CN115645414A - Antibacterial pharmaceutical composition and application thereof - Google Patents

Abstract

The invention provides an antibacterial pharmaceutical composition and application thereof. The invention investigates the antibacterial and anti-biofilm activity of tripterine on enterococcus and the ability of the tripterine to assist in restoring the sensitivity of VRE to vancomycin. The research result shows that the tripterine has obvious antibacterial and anti-biofilm activity on enterococcus including VRE. The sub-minimum inhibitory concentration of tripterine restores the activity of vancomycin on VRE in vitro and in vivo. The combination of vancomycin and tripterine generates a synergistic effect, and the tripterine is expected to be used as a new antibacterial agent or a sensitizer of vancomycin, so as to provide a selection scheme for the treatment of VRE.

Description

Antibacterial pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of biological medicines, in particular to an antibacterial pharmaceutical composition and application thereof.

Background

Enterococci are commensal bacteria that are predominantly present in the gastrointestinal tract of humans and animals and are considered one of the major causes of nosocomial infections (Agudelo Higuita et al, 2014, kim et al, 2019). More than 20 species of Enterococcus have been identified, of which Enterococcus faecalis (Enterococcus faecalis) and Enterococcus faecium (Enterococcus faecalis) are the most clinically relevant species, and the most common infections caused thereby are urinary tract infections, bacteremia, endocarditis and surgical site infections. The mortality rate of severe enterococcal infections is as high as 20-40% (Miro et al, 2013). Studies have shown an increasing trend in nosocomial infections with ampicillin and vancomycin resistant enterococci over the past decade (Haque et al, 2018). The emergence and spread of multidrug resistant enterococci, especially Vancomycin Resistant Enterococci (VRE), poses a serious threat to clinical therapy. The world health organization listed vancomycin-resistant enterococcus faecium on the "high priority pathogen" list (WHO, 2017). Therefore, there is a need to develop new antibacterial agents with new frameworks or to discover new strategies to treat VRE-caused infections.

The natural product provides abundant medicine sources for people since ancient times, and active compounds such as emetine, artemisinin, houttuynine sodium bisulfite and berberine are all representatives of anti-infective medicine lead compounds from plant sources. The monomer compound obtained by separating from natural products has a mother nucleus structure and an active group formed by long-term natural selection, and the biological activity expressed in the screening process has incomparable advantages compared with artificially synthesized compounds. Therefore, natural products and derivatives thereof with various structures have an extremely important position in the research of innovative medicines.

Celastrol (CEL) is a major bioactive component of tripterygium wilfordii and has received wide attention due to a variety of promising bioactivities, including anticancer, anti-inflammatory, weight-loss, cardioprotection, antibacterial, antioxidant, antiallergic, neuroprotective, antithrombotic, anti-osteoarthritis, and anti-alzheimer's disease, etc. (Hou et al, 2020). Tripterine has been reported to have anti-planktonic and anti-biofilm activity against staphylococci (Ooi et al, 2015; woo et al, 2017). At present, no relevant research report aiming at the activity of the tripterine against enterococcus exists.

Disclosure of Invention

The invention aims to provide a novel antibacterial pharmaceutical composition and application thereof.

In order to achieve the object of the present invention, in a first aspect, the present invention provides use of tripterine for preparing an antibacterial drug or composition; wherein the bacteria are enterococcus and drug-resistant bacteria thereof.

In the present invention, the enterococcus is preferably enterococcus faecalis (e.faecalis) and enterococcus faecium (e.faecalis), and a drug-resistant bacterium thereof.

Further, the drug-resistant bacteria may be selected from vancomycin-resistant enterococci (VRE) and ampicillin-resistant enterococci, such as vancomycin-resistant enterococcus faecalis and vancomycin-resistant enterococcus faecium.

In a second aspect, the invention provides an application of tripterine in preparing FtsZ protein inhibitors.

Preferably, the FtsZ protein is derived from enterococcus and resistant bacteria thereof.

In a third aspect, the invention provides a novel antibacterial pharmaceutical composition, the active ingredients of which are tripterine and vancomycin.

Furthermore, the mass ratio of the tripterine to the vancomycin is 1.

In a fourth aspect, the present invention provides the use of said composition for the preparation of an antibacterial biological product; wherein the bacteria are enterococcus and drug-resistant bacteria thereof.

