Use of gramine and its combination with antibiotics in the preparation of drugs for the treatment of bacterial infectious diseases

Abstract

The application discloses application of arundine and the antibiotic in preparation of a medicine for treating a bacterial infectious disease. The arundine has synergistic activity with a plurality of antibiotics in vivo and in vitro, and the combined use has strong bacteriostatic and bactericidal activity on a plurality of drug-resistant bacteria. The effectiveness in vivo and in vitro, the stability and the safety are systematically evaluated, which is helpful to develop a new type of antibiotic adjuvant candidate for resisting multiple drug-resistant bacteria, and to relieve the problem of the increasingly serious multiple drug-resistant bacteria (MDR).

Description

Technical Field

[0001] This invention relates to the medicinal uses of arundin, and particularly to the use of arundin and its combination with antibiotics in the preparation of drugs for treating bacterial infectious diseases. Background Technology

[0002] Antibiotics play a crucial role in modern medicine, and their effective treatment of bacterial diseases has saved countless lives. However, in recent years, the large-scale and irrational use of antibiotics has led to the widespread emergence, diffusion, and transmission of antibiotic resistance, posing a significant threat to human safety and the healthy development of the livestock and poultry farming industry. New antimicrobial strategies are urgently needed to address the increasingly serious antibiotic crisis.

[0003] Although traditional Chinese medicine compounds have received increasing attention in recent years due to their good antibacterial activity and unique antibacterial mechanism, many factors still restrict their large-scale clinical use. These include relatively backward screening and synthesis methods, high synthesis costs, poor stability, non-specific toxic side effects, and the fact that some antimicrobial peptides have good in vitro activity but lack in vivo efficacy. Therefore, there is an urgent need to screen for safer, more efficient and stable plant-derived monomeric compounds to address infectious diseases caused by multidrug-resistant bacteria. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide the application of arundin in the preparation of drugs for treating bacterial infectious diseases.

[0005] Another objective of this invention is to provide the application of aconitine in combination with antibiotics in the preparation of drugs for treating bacterial infectious diseases.

[0006] Technical solution: This invention provides the application of arundin in the preparation of drugs for treating bacterial infectious diseases.

[0007] Furthermore, the bacteria are bacteria in different metabolic states.

[0008] Furthermore, the bacteria in different metabolic states include biofilms and persistent bacteria.

[0009] Furthermore, the bacteria include Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, or Proteus mirabilis.

[0010] The application of aconitine in combination with antibiotics in the preparation of drugs for treating bacterial infectious diseases.

[0011] Furthermore, the antibiotics include tigecycline and ciprofloxacin.

[0012] Furthermore, the bacteria are bacteria in different metabolic states.

[0013] Furthermore, the bacteria in different metabolic states include biofilms and persistent bacteria.

[0014] Furthermore, the bacteria include Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, or Proteus mirabilis.

[0015] Beneficial effects: This invention provides an antibacterial adjuvant that exhibits synergistic activity with multiple antibiotics both in vitro and in vivo, and has strong antibacterial and bactericidal activity against multiple drug-resistant bacteria when used in combination. The efficacy, stability, and safety of the adjuvant have been systematically evaluated in vitro and in vivo. This invention helps to develop a new class of antibiotic adjuvant candidates to combat multidrug-resistant bacteria and alleviate the increasingly serious problem of multidrug-resistant bacteria (MDR). Attached Figure Description

[0016] Figure 1 The time-killing curves of arundin combined with antibiotics were shown. The control group was nutrient-free phosphate buffer with arundin concentration of 128 μg / mL and tigecycline concentration of 16 μg / mL.

[0017] Figure 2 As shown in the anti-biofilm experiment, arundin can inhibit the formation of biofilm and remove existing biofilms.

[0018] Figure 3 The persistent bacteria experiment showed that arundin combined with antibiotics can effectively kill different types of persistent bacteria;

[0019] Figure 4 The outer membrane integrity test and the inner membrane permeability test showed that arundin can disrupt the outer membrane integrity of Klebsiella pneumoniae and increase the inner membrane permeability.

[0020] Figure 5 The PMF experiment showed that arundin can disrupt the proton kinetic potential of bacteria.

[0021] Figure 6 As a motility experiment, it was found that arundin can disrupt the motility of Klebsiella pneumoniae;

[0022] Figure 7 As shown in the efflux pump function experiment, purslane can inhibit the function of the TmexCD1-ToprJ1 efflux pump and increase the accumulation of intracellular tigecycline.

