Application of QseC inhibitor fritzin in the preparation of anti-Klebsiella pneumoniae products

Abstract

This invention discloses the application of the QseC inhibitor ferrugin in the preparation of anti-Klebsiella pneumoniae products. Compared with existing technologies, this invention demonstrates that QseC can serve as a drug target against Klebsiella pneumoniae, and ferrugin can bind to QseC, effectively inhibiting the QseC protein. Therefore, ferrugin can act as a QseC inhibitor, effectively inhibiting Klebsiella pneumoniae. This compound possesses the advantages of low toxicity, safety, and significant antibacterial effect, providing solid theoretical support and candidate compounds for the subsequent development of novel targeted antibacterial drugs against Klebsiella pneumoniae, and also offering new ideas for the development of antibiotic alternatives to combat bacterial resistance.

Description

Technical Field

[0001] This invention belongs to the field of anti-Klebsiella pneumoniae drug technology, and particularly relates to the application of the QseC inhibitor zedoariacin in the preparation of anti-Klebsiella pneumoniae products. Background Technology

[0002] As one of the most common Gram-negative opportunistic pathogens in clinical practice, Klebsiella pneumoniae is drawing increasing attention due to its growing harmfulness. In poultry such as chickens and ducks, it can cause respiratory diseases and yolk peritonitis, reducing survival and egg production rates. In livestock such as pigs and cattle, it easily induces pneumonia, mastitis, and urinary tract infections, hindering growth and development and increasing losses at slaughter. More importantly, the spread of highly virulent drug-resistant strains not only increases the cost of treatment in livestock farming, but their resistance genes may also spread through livestock products or the environment, posing a potential risk to food safety and public health. Furthermore, it exacerbates the pressure on disease control in the livestock industry, impacting the industry's economic benefits and sustainable development.

[0003] Therefore, further research is warranted on screening new drugs that can effectively combat Klebsiella pneumoniae from existing technologies. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, this invention provides the application of the QseC inhibitor, zeaxanthin, in the preparation of anti-Klebsiella pneumoniae products.

[0005] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the application of zedoaria in the preparation of anti-Klebsiella pneumoniae products.

[0007] The molecular formula of the fringe acid is C. 27 H 45 NO2, molecular weight 415.65, CAS number: 98243-57-3, chemical structure formula is shown below:

[0008]

[0009] As a specific implementation scheme, the lake fritillary glycoside can inhibit Klebsiella pneumoniae by inhibiting QseC protein.

[0010] As one implementation scheme, the use of the clam extract as the sole active ingredient in the preparation of an anti-Klebsiella pneumoniae product.

[0011] As a specific implementation plan, the product is an anti-Klebsiella pneumoniae drug, which is selected from injections, tablets, capsules, powders, granules or oral liquids.

[0012] Secondly, this invention provides the application of fringe acid in the preparation of QseC inhibitors.

[0013] As a specific implementation scheme, the QseC inhibition can inhibit Klebsiella pneumoniae.

[0014] Thirdly, the present invention provides the use of compositions containing chrysanthin in the preparation of anti-Klebsiella pneumoniae products.

[0015] As a specific embodiment, the composition containing clamin can inhibit Klebsiella pneumoniae by inhibiting QseC protein.

[0016] In one embodiment, fritzin is the sole active ingredient in the composition.

[0017] Fourthly, the present invention provides the use of compositions containing frizacin in the preparation of QseC inhibitors.

[0018] This invention evaluated the in vitro and in vivo activity of fringexin, showing that the compound exhibits extremely low cytotoxicity to Vero normal cells and good biocompatibility; it also shows no significant hemolytic activity against mouse erythrocytes at concentrations of 2.048 mg / mL and below. Drug susceptibility testing revealed that the MIC of fringexin against Klebsiella pneumoniae was 512 μg / mL. Combined drug susceptibility testing and biofilm formation assays showed that fringexin does not exhibit synergistic effects with multiple antibacterial drugs, but it significantly inhibits Klebsiella pneumoniae biofilm formation at concentrations of 32 μg / mL and above. Further in vivo evaluation experiments were conducted in mice. A systemic infection model of Klebsiella pneumoniae KPB (highly toxic and drug-resistant) was established in BALB / c mice. Treatment results showed that fringexin effectively improved the survival rate of infected mice (from 16.7% to 33.4%), significantly reduced bacterial load in major target organs such as the spleen and lungs, and exhibited a sustained and stable antibacterial effect, similar to the therapeutic effect of the QseC inhibitor LED-209. The experimental results show that QseC has the potential to be a drug target. The selected citrinin, as a potential inhibitor of this target, has the characteristics of low toxicity, good safety and significant in vitro and in vivo antibacterial effects. It provides important theoretical basis and candidate compounds for the development of new antibacterial drugs for Klebsiella pneumoniae, and also provides new strategies and ideas for dealing with the problem of bacterial multidrug resistance.