Further, the tripterine and the vancomycin can be independently administered in no order, or the tripterine and the vancomycin can be simultaneously administered.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

compared with the prior art, the invention has at least the following advantages:

the invention investigates the antibacterial and anti-biofilm activity of tripterine on enterococcus and the ability of the tripterine to assist in restoring the sensitivity of VRE to vancomycin. The research result shows that the tripterine has obvious antibacterial and anti-biofilm activity on enterococcus including VRE. In vitro and in vivo, sub-MIC concentrations of tripterine (sub-minimum inhibitory concentrations) restored vancomycin activity to VRE. The combination of vancomycin and tripterine generates a synergistic effect, and the tripterine is expected to be used as a new antibacterial agent or a sensitizer of the vancomycin, so that a selection scheme is provided for the treatment of VRE.

Drawings

FIG. 1 is a graph showing the activity of CEL at various concentrations on enterococcus strains in the sterilization curve method in the preferred embodiment of the present invention. A: enterococcus faecalis ATCC700802 (MIC =4 μ g/mL); b: enterococcus faecalis ATCC51575 (MIC =2 μ g/mL); c: enterococcus faecium ATCC700221 (MIC =2 μ g/mL); d: enterococcus faecium ATCC51559 (MIC =2 μ g/mL).

Fig. 2 is a graph showing the concentration-dependent activity of CEL, vancomycin and ampicillin to clear biofilms in a preferred embodiment of the invention (n =3, P < 0.001).

FIG. 3 is a graph showing the activity of CEL and vancomycin at sub-MIC concentrations, alone and in combination, on VRE strains in preferred embodiments of the invention. A: enterococcus faecalis ATCC700802, 1/8MIC (0.5 μ g/mL) CEL and <1/128MIC (2 μ g/mL) vancomycin (CEL MIC =4mg/mL, vancomycin MIC =16 μ g/mL); b: enterococcus faecium ATCC700221, 1/4MIC CEL (0.5. Mu.g/mL) and <1/64MIC (16. Mu.g/mL) vancomycin (MIC =2mg/mL for CEL, MIC > 256. Mu.g/mL for vancomycin).

FIG. 4 is a graph showing the protective effect of CEL and vancomycin alone and in combination on enterococcus faecalis larvae of galleria mellonella in a preferred embodiment of the present invention. A: enterococcus faecalis ATCC 700802; b: enterococcus faecium ATCC 700221. Each group contained 10 larvae. The infection dose is 2X 10 ⁶ CFU/only.

FIG. 5 is a scanning electron micrograph of enterococcus faecalis ATCC700802 and Bacillus subtilis ATCC 21332 after 4 hours of non-drug treatment and CEL treatment in a preferred embodiment of the present invention. A1: enterococcus faecalis ATCC700802, control, 5000-fold magnification; a2: enterococcus faecalis ATCC700802, control, magnification 20000 times; b1: enterococcus faecalis ATCC700802, CEL treatment of 2 mu g/mL, amplification of 5000 times; b2: enterococcus faecalis ATCC700802, CEL treatment of 2 μ g/mL, magnification 20000 times; c: bacillus subtilis ATCC 21332, control; d: bacillus subtilis ATCC 21332. Mu.g/mL CEL treatment.

FIG. 6 is the docking results of CEL and FtsZ proteins in a preferred embodiment of the invention, involving the interaction of the different amino acids of CEL and FtsZ.

FIG. 7 is a graph of the surface plasmon resonance measurements obtained on FtsZ coated chips at different concentrations of CEL in a preferred embodiment of the invention.

FIG. 8 is a dose-response curve of CEL inhibiting GTPase activity of enterococcus faecalis FtsZ in a preferred embodiment of the present invention. Each point represents three independent experiments.

Detailed Description

The invention aims to research the antibacterial and anti-biofilm activity of tripterine on enterococcus and the ability of tripterine to assist in restoring the sensitivity of VRE to vancomycin.

The in-vitro antibacterial activity of the tripterine is researched by adopting Minimum Inhibitory Concentration (MIC) measurement, a biomembrane removal experiment and a time sterilization curve experiment. The synergy between the tripterine and the vancomycin is determined by adopting a chessboard method and a sterilization curve method. In vivo studies were performed on a model of infection by galleria mellonella larvae. Through molecular docking, biomolecule binding interaction and enzyme inhibition of tripterine on bacterial division protein FtsZ, the potential bacteriostatic mechanism of tripterine is discussed.

