ultra-thick filter paper sheet, transferring the bacteria into a 2ml sterile test tube, and storing the sterile test tube in a refrigerator at the temperature of minus 20 ℃ for later use; (3) selecting a monoclonal strain, inoculating the monoclonal strain into a BHI liquid culture medium for enrichment, and then extracting genome DNA; (4) taking the genomic DNA extracted in the step (3) as a template, and further confirming the genus by adopting a conventional PCR (polymerase chain reaction) 16SrRNA amplification and sequencing method; and meanwhile, detecting the amplified mcr gene.

The antibacterial activity of AMG or IBC flavonoid compounds was tested by broth dilution (Clinical and Laboratory Standards Institute, CLSI, 2019). The minimum inhibitory concentration of each strain to be detected is detected by the same steps as the method 1 of the embodiment 1:

the results are shown in Table 3. The results show that the compound AMG or IBC has good antibacterial activity on gram-positive bacteria (such as staphylococcus aureus and enterococcus faecalis) and has no antibacterial activity on gram-negative bacteria (escherichia coli). The minimum inhibitory concentration of the compound AMG or IBC to gram-positive bacteria (containing drug-resistant bacteria) is 0.5-1 mug/mL and 4-8 mug/mL respectively.

TABLE 3

MRSA, methicillin-resistant staphylococcus aureus; VRE, vancomycin-resistant enterococcus; AMG is alpha-mangostin; IBC is isobavachalcone; VAN vancomycin e. coli B2, clinical isolates carrying multiple antibiotic resistance genes, including bla_NDM-5,bla_TEM-1BMcr-1, tet (A), mdfA, arr-2, aph (4) and fosA3.

Example 4 synergistic Effect of flavone antibacterial Compounds AMG or IBC and antibacterial drugs

Tetracycline and tigecycline belong to tetracycline drugs. Florfenicol belongs to the group of chloramphenicol drugs. Ampicillin, ceftriaxone and meropenem belong to beta-lactam drugs. Erythromycin belongs to the class of macrolide drugs. Gentamicin belongs to the aminoglycoside class of compounds. Ofloxacin belongs to quinolone drugs. Polymyxins belong to cyclic peptide drugs. Rifampicin belongs to the class of rifamycins.

Synergistic effect of flavone antibacterial compound AMG or IBC and different antibiotics

First, in order to determine whether the compounds AMG and IBC have an enhancing effect on various classes of antibiotics, the multidrug-resistant bacterium e.coli B2 was tested as a model strain against antibiotics including tetracycline which inhibits protein synthesis, ofloxacin (quinolone antibiotic) which prevents DNA replication, rifampin which inhibits RNA synthesis, and ampicillin (a β -lactam antibiotic). Secondly, to verify the synergistic antibacterial effect, we further evaluated the synergistic effect against 87 drug-resistant clinical isolates of escherichia coli, 77 isolates derived from human and 10 isolates derived from animal. To further investigate the prevalence of this combination against different bacteria, we subsequently determined FIC values for 11 gram-negative bacteria, almost all of which tested had the mcr gene present.

The antibacterial agent is tetracycline, tigecycline, florfenicol, ampicillin, ceftriaxone, meropenem, erythromycin, gentamicin, ofloxacin, polymyxin, and rifampicin.

The synergistic effect of the compound AMG or IBC and different antibacterial drugs is detected by adopting a checkerboard analysis method, the test strain is Escherichia coli (Escherichia coli) B2(NDM-5+ mcr-1), and the specific steps of the checkerboard analysis method are the same as those of the method 2 in the example 1.

The test results are shown in FIG. 1. The results show that the FIC index of the compound AMG or IBC and the tested antibacterial drugs (tetracycline, tigecycline, florfenicol, ampicillin, ceftriaxone, meropenem, erythromycin, gentamicin, ofloxacin and rifampicin) are both greater than 0.5, but the FIC index of the compound AMG or IBC and the polymyxin is 0.04, so that the compound AMG or IBC and the polymyxin have good synergistic effect.

Secondly, the sensitivity of the gram-negative bacteria with the flavonoid antibacterial compound AMG or IBC for restoring the drug resistance of polymyxin to the polymyxin

The synergistic effect of the compound AMG or IBC and the antibacterial agent on mcr positive gram negative bacteria is determined by a chessboard analysis method, and the specific experimental method is the same as the method 2 of the example 1.

The results are shown in Table 4. The results show that the FIC index of the compound AMG or IBC combined with polymyxin to gram-negative bacteria sensitive or resistant to the drug is 0.03-0.26 and is far lower than 0.5. The compounds AMG or IBC thus restore the sensitivity of polymyxin-resistant gram-negative bacteria to polymyxins.

TABLE 4

Example 5 hemolytic test of flavone antibacterial Compounds AMG or IBC

The PBS buffer was 0.01M PBS buffer, pH 7.4.