[0023] Figure 8 RT-PCR experiments showed that arundin can inhibit the expression of the tmexCD1-toprJ1 gene;

[0024] Figure 9 As shown in the bacterial respiratory level experiment, arundin can promote the respiratory level of Klebsiella pneumoniae.

[0025] Figure 10 The experiment on intracellular ATP accumulation showed that arundin can increase intracellular ATP production in Klebsiella pneumoniae.

[0026] Figure 11 The safety evaluation of arundin showed that arundin did not have any toxic side effects at the tested dosage.

[0027] Figure 12 The combined use of arundin and antibiotics to treat bacterial infections in larvae of the large wax moth showed that the combined use of arundin and tigecycline could significantly improve the survival rate of the large wax moth.

[0028] Figure 13 , 14 The combined use of arundin and antibiotics to treat peritonitis infection in mice showed that arundin can effectively clear bacteria from the mice and significantly improve their survival rate. Detailed Implementation

[0029] 1. Determination of the antibacterial spectrum and minimum inhibitory concentration of arundin

[0030] The antibacterial activity of arundin against bacteria was determined by the micro-broth dilution method. The tested strains are shown in Table 1. The arundin showed certain antibacterial activity against Gram-positive, Gram-negative, and multidrug-resistant bacteria carrying different resistance genes, especially the most serious clinically prevalent methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae carrying the TmexCD1-ToprJ1 efflux pump.

[0031] Table 1

[0032]

[0033] Note: Reference 1 is by Liu Y, Ding S, and Dietrich R. E, Zhu KA biosurfactantinspired heptapeptide with improved specificity to kill MRSA[J]. AngewandteChemie International Edition, 2017, 56(6), 1486-1490.

[0034] Document 2 is Meirong Song, Yuan Liu, Xiaoyong Huang, Shuangyang Ding, Yang Wang, Jianzhong Shen and Kui Zhu. A broad-spectrum antibiotic adjuvantreverses multidrug-resistant Gram-negative pathogens. Nat. Microbiol., 2020, 5(8): 1040-1050.

[0035] Document 3 is Peng K, Wang Q, Yin Y, et al. Plasmids shape the currentprevalence of tmexCD1-toprJ1 among Klebsiella pneumoniae in food productionchains[J].Msystems, 2021, 6(5):e00702-21.

[0036] Document 4 is ANYANWU MU, NWOBI OC, OKPALA COR, et al. Mobile tigecyclineresistance: An emerging health catastrophe requiring urgent One Health global intervention [J]. Frontiers in Microbiology, 2022, 2200.

[0037] Document 5 is Wang Q, Peng K, Liu Y, et al. Characterization of TMexCD3-TOprJ3, an RND-type efflux system conferring resistance to tigecycline in Proteusmirabilis, and its associated integrative conjugative element[J]. Antimicrobialagents and chemotherapy, 2021, 65(7): e02712-20.

[0038] The specific steps for the micro-broth dilution method are as follows:

[0039] (1) The test strains were suspended in MHB broth medium to obtain bacterial suspensions with a concentration of 1×106 CFU / mL.

[0040] (2) Take a 96-well plate, add 100 μL of MHB broth medium to each well, add 100 μL of antibacterial drug solution to each well in the first column, and serially dilute from the first column to the tenth column; then add 100 μL of the bacterial suspension prepared in step (1) to each well, and incubate at 37℃ for 16-20 h to observe the lowest concentration of aconitine that inhibits bacterial growth. Set up positive control wells, and add 100 μL of MHB broth medium and 100 μL of the bacterial suspension prepared in step (1) to each positive control well.

[0041] Table 2

[0042]

[0043] Example 2: Synergistic antibacterial activity of arundin with different types of antibiotics

[0044] The synergistic antibacterial activity of arundin and different antibiotics against TmexCD-ToprJ efflux pump-mediated Klebsiella pneumoniae was determined using checkerboard analysis.

[0045] (1) The test strain was suspended in MHB broth medium, and the bacterial concentration was 1×10⁻⁶. 6 CFU / mL bacterial suspension.

[0046] (2) Take different types of antibiotics, dissolve them in water and dilute them with MHB broth medium to obtain antibiotic solutions with a concentration of 32 μg / mL.