Research results show that the tripterine inhibits all tested enterococcus faecalis and enterococcus faecium strains, the MIC range is 0.5-4 mu g/mL, and the tripterine inhibits the bacterial growth in a concentration-dependent manner in the bactericidal curve analysis. Tripterine can clear more than 50% of the biofilm after 24 hours of exposure at a concentration of 16 μ g/mL. The combined use of tripterine and vancomycin showed synergistic effect on all 23 tested strains, and the median Fractional Inhibitory Concentration Index (FICI) in the chessboard test was 0.25. The combined use of sub-MIC levels of celastrol and vancomycin showed a synergistic effect in the bactericidal curve experiment and a significant protective effect in the galleria mellonella larva infection model compared to either drug alone. Tripterine has strong binding and inhibiting ability to FtsZ, kd and IC ₅₀ 1.75. + -. 0.06. Mu.M and 1.04. + -. 0.17. Mu.g/mL, respectively.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Abbreviations and terms referred to in this invention are as follows:

and VRE: vancomycin-resistant enterococci;

CEL: tripterine;

MIC: a minimum inhibitory concentration;

and (3) CFU: a colony forming unit;

FICI: fractional inhibitory concentration index;

VSE: vancomycin-sensitive enterococci;

MHA: mueller-Hinton agar;

CAMHB: cation-conditioning Mueller-Hinton broth;

BHI: brain and heart infusion;

PBS: phosphate buffered saline;

SD: standard deviation;

CLSI: the american clinical and laboratory standards institute;

SEM: scanning an electron microscope;

SPR: surface plasmon resonance.

Example 1 Tripterine bacteriostasis study

1. Materials and methods

1.1 strains and growth conditions

All strains used in the present invention are derived from the pathogenic microorganism strain collection center of the academy of Chinese medical sciences (CAMS-CCPM-A), and include 5 ATCC standard VRE strains (2 enterococcus faecium and 3 enterococcus faecalis), 18 clinical VRE strains (17 enterococcus faecium and 1 enterococcus faecalis), 5 ATCC standard vancomycin-sensitive enterococci (VSE) (2 enterococcus faecium and 3 enterococcus faecalis), and 72 clinically isolated VSE strains (31 enterococcus faecium and 41 enterococcus faecalis). The strains are usually grown in cation-regulated Mueller-Hinton broth (CAMHB) or Mueller-Hinton agar (MHA) at 37 ℃.

1.2 Chemicals and reagents

Vancomycin, ampicillin and tripterine were purchased from the pharmaceutical biologicals assay institute of china (china, beijing). Vancomycin, tripterine and ampicillin were dissolved in distilled water, dimethyl sulfoxide and phosphate buffer (pH 8.0) respectively as stock solutions at a concentration of 10mg/mL, filtered through a 0.22 μm filter and stored at-20 ℃. CAMHB, MHA and Brain Heart Infusion (BHI) for bacterial culture and antimicrobial susceptibility testing were purchased from BD corporation (Franklin Lakes, NJ, USA).

The Cytophos phosphate assay Biochem kit and recombinant enterococcus faecalis FtsZ protein (FTZ 04-B) were purchased from Cytoskeleton (Denver, colorado, USA).

1.3 sensitivity test

All strains were stored at-80 ℃ and streaked onto MHA plates for overnight culture prior to testing. The Minimum Inhibitory Concentrations (MIC) of tripterine and vancomycin were determined by broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2021) with ampicillin as the quality control antibiotic. The compound at the starting concentration of 256 μ g/mL was added to the wells of a 96-well microtiter plate and serial double dilutions were made in CAMHB medium. Add 10. Mu.L of bacterial suspension to achieve a final inoculum size of 5X 10 ⁵ CFU/mL, plates were incubated at 37 ℃ for 24 hours before MIC reading. MIC was defined as the lowest concentration of compound that inhibited bacterial growth. The vancomycin MIC break points are specified as follows: is less than or equal to 4 mu g/mL and is sensitive; 8-16. Mu.g/mL, mediator; not less than 32 mu g/mL, and drug resistance.