The preparation method of the 8% erythrocyte suspension comprises the following steps: (1) centrifuging 10mL of defibrinated sheep blood at 3000g for 10min, and removing supernatant to obtain precipitate; (2) taking the precipitate obtained in the step (1), and washing twice by using a PBS (phosphate buffer solution) to obtain a 100% erythrocyte suspension; (3) 8mL of 100% erythrocyte suspension and 92mL of PBS buffer were mixed to obtain 8% erythrocyte suspension.

Taking the compound AMG or IBC, dissolving the compound AMG or IBC in DMSO, and then diluting the compound AMG or IBC with PBS buffer solution to obtain a compound AMG or IBC solution with the concentration of 1024 mu g/mL.

1. 100 ul/well of PBS solution was added to the first 11 columns of the 96-well plate, and 100ul of 0.2% Triton X-100 was added to the last column as a positive control.

2. After the step 1 is finished, taking the 96-well plate, adding 100 mu L of compound AMG or IBC solution into the first well, and sequentially diluting to the 10 th column in a multiple ratio manner; column 11 was used as a negative control. The concentrations of the first to tenth wells are: 512 μ g/mL, 256 μ g/mL, 128 μ g/mL, 64 μ g/mL, 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL.

3. After completing step 2, the 96-well plate was taken, 100. mu.L of 8% erythrocyte suspension was added to each well, incubated at 37 ℃ for 1h, centrifuged at 3000g for 10min, and 100. mu.L of supernatant was pipetted into a new 1.5ml centrifuge tube.

4. Measuring OD of the supernatant obtained in the step 3 by using a multifunctional microplate reader_576nmValue, i.e. OD of solution of compound AMG or IBC_576nmThe value is obtained.

5. Replacing step 2 with step a, and obtaining negative control OD without changing other steps_576nmThe value is obtained. The step a is as follows: the 96-well plate was taken and 100. mu.L of PBS buffer was added to each well.

6. Replacing step 2 with step b, and obtaining positive control OD without changing other steps _576nmThe value is obtained. The step b is as follows: the 96-well plate was taken and 100. mu.L Triton X-100 was added to each well.

The hemolysis rate and the half-hemolysis concentration Hly were calculated according to the following formula₅₀：

Hemolysis rate (Compound AMG or IBC solution OD 576)_nmValue-negative control OD_576nmvalue)/(Positive control OD_576nmValue-negative control OD_576nmValue) × 100%.

The results of some of the experiments are shown in FIG. 2. The results show that the Hly of the compounds AMG and IBC₅₀Are respectively as41.89. mu.g/mL and 391.1. mu.g/mL, with therapeutic indices of 83.78 and 97.78, respectively, both compounds have safer therapeutic indices.

EXAMPLE 6 treatment of skin infections in mice with Compounds AMG or IBC

1. Concrete steps of experimental operation

(1) Weighing and grouping the mice, wherein each group contains 10 mice;

(2) BALB/c mice were anesthetized by 1% sodium pentobarbital intraperitoneal injection at 100uL, shaved and shaved.

(3) Drawing a circle with the diameter of 0.5cm by using a pencil; one mouse had 2 wound replicates on the back; when the hair grows out near the wound, the hair is cleaned in time, and the surrounding of the wound is ensured to be clean and have no hair.

(4) Dripping 50uL of MRSA at the wound with bacteria concentration of 2X 10⁸CFU/mL. The final amount of bacteria dropped on the wound is 1 × 10⁷CFU。

(5) 1h after the challenge treatment, 10% DMSO, 2mg/kg AMG, 8mg/kg IBC, Van 1mg/kg (50 uL per drop for treatment)

(6) The wound suppuration after one day of infection is successful in molding;

(7) photographs were taken 3, 5, 7, 10, 15 days post infection, wound skin (four mice each time, four wounds) was removed, ground and colonies counted.

2. Wound sizes were counted at 3, 5, 7, 10, 15 days post infection and wound colony counts were performed.

The results of the experiment are shown in FIG. 3. The result shows that AMG and IBC can promote the healing of skin wounds caused by MRSA, have the treatment effect on a mouse skin infection model, and have the in-vivo antibacterial action similar to vancomycin.

Example 7 Compounds AMG or IBC are effective in clearing away VRE intestinal colonization

Grouping: a PBS group; AMG (1, 4mg/kg) treatment group; IBC (16, 64mg/kg) treatment group; control treatment group with tiamulin (2 mg/kg).