[0047] (3) Take alubromine, dissolve it in DMSO and dilute it with MHB broth medium to obtain an antibacterial drug solution with a concentration of 256 μg / mL.

[0048] (4) Take a 96-well plate, add 100 μL of MHB broth medium to each well, add 100 μL of the antibiotic solution prepared in step (2) to each well in the last row, serially dilute from the eighth row to the second row; add 100 μL of the antibacterial drug solution prepared in step (3) to each well in the first column, serially dilute to the seventh column, then add 100 μL of the bacterial suspension prepared in step (1) to each well, incubate at 37℃ for 16-20 h, and observe the lowest concentration combination of aconitine and various antibiotics when used together to inhibit bacterial growth. Set up positive control wells, add 100 μL of MHB broth medium and 100 μL of the bacterial suspension prepared in step (1) to each positive control well. The graded inhibition concentration FIC index is calculated according to the following formula:

[0049] FIC = MIC(A combined application) / MIC(A alone) + MIC(B combined application) / MIC(B alone)

[0050] The results showed that arundin significantly enhanced the antibacterial activity of tigecycline and ciprofloxacin against Klebsiella pneumoniae carrying different Tmex variants, with a synergistic effect ranging from 16 to 32 times. The fractional inhibitory concentration index (FIC index) of the combined use was less than 0.5, indicating a significant synergistic effect.

[0051] Table 3 Synergistic activity of arundin combined with different types of antibiotics against RGF105-1

[0052] antibiotic <![CDATA[MIC a (μg / mL)]]> FIC index <![CDATA[MIC b (μg / mL)]]> <![CDATA[Fold increase c > Tigecycline 32 0.078 1 32 tetracycline ＞128 1.5 ＞128 - Ciprofloxacin 16 0.26 1 16 Kanamycin ＞128 2 ＞128 -

[0053] a. Minimum inhibitory concentration (MIC) of an antibiotic against resistant bacteria when used alone; b. MIC of different antibiotics against resistant bacteria after the addition of arundin; c. Factor by which the antibacterial activity of an antibiotic is increased.

[0054] Example 3: Time-based bactericidal curve of arundin

[0055] Klebsiella pneumoniae RGF105-1 monoclonal strains were inoculated into MHB broth and cultured for 4-5 hours, after which the bacterial concentration was adjusted to 10. 6 CFU / mL, then treated with tigecycline or arundin alone or in combination. Subsequently, at 0h, 4h, 8h, 12h, and 24h, 50 μL of the mixture of the above compounds and bacterial culture was taken, suspended in PBS and serially diluted, dropped onto LB agar plates, and incubated overnight before colony counting.

[0056] The experimental results are shown in Figure 1 The results showed that arundin significantly enhanced the bactericidal activity of tigecycline in tmexCD1-toprJ1 positive strains.

[0057] Example 4: The inhibitory effect of arundin on biofilms and its activity in scavenging and killing persistent bacteria.

[0058] Biofilm inhibition assay: RGF105-1 was mixed with sub-inhibitory (MIC and below) concentrations of arundin and different concentrations of tigecycline in 96-well plates and incubated at 37°C for 36 h. The bacterial culture was then discarded, the plates were washed with PBS and discarded, and 50 μL of methanol was added for fixation for 15 min. After 15 min, the fixative was aspirated and air-dried. 100 μL of 0.1% crystal violet solution was added for staining for 15 min. After staining, the staining solution was aspirated, the plates were washed with PBS and air-dried. 33% acetic acid was added and the plates were incubated at 37°C for 30 min to dissolve the crystal violet. Finally, the absorbance at 576 nm was measured using a microplate reader. The experimental results are shown below. Figure 2The results showed that arundin had a concentration-dependent inhibitory effect on the formation of RGF105-1 biofilm. Specifically, the addition of arundin at 128 μg / mL significantly inhibited the formation of RGF105-1 biofilm.

[0059] Biofilm clearance experiment: RGF105-1 CFU = 10 6 The bacterial culture and MHB medium were incubated in 96-well plates at 37°C for 36 hours. Afterward, the bacterial culture was aspirated and discarded. 100 μL of different concentrations of arundin (64, 128, 256 μg / mL) were added to each well and incubated at 37°C for 2 hours. Following incubation, the plates were sonicated for 15 minutes to remove attached bacteria, and a droplet count was performed. The experimental results are shown below. Figure 2 The results showed that arundin exhibited a concentration-dependent inhibitory effect on the removal of RGF105-1 biofilm, and the addition of arundin (64 μg / mL) significantly removed the formed biofilm.