[0019] Beneficial Effects: Compared with existing technologies, this invention demonstrates that QseC can serve as a drug target against Klebsiella pneumoniae, and that fritigin can bind to QseC, effectively inhibiting the QseC protein. Therefore, fritigin can act as a QseC inhibitor, effectively inhibiting Klebsiella pneumoniae. This compound possesses the advantages of low toxicity, safety, and significant antibacterial effect, providing solid theoretical support and candidate compounds for the subsequent development of novel targeted antibacterial drugs against Klebsiella pneumoniae, and also offering new ideas for the development of antibiotic alternatives to combat bacterial resistance. Attached Figure Description

[0020] Figure 1 Visualize molecular docking.

[0021] Figure 2 This is a balance diagram of temperature fluctuations and total energy fluctuations.

[0022] Figure 3 The image shows the RMSD results for the complex.

[0023] Figure 4 The image shows the RMSF results for the complex.

[0024] Figure 5 The FIC index was used for combined drug sensitivity screening.

[0025] Figure 6 These are the results of a biofilm formation experiment.

[0026] Figure 7 This describes the toxic effects of fritter extract on Vero cells.

[0027] Figure 8 This refers to the hemolytic effect of fritter extract on erythrocytes.

[0028] Figure 9 Survival curves for KPB-infected mice.

[0029] Figure 10 The treatment survival curves for mice are shown.

[0030] Figure 11 This study measured the bacterial load in mice infected with bacteria. A: Bacterial load in liver tissue; B: Bacterial load in spleen tissue; C: Bacterial load in lung tissue.

[0031] Figure 12 The results are shown in the smear images of mouse lung tissue. A: Smear image of lung tissue at a 10⁻⁵ dilution in the control group; B: Smear image of lung tissue at a 10⁻⁵ dilution in the 10 mg / kg Hupehine group; C: Smear image of lung tissue at a 10⁻⁵ dilution in the 20 mg / kg LED-209 group. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. All equivalent substitutions, simple modifications, or conventional technical adjustments made based on the concept and principles of the present invention should fall within the scope of protection of the present invention.

[0033] This invention selected four representative Klebsiella pneumoniae strains (highly toxic and drug-resistant strain KPB, low-toxicity and drug-resistant strain ATCC 700603, highly toxic and drug-resistant strain 20k-243, and low-toxicity and drug-resistant strain 20k-58) for in-depth research based on drug susceptibility testing and virulence level detection. Complete proteomic sequences of these strains were obtained through NCBI retrieval and sequencing. Based on bioinformatics analysis, by removing paralogous proteins, identifying human non-homologous proteins, comparing metabolic pathways, identifying essential proteins, and performing subcellular localization analysis, the potential drug target QseC for Klebsiella pneumoniae was screened. A three-dimensional model of the QseC protein was constructed using SWISS MODEL homology modeling combined with GalaxyRefine optimization. After determining the optimal binding pocket, a ligand library containing 270,549 small molecules was constructed based on the ZINC15 natural product database. Through molecular docking, ADMET property screening, drug-likeness analysis, and molecular dynamics simulation, eight candidate small molecules were screened from the library. Finally, claminin was selected for in vitro and in vivo activity evaluation.

[0034] Example 1: Molecular Dynamics Simulation

[0035] This embodiment visualizes the molecular docking between fringe quinone and the target protein QseC, as shown in Figure 1. Fringe quinone has hydrogen bonds and hydrophobic bonds with amino acids GLN258, PRO251, LEU289, and ARG285.

[0036] The stability and dynamic changes of the QseC-clayoid complex were verified using molecular dynamics simulations. Figure 2 As shown in Figure 3, the time-series analysis of system temperature and total energy indicates that the system temperature stably fluctuates around the target value of 300 K, with a fluctuation range of approximately ±1.5 K. The temperature curve shows no significant drift, and the system remains in thermodynamic equilibrium throughout the simulation, demonstrating stable temperature control and energy conservation. In the overall RMSD curve of the composite, the system reaches conformational equilibrium after 25 ns, and no large-scale conformational rearrangement or dissociation occurs in subsequent simulations, proving that the composite exhibits good structural stability.