1. Concrete steps of experimental operation

(1) After five days of 0.5g/L AMP drinking water treatment; stopping the medicine for one day; taking feces, weighing and VRE counting (CFU/g);

(2)1×10⁹the CFUs VRE were gavaged once, 200ul of the bacterial solution was gavaged for each mouse; after 24h (i.e., before treatment), the feces were removed, weighed and VRE number (CFU/g) determined. Taking 3 per group, taking feces and intestine sections, and sending to a company for testing the flora abundance;

(3) 24h after the administration of VRE, the administration was performed by gavage (200ul), and 6 groups of mice were given PBS, AMG (1 and 4mg/kg), IBC (16 and 64mg/kg) and tiamulin (2mg/kg), respectively.

(4) At a fixed time each day, feces were taken, weighed and VRE number was measured for a total of 7 days. Counting the contents of ileum, caecum and

colon

1 and 7 days after administration, and fixing part of each intestinal segment with 4% paraformaldehyde and freezing at-80 deg.C (for remarks: administering half of the mice dissected on the first day, collecting intestinal segment, administering the rest half of the mice dissected on the seventh day, collecting intestinal segment).

(5) Feces collected before treatment, feces collected 1, 3 and 7 days after treatment and

intestinal segments

1 and 7 days after administration treatment are sent to a Xenocy lily company for 16s metagenomic sequencing, and the relative abundance and diversity of intestinal flora are measured (remark: feces or contents can be collected a little more, for example, three to four feces are collected from each mouse, and the contents in the intestinal segments are collected as much as possible).

2. Test animal preparation

Experimental animals: ICR mice 6 weeks old. Sterile water and normal rat grain are used in the experiment. The test was divided into 6 groups of 8 mice each, and 48 mice were required for the official experiment.

3. Statistics of results of experiments

Feces collected before treatment,

feces

1, 3, and 7 days after treatment, and

intestinal segments

1 and 7 days after treatment were sent to the Xenoy lily company for 16s metagenomic sequencing.

The results of the experiment are shown in FIG. 4. Oral administration of AMG or IBC results in a decrease in the concentration of colonized VRE in the gut. These results indicate that AMG or IBC can rapidly kill VRE in vivo.

EXAMPLE 8 Compound AMG or IBC preservation decay model

Next, AMG and IBC were used as food preservatives in the spoilage model.

1. Concrete steps of experimental operation

(1) 28 pork pieces (5 g each) were randomly divided into 7 groups; one group was negative control and was not treated; 200. mu.L of Staphylococcus aureus ATCC 292136X 10 was added dropwise to each of the remaining 6 groups³CFUs/mL, then 100. mu.L of AMG or IBC with different concentrations are respectively added dropwise, and the specific concentrations are as follows: 0 mug/mL of medicine; 2mg/kg of AMG; ③ 5mg/kg of AMG; IBC8 mg/kg; IBC 20 mg/kg; the concentrations of the compounds are respectively 2 × MIC and 5 × MIC; sixthly, comparison: potassium sorbate (0.075 g/kg);

(2) placing in a 37 deg.C incubator for 24 hr, observing meat quality, morphological change and odor;

(3) homogenizing pork, diluting in a gradient manner, and counting colonies by using a staphylococcus aureus chromogenic medium.

The results of the experiment are shown in FIG. 5. The result shows that AMG or IBC can effectively reduce the amount of staphylococcus aureus in a pork decay model, and the quality and the smell of meat treated by AMG or IBC are well preserved.

EXAMPLE 9 Compound AMG or IBC Sterilization model

To evaluate the effect of AMG and IBC as tableware disinfectant, the disinfecting effect of AMG and IBC on the lunch box containing MRSA was studied.

1. Concrete steps of experimental operation

(1) 28 lunch boxes are randomly divided into 7 groups; one group was negative control and was not treated; the bottom of each lunch box of the other 6 groups is coated with a lunch box containing Staphylococcus aureus ATCC 292136X 10⁶Suspending 200 mu L of CFUs/mL, and standing for 30 min;

(2) then respectively dripping 500 mu L of AMG or IBC with different concentrations, wherein the specific concentration is as follows (0 mu g/mL); 2mg/L of AMG; ③ 5mg/L of AMG; IBC 8 mg/L; IBC 20 mg/L; the concentrations of the compounds are respectively 2 × MIC and 5 × MIC; sixthly, comparison: benzalkonium bromide (2 g/L).

(3) After standing at room temperature for 2min, colony counting is carried out by using a staphylococcus aureus chromogenic medium.

The results of the experiment are shown in FIG. 6. The results show that concentrations of AMG or IBC of 2. mu.g/mL and 8. mu.g/mL, respectively, significantly reduced the number of Staphylococcus aureus on the plastic lunch box at 2 minutes and eliminated all Staphylococcus aureus in 10 minutes, similar to the effect of benzalkonium bromide. Therefore, the compound AMG or IBC has better disinfection effect.

Claims

1. Use of a composition for the manufacture of an antibacterial medicament, said composition comprising isobavachalcone and Colistin; the fungus is characterized by mcr + bla_NDM-5Coli B2.