[0060] Persistent bacterial culture experiment: RGF105-1 monoclonal antibodies were inoculated into MHB broth and cultured on a shaker at 37°C for 16 h. Then, ciprofloxacin (20 MIC) or tigecycline (20 MIC) was added and the mixture was incubated at 37°C for 4 h to obtain persistent bacteria. After antibiotic treatment, the cells were centrifuged, resuspended in PBS until OD600 = 0.5, and then incubated at 37°C for 6 h. CFU was then determined by drop plate assay. Experimental results are shown below. Figure 2 The results showed that arundin combined with ciprofloxacin and tigecycline had significant bactericidal activity against persistent bacteria.

[0061] Killing effect on starvation-induced persistent bacteria: RGF105-1 monoclonal strains were incubated in MHB broth at 37°C in a shaker until the exponential phase, then washed twice with PBS, and resuspended in physiological saline and incubated at 37°C for 24 h to induce a starvation effect. Different concentrations of arundin were added and incubated at 37°C for 6 h, followed by CFU measurement by drop plate analysis. The experimental results are shown below. Figure 2 The results showed that arundin combined with ciprofloxacin and tigecycline also had significant bactericidal activity against starvation-mediated persistent bacteria.

[0062] Example 5: Analysis of Outer Membrane Integrity

[0063] Using the fluorescent probe n-phenylnaphthylamine (NPN) (10×10) -6 m) The outer membrane integrity of Klebsiella pneumoniae RGF105-1 treated with resveratrol was assessed. Fluorescence intensity was measured using an excitation wavelength of 350 nm and an emission wavelength of 420 nm. Experimental results are shown below. Figure 3 The results showed that arundin combined with tigecycline disrupted bacterial outer membrane permeability.

[0064] Example 6: Inner membrane permeability measurement

[0065] Using the fluorescent probe propidium iodide (PI) (10×10) -9 m) The inner membrane permeability of Klebsiella pneumoniae RGF105-1 treated with resveratrol was assessed. Fluorescence intensity was measured using an excitation wavelength of 535 nm and an emission wavelength of 615 nm. Results are as follows: Figure 3 The observed significant increase in fluorescence indicates that the combination of arundin and tigecycline disrupts the integrity of the bacterial inner membrane.

[0066] Example 7, PMF test

[0067] Overnight cultured Klebsiella pneumoniae RGF105-1 was diluted 1:100 in blank MH broth and incubated at 37°C and 200 rpm for 4 hours before cell collection. The suspension was then washed with PBS and resuspended to an OD600 of 0.5. A final concentration of 0.5 μM of the fluorescent dye DiSC3(5) was added, and the mixture was incubated at 37°C for 30 minutes. The probe-labeled bacterial cells were seeded into 96-well plates, and the combined drug (16 μg / mL tigecycline + different concentrations of arundin) was added. Fluorescence units at an excitation wavelength of 622 nm and an emission wavelength of 670 nm were measured using a microplate reader. Results are as follows: Figure 4 As shown, arundin can disrupt the proton kinetic potential of bacteria.

[0068] Simultaneously using the pH-sensitive fluorescent probe BCECF-AM (20×10⁻⁶) -6 m) The intracellular pH of Klebsiella pneumoniae treated with resveratrol was tested. The method was as described above, and the results were as follows: Figure 4 As shown, the combined use of arundin and tigecycline increased the intracellular ΔpH of the lung, which could promote the uptake of tigecycline by bacteria.

[0069] Example 8, Motion Analysis

[0070] Klebsiella pneumoniae RGF105-1 was cultured at 37℃ and 200 r / min for about 6 h, and the bacterial concentration was adjusted to OD. 600 To ensure the bacteria are in the logarithmic growth phase, the bacterial culture should be kept at approximately 0.5 μL. Prepare fresh 0.3% agar plates containing different concentrations of arundin, and inoculate 2 μL of the bacterial culture into the center of the plate using a pipette tip to puncture the agar plate. Set up three replicates, repeating the experiment three times. After incubating at 37°C for 48 hours, a bacterial zone should form around the inoculation site on the plate, indicating motility. The size of the bacterial zone represents the strength of the bacterial community's motility. Figure 5 As shown, with increasing doses of arundin, lung function was impaired.