[0037] Residue-level RMSF analysis further revealed that the protein core domain remained rigid, with only localized flexibility fluctuations in the surface loop region. Furthermore, the residue fluctuations in the ligand-binding pocket region were within a reasonable range, suggesting that the ligand-protein binding mode remained stable during the simulation (Figure 4). These results collectively confirm that the QseC-claysin complex exhibits good dynamic stability under physiological conditions at 300 K, providing a reliable structural basis for subsequent binding mechanism analysis and drug optimization.

[0038] Example 2: In vitro activity evaluation of pekinensis

[0039] 1. Drug sensitivity test

[0040] (1) Strawberry strains KPB, ATCC700603, 20k-243 and 20k-58 were placed on LB agar plates and incubated at 37°C for 10 h. After that, a single strain was picked and incubated in 3 mL of fresh LB broth at 37°C for 5 h.

[0041] (2) Take 100 μL of bacterial solution and add it to 900 μL of fresh LB broth. Mix well, then take 20 μL of bacterial solution and dilute it 1,000 times in a 90 mm plate containing 20 mL of MHB broth. Use an eight-pipette to mix well.

[0042] (3) Use an eight-piece pipette to draw bacterial culture from the plate and add it to a 96-well plate, 50 μL per well, up to column 11. Add MHB blank broth to column 12 as a negative control.

[0043] (4) Add 10 μL of FFC or other drugs to the first column of the 96-well plate to make a final concentration of 512 μg / mL. Mix well by pipetting and then transfer 50 μL of the mixture to the next column for serial dilution. After dilution to the 10th column, leave 11 columns as positive controls.

[0044] (5) Seal the 96-well plate with sealing film and place it in a 37°C incubator for 20 h.

[0045] (6) Observe the results and record the minimum inhibitory concentration.

[0046] The results are shown in Table 1. Significant differences in resistance to multiple antibiotics were observed among different strains. Clinical strain KPB was sensitive to GEN, CIP, and AMP, but resistant to FFC, ERY, and MER. Strains 20k-243 and 20k-58 showed high resistance to most tested drugs, retaining sensitivity only to a few drugs such as FFC and TET. The standard strain ATCC700603 showed intermediate or resistant resistance to multiple drugs. Both fringexin and LED-209 had a MIC value of 512 μg / mL against all four strains, indicating weak antibacterial activity when used alone. This suggests that fringexin has limited antibacterial activity when used alone in vitro.

[0047] Table 1 Drug sensitivity results

[0048]

[0049] 2. Checkerboard method for drug sensitivity testing

[0050] (1) Strawberry strains KPB, ATCC700603, 20k-243 and 20k-58 were placed on LB agar plates and incubated at 37°C for 10 h. After that, a single strain was picked and incubated in 3 mL of fresh LB broth at 37°C for 5 h.

[0051] (2) Take 100 μL of bacterial solution and add it to 900 μL of fresh LB broth. Mix well, then take 20 μL of bacterial solution and dilute it 1,000 times in a 90 mm plate containing 20 mL of MHB broth. Use an eight-pipette to mix well.

[0052] (3) Use an eight-pipette to draw bacterial culture from the plate and add it to a 96-well plate, 50 μL per well, for a total of 11 columns.

[0053] (4) Add 10 μL of FFC and other drugs to the first column of the 96-well plate to make the final concentration 512 μg / mL. After mixing by pipetting, transfer 50 μL of the mixture to the next column and serially dilute to the 11th column. Set the concentration of lindane to 7 gradients and use 2 mL EP tubes to serially dilute to obtain 7 concentrations from largest to smallest.

[0054] (5) Add the corresponding high to low concentration of Fritillaria cirrhosa extract to each layer of the 96-well plate from top to bottom, for a total of 7 rows and 11 columns. At this time, the 12th column of the 96-well plate is the positive control of Fritillaria cirrhosa extract alone, and the 8th row is the positive control of drugs such as FFC.

[0055] (6) Seal the 96-well plate with sealing film and place it in a 37°C incubator for 24 h.

[0056] (7) Observe the results and record the MIC.