1.4 time sterilization Curve of Tripterine

Kinetics of bactericidal activity of celastrol were assessed by bactericidal curve experiments according to the method described by CLSI (CLSI, 1999). The experiments were performed on enterococcus faecalis ATCC700802, ATCC51575, enterococcus faecalis ATCC700221, ATCC 51559. Briefly, overnight-cultured broth was diluted to approximately 2X 10 with CAMHB ⁶ Final concentration of CFU/mL, aliquoted into sterile glass tubes, 10mL per tube. Different concentrations of tripterine (1/4 MIC, 1/2MIC, 2MIC, and 4 MIC) were obtained by adding a volume of tripterine stock solution to each tube, with tubes containing bacteria but no tripterine serving as growth controls. Bacterial counts of the inoculum were determined at 0, 2, 4, 6, 8 and 24 hours of culture. After 10-fold serial dilution of the bacterial suspension, 10. Mu.L of the sample was run down on MHA plates in triplicate and incubated at 37 ℃ for 24 hours, and viable counts were performed and recorded as log ₁₀ CFU/mL. The bactericidal activity is defined as a 3log reduction in colony counts compared to the initial inoculum size ₁₀ CFU/mL (99.9% clearance). Detection limit is 2log ₁₀ CFU/mL。

1.5 biofilm removal experiments

The study strains were subjected to the organisms described aboveMembrane removal experiments (Wang et al, 2019). Enterococcus faecalis ATCC700802 was inoculated into BHI broth and shaken overnight at 37 ℃. After diluting the bacterial suspension 1. The medium was gently removed and 100 μ L of fresh medium containing different concentrations of celastrol or antibiotic controls (3 biological replicates) were added. After another 24 hours of incubation, the media was removed, the biofilm was gently rinsed 3 times with Phosphate Buffered Saline (PBS), and heat-fixed at 60 ℃ for 1 hour. To each well was added 50 μ L of 0.06% crystal violet for 5 minutes and then washed repeatedly with distilled water to remove excess dye. Crystal violet was eluted from the stained biofilm by adding 200. Mu.L of 30% acetic acid to each well and transferred to another 96-well plate for OD ₅₉₅ And (6) reading.

1.6 statistical analysis

Statistical significance was determined using SPSS 16.0 one-way analysis of variance (ANOVA), with P values <0.05 considered statistically significant.

2. Results of the experiment

The antibacterial activity of the tripterine is as follows: MIC values of tripterine and vancomycin against 52 enterococcus faecalis and 48 enterococcus faecium are shown in Table 1. 23% of the strains (23/100) were resistant to vancomycin, including 4 enterococcus faecalis and 19 enterococcus faecium. The tripterine shows antibacterial action on all vancomycin sensitive and drug resistant strains, and the MIC range is 0.5-4 mug/mL, which possibly shows that the tripterine exerts antibacterial activity through an action mechanism different from vancomycin and is not easy to generate cross drug resistance with the vancomycin.

As shown in FIG. 1, tripterine has concentration-dependent antibacterial effect on enterococcus. Tripterine showed bactericidal effects on enterococcus faecalis ATCC700802, ATCC51575, enterococcus faecium ATCC700221, ATCC51559, respectively, at a concentration of 4MIC for all the strains tested, 24 hours after inoculation. Tripterine at a concentration of 2MIC for each strain showed bacteriostatic activity against these four strains and persisted for up to 24 hours. For MIC concentrations, enterococcus faecalis ATCC700802 was still inhibited by tripterine after 24 hours of culture, while bacterial growth was observed in the other three strains. Tripterine at 1/4 and 1/2MIC showed no significant antibacterial activity against all strains.

Biofilms are associated with refractory infections such as endocarditis, osteomyelitis, chronic wound infections, catheter-related infections, and artificial joint infections, and have also been used as models for studying drug resistance. The invention further evaluates the influence of the tripterine on the VRE biofilm. Ampicillin or vancomycin, which are commonly used antibiotics for clinical treatment of enterococcal infections, have little effect on biofilm removal. In contrast, tripterine at 16 μ g/mL cleared more than 50% of the enterococcus faecalis ATCC700802 biofilm after 24 hours of exposure (fig. 2).