[0071] Example 9: Functional Measurement of the External Drainage Pump

[0072] Overnight cultured Klebsiella pneumoniae RGF105-1 was diluted 1:100 in blank MH broth and incubated at 37°C and 200 rpm for 4 hours on a shaker before cell collection. The suspension was then washed with PBS and resuspended to an OD600 of 0.5. Ethidium bromide (EtBr) and different concentrations of arundin or a combination of these drugs were incubated for 30 minutes. The suspension was then centrifuged at 5000g for 10 minutes at 4°C, collected, and resuspended in PBS. Efflux of EtBr from the bacterial cells was monitored over 60 minutes using an excitation wavelength of 530 nm and an emission wavelength of 600 nm. Results are as follows: Figure 6 As shown, arundin can significantly inhibit the efflux of EtBr, and the TmexCD1-ToprJ1 efflux pump function is suppressed.

[0073] Example 10: Determination of intracellular accumulation of tigecycline

[0074] Overnight cultured Klebsiella pneumoniae RGF105-1 was diluted 1:100 in fresh LB broth and incubated at 37°C and 200 rpm for 6 hours. The bacteria were then centrifuged at 5000 rpm for 10 minutes at 4°C, and the supernatant was discarded. The precipitate was resuspended in PBS, and the bacteria were concentrated to a final concentration of 10. 10 CFU / mL was aliquoted into 2mL centrifuge tubes. Tigecycline was added to a final concentration of 16μg / mL, and the sample was incubated at 37℃ and 200 rpm for 1 hour on a shaker. After incubation, the bacteria were precipitated by centrifugation at 12,000 rpm for 10 minutes. The precipitate was dissolved in 1mL of water, and then subjected to three freeze-thaw cycles (3 minutes each) in liquid nitrogen, followed by lysis in a 55℃ water bath for 3 minutes. The sample was then centrifuged at 12,000 rpm for 10 minutes at room temperature, and the supernatant was collected. The remaining precipitate was resuspended in 1mL of acetonitrile, and the supernatant was collected by vortexing and centrifugation. The supernatant was combined with the previously collected supernatant. The sample was filtered through a 0.22μm syringe filter, and the filtrate was collected for LC-MS / MS analysis.

[0075] The content of tigecycline in the supernatant was quantitatively analyzed by LC-MS / MS. LC separation conditions: mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (acetonitrile solution); column: C18 column; flow rate: 0.5 mL / min; gradient elution ratios as follows: 0–0.5 min, 90% A; 0.5–1.5 min, 90–10% A; 1.5–5.0 min, 10% A; 5.0–5.5 min, 10–90% A; and 5.5–7.0 min, 90% A; injection volume: 10 μL. Intracellular antibiotic content was quantified using multiple reaction monitoring (MRM) in positive ion mode (m / z 586.4→513.3).

[0076] like Figure 6 As shown, arundin increases the accumulation of tigecycline in lung cells in a dose-dependent manner.

[0077] Example 11, RT-PCR analysis

[0078] Overnight cultured Klebsiella pneumoniae RGF105-1 was diluted 1:100 in fresh LB broth, and different concentrations of tigecycline and arundin were added. The mixture was incubated at 37°C and 200 rpm for 6 hours, with three replicates per group. Total bacterial RNA was extracted using the EASY spin Plus kit.

[0079] Before cDNA synthesis, RNA from all bacterial cells was adjusted to the same concentration. Reverse transcription of the RNA was performed using the PrimeScript™ RT kit (Takara).

[0080] The mRNA level of tmexCD1-toprJ1 gene relative to the control gene in RGF105-1 was determined by RT-PCR. RT-PCR was performed using the SYBR Green qPCR kit (Takara). Thermal cycling was performed using a two-step PCR amplification standard method: 95°C for 30 seconds and 40 cycles of 95°C for 5 seconds, 60°C for 34 seconds. Detection was performed using real-time quantitative PCR (PT-PCR) and 2- ΔΔct The method determines fold changes in gene expression.

[0081] like Figure 7 As shown, the addition of arundin can inhibit the expression of the tmexCD1-toprJ1 gene.