[0057] (8) Calculate the FIC index of antibacterial concentration: FIC = MIC (A combination) / MIC (antibiotic alone) + MIC (B combination) / MIC (compound alone).

[0058] (9) Result determination: FIC < 0.5 indicates a synergistic effect; FIC between 0.5 and 1 indicates an additive effect; FIC greater than 1 and less than 2 indicates an unrelated effect; FIC > 2 indicates an antagonistic effect.

[0059] The results are as follows Figure 5 When ferrugin was used in combination with GEN, FFC, ERY, CIP, TET, MER, and AMP, the FIC index of each combination did not meet the synergistic criteria, and no synergistic antibacterial effect was observed. The antibacterial effect of all combination groups was not significantly different from that of the single-drug groups, and no obvious additive or antagonistic effects were observed.

[0060] 3. Biofilm formation experiment

[0061] (1) Inoculate KPB onto LB agar plates and activate overnight at 37°C.

[0062] (2) Pick a single colony and inoculate it into LB broth. Incubate at 37°C and 200 rpm with shaking until the logarithmic growth phase (OD600 ≈0.4–0.6). Dilute the bacterial solution with fresh LB broth to a final concentration of 1×106 CFU / mL for later use.

[0063] (3) The inhibitory effect of the drug on KPB biofilm formation was detected by the crystal violet quantitative method in 96-well plates. Six groups were set up in the experiment: blank control group (100 μL PBS + 100 μL LB broth), positive control group (100 μL bacterial suspension + 100 μL LB broth), and drug gradient treatment group (100 μL bacterial suspension + 100 μL LB broth containing different concentrations of drug, with final concentrations of 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, and 512 μg / mL). Each group had 3 replicates.

[0064] (4) After gently mixing the 96-well plate, incubate it at 37°C for 48 h (to induce biofilm formation); after the incubation, gently discard the supernatant of each well (to remove airborne bacteria) to avoid disturbing the bottom biofilm; add 200 μL of sterile PBS buffer to each well and wash gently twice to remove unattached bacteria; after air drying at room temperature, add 200 μL of methanol to each well and fix at room temperature for 15 min. Discard the methanol, wash once with PBS, dry at 37°C, add 200 μL of 0.1% crystal violet staining solution to each well and stain at room temperature for 15 min; discard the staining solution, wash three times with PBS buffer to remove free dye, and air dry at room temperature.

[0065] (5) Add 125 μL of 30% glacial acetic acid to each well and elute by shaking at room temperature for 15-20 min to completely dissolve the crystal violet; measure the OD595 nm absorbance of each well using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the biofilm inhibition rate according to the following formula.

[0066] Biomembrane inhibition rate (%) = OD control group - OD treatment group / OD control group × 100%

[0067] The results are as follows Figure 6 As shown, using *KPB* as the target organism, the concentrations of ferruginous linteurine were set at 4 μg / ml, 8 μg / ml, 16 μg / ml, 32 μg / ml, 64 μg / ml, 128 μg / ml, 256 μg / ml, and 512 μg / ml to examine its effect on biofilm formation. The results showed that low concentrations of ferruginous linteurine (4 μg / ml, 8 μg / ml, and 16 μg / ml) had no significant inhibitory effect on bacterial biofilm formation. When the concentration increased to 32 μg / ml, biofilm formation began to decrease significantly. With further increases in concentration, the inhibitory effect gradually strengthened, showing a concentration-dependent relationship. The high concentration groups (256 μg / ml and 512 μg / ml) showed the most significant inhibitory effect on biofilm formation, with OD595 values ​​significantly lower than the control group. This indicates that ferruginous linteurine can effectively inhibit *KPB* biofilm formation, and the inhibitory ability is positively correlated with concentration.

[0068] 4. Cytotoxicity test

[0069] (1) Cell resuscitation: African monkey kidney cells (Vero) were taken out of the liquid nitrogen tank and immediately placed in a 37°C water bath to thaw until completely thawed. Then, the cells were gently transferred to a 10 mL EP tube containing 6 mL of culture medium using a pipette, centrifuged at room temperature (1,200 rpm, 3 min), the supernatant was carefully discarded, and 2 mL of fresh DMEM culture medium (containing 10% BI serum and 1% penicillin antibiotics) was added. The cells were gently resuspended by pipetting. Next, all the cells in the EP tube were transferred to a culture dish pre-filled with 6 mL of culture medium, and the dish was gently shaken to evenly disperse the cells at the bottom. Finally, the culture dish was placed in a cell culture incubator (5% CO2, 37°C) for further culture. The cells in the culture dish were shaken again in a figure-eight pattern to allow cell growth.