TABLE 1 minimum inhibitory concentrations of Tripterine and vancomycin against 100 enterococcus standard strains and clinical isolates

Example 2 in vitro synergy of Tripterine and vancomycin

1. Experimental methods

The synergy of celastrol and vancomycin was evaluated on 23 VRE strains (including 5 standard strains and 18 randomly selected clinical isolates) using microdilution checkerboard (Wang et al, 2019). Briefly, the bacterial suspension was added to wells of a 96-well plate containing serially diluted concentrations of tripterine and vancomycin at a bacterial load of 5 × 10 ⁵ CFU/mL. The tripterine and vancomycin diluents used in the chessboard method are respectively 0.125-8 mug/mL and 0.25-256 mug/mL. After incubation at 37 ℃ for 24 hours, the combined effect of tripterine and vancomycin was analyzed using the Fractional Inhibitory Concentration Index (FICI) calculated using the following equation, with three replicates:

FICI = (MIC for drug A combination/MIC for drug A alone) + (MIC for drug B combination/MIC for drug B alone)

When the FICI is less than or equal to 0.5, synergy is achieved; the FICI is not less than 0.5 and not more than 4, and the antagonism is realized when the FICI is more than 4.

A bactericidal profile experiment was performed on enterococcus faecalis ATCC700802 and enterococcus faecium ATCC700221 to evaluate the bactericidal kinetics of tripterine and vancomycin. The study of the time sterilization curve of the combined medication is similar to that of the single use of the tripterine. Each strain included sterile liquid as a growth control. Tripterine and vancomycin were tested at sub-MIC concentrations for each drug. Colony counts were determined at 0, 2, 4, 6, 8 and 24 hours. Synergy is defined as a 24 hour reduction of viable counts of the combination of greater than or equal to 2log compared to the more active single combination ₁₀ CFU/mL, and compared with the initial inoculation amount, the count of viable bacteria in 24 hours of the simultaneous combination group is reduced by more than or equal to 2log ₁₀ CFU/mL。

2. Results of the experiment

The results of the checkerboard test are shown in table 2. When vancomycin is used in combination with 1/8-1/4MIC (0.25-1. Mu.g/mL) of tripterine, the inhibitory concentration of vancomycin against the strain is significantly reduced to its 1/128-1/4MIC (2-16. Mu.g/mL). According to the FIC index, the tripterine and vancomycin show synergistic effect on 23 strains of enterococcus when used together.

The results of the bactericidal curves of tripterine and vancomycin when combined are shown in fig. 3. At sub-MIC concentrations, vancomycin alone had poor inhibitory activity on bacterial growth. The inhibition effect of the tripterine on the growth of bacteria is weak when the tripterine is used alone, and the regeneration of the bacteria is observed 6 to 24 hours after inoculation. CFU counts using vancomycin and tripterine alone were the same as growth control levels of the respective strains over 24 hours. In contrast, the combination of tripterine and vancomycin showed strong synergistic effect on 2 strains tested. After 24 hours of culture, compared with the single use of tripterine and vancomycin, the combined use can obviously reduce the viable count by more than 3log10 CFU/mL. After 24 hours, inhibition of growth of all strains was still observed. Furthermore, the combination of CEL-vancomycin reduced the viable count by 2log10 CFU/mL compared to the initial inoculum size.

TABLE 2 chessboard test results of 23 strains of enterococcus using tripterine and vancomycin

Example 3 in vivo synergy of Tripterine and vancomycin

1. Establishment of greater wax moth larva infection model

The larvae of the galleria mellonella are kept for adaptation for 24 hours at room temperature. Larvae with a weight of 270-330 mg and a size of about 2cm were selected for the test. Enterococcus faecalis ATCC700802 and enterococcus faecium ATCC700221 were streaked on BHI agar plates and propagated overnight in BHI. The overnight cultures of both strains were washed with PBS and adjusted to the appropriate density (about 2X 10) ⁸ CFU/mL), 10 μ L of the inoculum was injected into the right hind paw of the larvae with a glass microinjector. 1 hour after infection, the larvae were injected with vancomycin, tripterine, or a combination of vancomycin and tripterine via left paw, 10 per group. In addition, 10 larvae were injected with 10 μ L PBS as a negative control. The larvae were incubated at 35 ℃ and monitored continuously for 96 hours after infection. When the larvae were unresponsive to mechanical stimuli, they were judged dead and survival curves for each group were plotted.