[0082] Example 12: Cellular Respiration Level Test

[0083] Overnight cultured Klebsiella pneumoniae RGF105-1 was diluted 1:100 in blank MH broth and incubated at 37°C and 200 rpm for 4 hours. The bacterial cells were then collected. The cells were washed three times with PBS and resuspended in PBS to a DD 600 of 0.5. The bacterial culture was plated into 96-well plates, and different concentrations of arundin or combined drugs were added. Resazurin solution was then added to a final concentration of 0.01 mg / mL. Fluorescence units at an excitation wavelength of 550 nm and an emission wavelength of 590 nm were measured within 30 minutes using a microplate reader. Figure 8 As shown, arundin can increase the respiratory level of bacteria.

[0084] Example 13, ATP test

[0085] Intracellular ATP levels in Klebsiella pneumoniae RGF105-1 cells were detected using an ATP assay kit. Overnight cultured RGF105-1 cells were diluted 1:100 in blank MH broth and incubated at 37°C with a shaker at 200 rpm for 4 hours before cell collection. Cells were washed three times with PBS and resuspended in PBS to OD. 600 The solution was diluted to 0.5 and aliquoted into 2 mL centrifuge tubes. Next, different final concentrations of arundin (64, 128, 256, 512 g / mL), or a combination of drugs, were added. After incubation for 1 hour, the cells were washed three times with PBS, and 200 μL of lysis buffer was added to each tube. The cells were then vortexed thoroughly to lyse them. The cells were centrifuged at 12,000 rpm, 40 °C, for 5 minutes, and the supernatant was collected as a sample.

[0086] Preparation of standard curve: Dilute the ATP standard in the kit with lysis buffer to seven concentration gradients of 0.01, 0.03, 0.1, 0.3, 3 and 10 μM.

[0087] like Figure 9 As shown, arugula alkaloids can increase ATP production in Klebsiella pneumoniae RGF105-1 cells.

[0088] Example 14, Safety Evaluation

[0089] Preparation of 4% erythrocyte suspension: Fresh defibrinated sheep blood was added to PBS buffer (final concentration 4%) and centrifuged at 4°C, 1500 rpm for 10 min. After centrifugation, the supernatant was removed, and the sediment was washed repeatedly with the same volume of 1×PBS buffer by gently inverting the container. Centrifugation was continued at 4°C, 1500 rpm for 10 min, and repeated until the supernatant was no longer turbid. Finally, the supernatant was discarded, and an equal volume of 1×PBS buffer was added and gently mixed before use.

[0090] Determination of erythrocyte hemolysis rate: The procedure was performed in 96-well cell culture plates. 50 μL of PBS buffer was added to columns 2 to 10. 100 μL of the compound diluted to 1024 μg / mL was added to the first column. Three parallel groups were set up for each concentration. The mixture was serially diluted to the last column and the excess liquid was discarded. PBS buffer was added to the negative control group and distilled water was added to the positive control group.

[0091] 150 μL of red blood cell suspension was transferred to the corresponding well of a serially diluted 96-well plate. After incubation at 37°C for 1 h, the 96-well cell culture plate was removed, centrifuged for 5 min, and 100 μL of supernatant was transferred to a new 96-well plate. The absorbance at 540 nm was measured.

[0092] Result determination: Red blood cell hemolysis rate = (A-A0) / (A total-A0)×100%, where A is the absorbance of the test drug, A0 is the absorbance of the negative control group, and A total The absorbance is the value of the positive control group.

[0093] CCK8 cell proliferation experiment

[0094] The cytotoxic effect of aconitine on RAW264.7 (mouse mononuclear macrophage leukemia cells) was detected using the CCK8 assay kit. The experimental protocol is as follows:

[0095] Cells were resuscitated and cultured in DMEM at 37°C with 5% CO2. Cells in the logarithmic growth phase were collected for later use. Cell suspension (200 μL / well, 2000 cells / well) was seeded in 96-well plates and cultured for 48 h. The culture medium was then aspirated, and the plates were washed with PBS buffer. Different concentrations of arugula alkaloids were added to each well for testing, and the plates were incubated for 24 h. After the test time, 20 μL of CCK8 solution was added to each well. After adding CCK8, incubation continued for one hour. The absorbance of the solution at 450 nm was measured using a microplate reader.