[0070] (2) After the cells adhere and grow, discard the culture medium in the culture dish, rinse twice with 2 mL PBS, then gently remove the PBS, add 6 mL of culture medium and continue culturing. After 24 h, change the culture medium in time and continue culturing under the above culture conditions.

[0071] (3) Passaging: When the cells have adhered to the culture dish to about 80% confluence under a microscope, discard the culture medium, wash twice with 2 mL PBS, discard the PBS after washing, add 1 mL of trypsin (add along the wall), and gently shake to digest. After digestion for 2-3 minutes (control the digestion time well), observe the cells becoming round under a microscope. Then add 2 mL of culture medium containing 10% fetal bovine serum to the culture dish to stop the digestion. Gently blow off the digested cells while rotating the dish, and finally transfer them to a 10 mL EP tube. Centrifuge at 1,200 rpm for 3 min, discard the supernatant, add 1 mL of culture medium, and mix well by pipetting.

[0072] (4) Take 500 μL of cell suspension and add it to a culture dish that has been filled with 8-9 mL of culture medium (one can be transferred to three), mix it in a figure-eight pattern, and incubate it in a 37℃ incubator.

[0073] (5) Cell counting: Take the cell suspension, dilute it by the appropriate factor, and add 10 μL to the well of the counting plate. Count diagonally (4 regions or 2 regions).

[0074] (6) 96-well plate: If there are 10,000 cells per well, 100 μL of cell suspension is plated in each well. Set up 3-6 replicate wells. Only the middle 60 wells of the 96-well plate are used. When calculating, it can be counted as 70 wells (7 mL).

[0075] (7) Assuming the cell count is X, 70 × 1 × 1,000 = X × Y (volume of cell suspension in μL). Assuming Y is 166 μL of cell suspension, then take 166 μL of suspension + 6.834 mL of culture medium. Cell count in cell suspension / mL = average cell count X × 10⁴;

[0076] (8) Adding drugs: Aspirate the supernatant culture medium from the 96-well plate and dilute the lindane (directly diluted with the basal culture medium) to different concentrations (8.192 mg / mL, 4.096 mg / mL, 2.048 mg / mL, 1.024 mg / mL, 0.512 mg / mL, 0.256 mg / mL, 0.128 mg / mL, 0.064 mg / mL). After the cells adhere to the plate for 18-24 hours, add 100 μL of different concentrations of the drug by changing the medium. Make 3 replicates per group and incubate at 37°C.

[0077] (9) Adding CCK-8 and measuring OD value: About 24 h after adding the drug, add 10 μL of CCK-8 solution, gently tap the edge of the culture plate to promote mixing, and be careful to avoid generating air bubbles (air bubbles may affect the accuracy of subsequent absorbance detection). Then incubate in a 37℃ incubator for 1-4 h, use an ELISA reader to measure the OD450 nm value, and calculate the cell viability. The calculation method is: cell viability (%) = (OD450 of drug group - OD450 of culture medium group) / (OD450 of cell group - OD450 of culture medium group) × 100%.

[0078] As shown in Figure 7, the cell viability of Vero cells reached 96% when the concentration of fringexin was 0.512 mg / mL, while the cell viability reached 100% when the concentration of fringexin was 0.032 mg / mL or lower, and the cytotoxicity was negligible. This indicates that fringexin has good safety at effective concentrations.

[0079] 5. Hemolytic test of red blood cells

[0080] (1) Preparation of red blood cell (RBC) suspension: Centrifuge 5 mL of defibrinated sheep blood at 3,500 rpm for 10 min at 4°C, remove the supernatant, gently rinse the precipitate with PBS, and centrifuge at 3,500 rpm for 10 min at 4°C. Add 300 μL of the red blood cell precipitate to 5,700 μL of PBS to prepare a 5% red blood cell suspension.

[0081] (2) Take 5% red blood cell suspension, 100 μL and place it in a 96-well plate. Add 100 μL of compound with 8.192 mg / mL (final concentration) to the first column, 50 mg / mL to the second column, and so on up to the 10th column. Add 100 μL of PBS solution per well to the 11th column as a negative control. Add 100 μL of 0.1% Triton X-100 to the last column as a positive control. Make three parallel controls.