Multiple comparative analyses of survival data were performed by log rank test in conjunction with Bonferroni correction using the Kaplan-Meier survival analysis by GraphPad Prism 8.

2. Results

The combination of VRE strains enterococcus faecalis ATCC700802 and enterococcus faecium ATCC700221 with CEL-vancomycin with lethal infection amount is examined to show protective effect on larvae of the galleria mellonella. 2X 10 ⁶ After infection of larvae with CFU bacteria, 10. Mu.l PBS, 0.25mg/kg CEL, 40mg/kg vancomycin, or CEL-vancomycin combinations were injected.All larvae treated with PBS, CEL, or vancomycin alone died within 48 hours after VRE inoculation. The use of CEL or vancomycin alone was not sufficient to provide protection, whereas 80% of the larvae infected with enterococcus faecalis ATCC700802 and 90% of the larvae infected with enterococcus faecium ATCC700221 survived for more than 96 hours after combination, which significantly extended survival time (fig. 4). Therefore, the combined application of CEL and vancomycin has effective protection effect on lethal VRE infection.

Example 4 cellular mechanism of action study

1. Scanning Electron Microscope (SEM)

Overnight cultures of enterococcus faecalis ATCC700802 and bacillus subtilis ATCC 21332 were diluted 1 _600nm = 0.4), the bacteria were then cultured in medium with or without sub-MIC level CEL for 4h, washed with pbs, fixed with 2.5% glutaraldehyde for 24 h, dehydrated by ethanol gradient, dried, gold sprayed, and observed by scanning electron microscope (japanese hitachi SU 8020).

2. Docking analysis

Small molecule docking studies were performed using the 3D structure (PDB accession number: 5MN 4) of the FtsZ protein (ID: 60893384 in NCBI) using Discovery Studio 4.5 software. Proteins were regularized to determine the important amino acids in the predicted binding pocket. After energy minimization, all conformations of CEL were docked with the site of the selected active cavity using Libdock. Docking compounds are scored according to their binding pattern at the binding site. 3. Surface Plasmon Resonance (SPR) analysis

SPR analysis was performed on a BIAcore T200 biosensor system (Piscataway GE Healthcare Life Sciences, N.J.) at 25 ℃ using a CM5 chip. Different concentrations of CEL (25, 12.5, 6.25, 3.1, 1.6. Mu.M) were combined with FtsZ in 1 XPBS-P + (GE Healthcare Life Sciences) at a flow rate of 20. Mu.L/min for 120s. After each binding reaction, the signal was returned to baseline with a dissociation time of 60 s. Kd values were calculated using Biacore T200 evaluation software (Piscataway GE Healthcare Life sciences version 2.0, N.J.). 4. GTPase Activity assay

The GTPase activity of recombinant enterococcus faecalis FtsZ protein was determined as described in the literature using the Cytophos phosphate detection Biochemical kit (Cytoshieton, USA) (Sun et al, 2017). Enterococcus faecalis FtsZ protein (0.5. Mu.M) was incubated with solvent (1% dimethyl sulfoxide) or CEL (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32. Mu.g/mL) at different concentrations in 20mM Tris buffer (pH 7.5) for 10min at room temperature. Then 5mM MgCl was added ₂ And 200mM KCl. The reaction was started by adding 500. Mu.M GTP and reacted at 37 ℃. After 30 minutes, the reaction was stopped by adding 140. Mu.L of Cytophos reagent and incubated for 10 minutes. The inorganic phosphate was quantified by measuring the absorbance at 650nm using a microplate reader (Bio Rad laboratories, inc., UK). Relative IC was determined by non-linear regression using sigmoidal concentration response curves in GraphPad Prism 9 (GraphPad Software, la Jolla, calif.) ₅₀ The value is obtained. Three independent analyses were performed for all experiments.

5. Results of the experiment

Changes in bacterial cell morphology can often provide valuable clues to the mechanism of antibacterial action and are often used in preliminary mechanism of action studies. The mechanism of the CEL antibacterial action is deeply discussed for the first time through the observation of a scanning electron microscope on the cell morphology of bacteria. As shown in fig. 5, treatment with CEL did not cause any observable bacterial surface abnormalities compared to untreated cells. However, in the CEL treatment group, significant growth of the bacteria was observed, in the "candied gourd" shape, indicating that bacterial division was inhibited. To further confirm our hypothesis, we performed cytomorphological studies with B.subtilis. The length of bacillus subtilis increased significantly after addition of CEL compared to untreated cells. Thus, we speculate that CEL exhibits its antibacterial activity by inhibiting bacterial division.