[0082] (3) After being placed at 37°C for 1 h, centrifuged at 3,500 rpm for 10 min, 100 μL of supernatant was taken into a new 96-well plate and its absorbance at 540 nm was measured. The hemolysis rate of red blood cells was calculated as follows: Hemolysis rate (%) = (OD540 of drug-treated group – OD540 of 5% blank red blood cell suspension) / (0.1% Triton X-100 OD540 – 5% blank red blood cell suspension OD540) × 100%.

[0083] The results showed that hemolysis occurred at a concentration of 4.096 mg / mL, with a hemolysis rate of 56%; while at concentrations of 2.048 mg / mL and below, the hemolysis rate was 0. Therefore, it can be concluded that when used at concentrations of 2.048 mg / mL and below, fringexin does not cause hemolysis and has a high safety profile. Figure 8 As shown.

[0084] Example 3: In vivo activity evaluation of peony extract

[0085] Lethal dose test of strain 1 in mice

[0086] KPB strain was inoculated into LB broth and incubated at 37°C and 200 rpm for 4 hours until the bacteria reached the logarithmic phase. After incubation, the bacterial cells were collected by centrifugation at 4,000 × g for 10 min. The supernatant was discarded, and the bacterial pellet was resuspended in sterile PBS buffer, followed by centrifugation, washing, and discarding of the supernatant again. The bacterial concentration was adjusted to 2.8 × 10⁻⁶ based on the OD600 absorbance and plate count method. 8 CFU / mL. SPF-grade mice were randomly divided into 7 experimental groups, with 6 mice in each group. Except for the blank control group, the other 6 groups were injected intraperitoneally. Using a microsyringe, 100 μL of serially diluted bacterial solution was injected into each group of mice at the following concentrations: 2.8 × 10⁻⁶ CFU / mL. 6 2.8×10 5 2.8×10 4 The concentrations of the infectious bacterial solution were 2.8 × 10³, 2.8 × 10², and 2.8 × 10¹ CFU / mL. The blank control group received only 100 μL of sterile PBS. After injection, the mice were isolated and housed in a constant temperature and humidity environment. The survival status of the mice was observed and recorded 24 hours a day for 168 hours (7 days). The validity criterion for the experiment was that the number of dead mice in the blank control group did not exceed 2. This experiment aimed to determine the optimal concentration of the infectious bacterial solution that would result in a mortality rate of 80% in mice. Based on the number of mice that survived within 168 hours, survival curves were plotted using Graphpad software, and the results were analyzed.

[0087] Different bacterial strains had different lethal doses in mice. The survival curves of mice infected with the KPB strain are shown in Figure 9. When the injected bacterial solution reached 2.8 × 10⁻⁶... 4 When CFU was administered, all mice died within 48 hours; when the injected bacterial solution reached 2.8 × 10⁻⁶, the mice died. 3 When CFU was administered, all mice died within 96 hours; when the injected bacterial solution reached 2.8 × 10⁻⁶, the mice died. 2When CFU was administered, all mice died within 120 hours; when the injected bacterial solution reached 2.8 × 10⁻⁶, the mice died. 1 During CFU treatment, the survival rate of mice reached 30% at 168 h. Based on the survival curve of the mouse infection model, the bacterial concentration used in the treatment trial was determined to be 2.8 × 10⁻⁶. 1 CFU / ml.

[0088] 2. Mouse treatment trial

[0089] Based on the lethal dose results in mice, the bacterial concentration for infection and treatment was determined to be 2.8 × 10¹ CFU / mL.

[0090] A KPB bacterial suspension concentration of 2.8 × 10¹ CFU / mL was selected as the challenge dose. Animal grouping and treatment: Mice were randomly divided into 6 groups of 6 mice each. Except for the control group, all mice in each group were infected by intraperitoneal injection of 0.1 mL of the above-mentioned bacterial suspension. Two hours later, the following treatment protocols were applied: Control group: no infection, injected with an equal volume of PBS; Positive control group: injected with 0.9% NaCl saline; Drug treatment groups: intraperitoneal injection of 2.5, 5, and 10 mg / kg of Hubeienine, respectively; Negative control group: injected with 20 mg / kg LED-209. After infection, mice were returned to a constant temperature and humidity environment, and survival was observed and counted daily for 24 hours, with an observation period of 168 hours. Survival data were collected, and survival curves were plotted using GraphPad Prism software to analyze the effect of drugs on survival rate.