Due to the important role and high degree of conservation in all bacteria of the filamentous temperature sensitive mutant Z (FtsZ), it has become a target of interest for new antibacterial drugs (Schaffner Barbero et al, 2012). FtsZ self-assembles into protofilaments by GTP-dependent polymerization and further into the Z-ring, a key organelle for bacterial cell division, recruiting other proteins to drive bacterial division and the formation of new cell poles together.

Based on the findings under Scanning Electron Microscopy (SEM) and the important role of FtsZ proteins in bacterial cell division, we propose the hypothesis that CEL might be antibacterial by inhibiting the function of FtsZ and its associated biological activity. We predict that FtsZ may be one of the targets of CEL antibacterial action.

The present invention provides for the first time molecular docking between CEL and FtsZ. The Discovery Studio 4.5 software was used for molecular docking. As shown in FIG. 6, CEL fits well into the active pocket of FtsZ protein with a docking score of 113.1. The predicted interactions of small molecules and proteins include two hydrogen bonds between the oxygen atom on the x-carbonyl or x-carboxyl group and the ARG143 or THR133 residues, and one Pi anionic bond between ring a and GLU 139. These interactions together promote binding, suggesting that the FtsZ protein may be a direct target for CEL.

To further demonstrate the interaction between CEL and FtsZ, the binding between the two was analyzed by SPR. The results show that CEL binds FtsZ dose-dependently with a Kd of 2.454 μ M (fig. 7), indicating that it binds FtsZ strongly.

Since the kinetics of FtsZ assembly is thought to be modulated by its GTPase activity, the inhibitory effect of CEL on the gtsz GTPase activity of enterococcus faecalis was evaluated. CEL was found to significantly inhibit the GTPase activity, IC, of the enterococcus faecalis FtsZ protein ₅₀ The value was 1.04. + -. 0.17. Mu.g/mL (FIG. 8). The good correlation between GTPase inhibitory activity and antibacterial activity indicates that the compound interferes with bacterial growth through binding to FtsZ and GTPase function inhibition mechanisms. The antibacterial mechanism by CEL, which is different from antibiotics, also explains its synergistic effect with vancomycin.

Taken together, CEL has antibacterial and anti-biofilm activity against enterococci (including VRE strains) and restores vancomycin activity against VRE in vitro and in vivo. Overall, CEL is expected to be a new antibacterial agent and adjuvant, providing a new therapeutic option against VRE.

Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims (10)

1. the application of tripterine in preparing antibacterial drugs or compositions; wherein the bacteria are enterococcus and drug-resistant bacteria thereof.

2. The use according to claim 1, wherein said Enterococcus is selected from the group consisting of Enterococcus faecalis (Enterococcus faecalis) and Enterococcus faecium (Enterococcus faecalis), and resistant bacteria thereof.

3. Use according to claim 1 or 2, wherein the drug-resistant bacteria are selected from vancomycin (vancomycin) resistant enterococci and ampicillin resistant enterococci.

4. Application of tripterine in preparation of FtsZ protein inhibitor is provided.

5. The use of claim 4, wherein the FtsZ protein is derived from enterococcus and resistant bacteria thereof.

6. The antibacterial pharmaceutical composition is characterized in that the active ingredients are tripterine and vancomycin.

7. The composition according to claim 6, wherein the mass ratio of the tripterine to the vancomycin is 1.

8. Use of a composition according to claim 6 or 7 for the preparation of an antimicrobial biological product; wherein the bacteria are enterococcus and drug-resistant bacteria thereof.

9. The use of claim 8, wherein said enterococcus is selected from the group consisting of enterococcus faecalis and enterococcus faecium, and drug-resistant bacteria thereof.

Preferably, the drug-resistant bacteria are vancomycin-resistant enterococcus faecalis and vancomycin-resistant enterococcus faecium.

10. The use according to claim 8 or 9, wherein tripterine and vancomycin are administered separately in no order, or tripterine and vancomycin are administered simultaneously.

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