[0091] Survival curves of mice infected with KPB strain after treatment are shown in the figure. Figure 10 The results showed that the survival rate of mice in the 0.9% NaCl control group was low; the survival rates of the 2.5 and 5 mg / kg fritillaria cirrhosa groups were slightly improved, but by day 7, the survival rates were consistent with those of the 0.9% NaCl control group; the survival curves of the 10 mg / kg fritillaria cirrhosa group and the 20 mg / kg LED-209 group overlapped, and the survival rate of the 10 mg / kg fritillaria cirrhosa group increased from 16.7% to 33.4% compared with the 0.9% NaCl control group, showing a significant difference. No obvious toxic side effects were observed in any group, and the animals were in good condition. This indicates that fritillaria cirrhosa can effectively improve the survival rate of KPB-infected mice in vivo.

[0092] 3. Bacterial load test in mice

[0093] Based on the lethal dose results in mice, the bacterial concentration for infection and treatment was determined to be 2.8 × 10¹ CFU / mL.

[0094] A KPB bacterial suspension concentration of 2.8 × 10¹ CFU / mL was selected as the challenge dose. Animal grouping and treatment: Mice were randomly divided into 6 groups, with 6 mice in each group. Except for the blank control group, all mice in each group were infected by intraperitoneal injection of 0.1 mL of the above-mentioned bacterial suspension. Two hours later, the following treatment protocols were followed: Blank control group: no infection, injected with an equal volume of PBS; Positive control group: injected with 0.9% NaCl saline; Drug treatment groups: intraperitoneal injection of 2.5, 5, and 10 mg / kg of Hubeienine, respectively; Negative control group: injected with 20 mg / kg LED-209. At 48 h post-infection, the survival rate of mice in each group showed significant differences. To assess the early tissue bacterial load, one surviving mouse from each group was randomly selected for dissection. The liver, spleen, and lung tissues were aseptically isolated, accurately weighed, and thoroughly homogenized in a sterile homogenizer to prepare tissue homogenates. The tissue homogenates were serially diluted 10-fold (dilution factor 10). -3 10 -4 10 -5 100 μL of bacterial suspension at each dilution was spread onto LB agar plates and incubated at 37°C for 18–24 h. Colony counts (CFU / g) were then performed. Based on the plate count results, the number of viable bacteria per unit mass of tissue was calculated. GraphPad Prism software was used for visualization and statistical analysis of the bacterial load data for each group.

[0095] The results of bacterial load in mice infected with KPB strain after administration are as follows: Figure 11 As shown in Figures 1 and 12, the results indicated that at 24 h after administration, the bacterial load in the Fritillaria cirrhosa group was significantly lower than that in the PBS control group, with a statistically significant difference. At 48 h, anatomical results showed that although there was no significant change in the bacterial load in the liver tissue of each group, the bacterial loads in the spleen and lung tissues of the 10 mg / kg Fritillaria cirrhosa group and the 20 mg / kg LED-209 group were similar, both significantly lower than the blank control and low-dose groups. The plate count results were consistent with the tissue bacterial load trend, demonstrating that the drug can effectively reduce the bacterial load in vivo over a long period.

[0096] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. Application of Fritillaria cirrhosa extract in the preparation of anti-Klebsiella pneumoniae products.

2. The application according to claim 1, characterized in that, The lake fritillary glycoside can inhibit Klebsiella pneumoniae by inhibiting the QseC protein.

3. The application according to claim 1, characterized in that, The application of the lake fritillary glycoside as the sole active ingredient in the preparation of anti-Klebsiella pneumoniae products.

4. The application according to claim 1, characterized in that, The product is an anti-Klebsiella pneumoniae drug, which is selected from injections, tablets, capsules, powders, granules or oral liquids.

5. Application of Fritillaria cirrhosa extract in the preparation of QseC inhibitors.

6. The application according to claim 5, characterized in that, The QseC inhibitor can inhibit Klebsiella pneumoniae.

7. The use of compositions containing zeaxanthin in the preparation of anti-Klebsiella pneumoniae products.

8. The application according to claim 7, characterized in that, The composition containing clamin is able to inhibit Klebsiella pneumoniae by inhibiting QseC protein.

9. The application according to claim 7, characterized in that, In the composition, fringe acid is the sole active ingredient.

10. The use of compositions containing fringe acid in the preparation of QseC inhibitors.

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