[0096] The results are as follows Figure 10 As shown, the hemolysis rate of arundin was below 5%, indicating high safety. Arundin showed no cytotoxicity to RAW264.7 cells at concentrations below 512 μg / mL.

[0097] Example 15: Treatment of bacterial infection in larvae of the large wax moth using a combination of arundin and tigecycline.

[0098] Arugula alkaloid 50mg / kg: 58.6μL of the drug with a stock solution concentration of 25.6mg / mL was dissolved in 1mL of PBS.

[0099] Tigecycline 50 mg / kg: Dissolve 1500 μg of tigecycline in 1 mL of water.

[0100] Arugula alkaloid + tigecycline 50mg / kg + 50mg / kg (1500μg / mL + 1500μg / mL).

[0101] Bacterial suspension: RGF105-1 was resuspended in PBS buffer to obtain a bacterial suspension; the bacterial concentration was adjusted to 1.0×107 CFU / mL.

[0102] 1. Grouping and treatment of large wax moth larvae

[0103] Forty larvae of the large wax moth, each weighing 300 mg, were randomly divided into four groups: Vehicle group, treated with arundin 50 mg / kg, treated with tigecycline 50 mg / kg, and treated with tigecycline + arundin 50 mg / kg (n=10 per group). The following treatments were administered:

[0104] RGF105-1:

[0105] Vehicle group: The last abdominal leg on the lower left of the larvae of the large wax moth was injected with 0.01 mL of LGF105-1 bacterial suspension; 1 hour later, the last abdominal leg on the lower right was injected with 0.01 mL of PBS buffer.

[0106] Treatment group: 0.01 mL of LGF105-1 suspension was injected into the last abdominal leg on the lower left side of the larvae of the large wax moth; 0.01 mL of LI14 solution with a concentration of 1500 μg / mL was injected into the last abdominal leg on the lower right side 1 hour later.

[0107] Tigecycline 50 mg / kg (1500 μg / mL) treatment group: 0.01 mL of LRGF105-1 suspension was injected into the last abdominal leg on the lower left side of the larvae of the large wax moth; 1 hour later, 0.01 mL of LI14 solution with a concentration of 1500 μg / mL was injected into the last abdominal leg on the lower right side.

[0108] Synergistic treatment group: 0.01 mL of RGF105-1 suspension was injected into the last abdominal leg on the lower left side of the larvae of the large wax moth; 1 hour later, 0.01 mL of tigecycline + 50 mg / kg (1500 μg / mL + 1500 μg / mL) mixture of aconitine was injected into the last abdominal leg on the lower right side.

[0109] 2. Statistical survival rate

[0110] The survival rate of the large wax moth larvae was counted on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th days after completing step 1.

[0111] The experimental results are shown in Figure 11 The results showed that all larvae in the arundin monotherapy group died within two days, and the survival rate in the tigecycline treatment group was also less than 20%. However, in the synergistic treatment group, the survival rate of the giant wax moth was significantly higher than that of tigecycline (50 mg / kg) and arundin (50 mg / kg) monotherapy, with a survival rate of 100%.

[0112] Example 16: Treatment of mouse peritonitis with arundin

[0113] 1. Grouping and treatment of mice

[0114] Twenty-four mice were randomly divided into four groups: vehicle group, arundin 20 mg / kg, tigecycline 20 mg / kg, arundin + tigecycline 20 mg / kg + 20 mg / kg (n=6 per group). All mice were intraperitoneally injected with 0.2 mL of RGF105-1 bacterial suspension (7 × 10⁻⁶ mg / kg). 8 CFUs). One hour after infection, patients were treated with PBS, arundin (20 mg / kg), tigecycline (20 mg / kg), or arundin + tigecycline (20 mg / kg + 20 mg / kg), respectively.

[0115] 2. Statistical analysis of survival rate and measurement of bacterial load.

[0116] The experimental results are shown in Figure 12 It was found that the survival rates of mice in the arundin-treated group and the tigecycline-treated group were both below 30%. However, the combined use of arundin and tigecycline significantly improved the survival rate, reaching 80%. Furthermore, the bacterial load in the internal organs (heart, liver, spleen, lungs, and kidneys) of mice was measured after 48 hours. Figure 13 The results showed that the bacterial counts in all organs of the arundin group and the tigecycline group were all in the 9th power of Log10. However, the bacterial counts in all internal organs of the combined administration group were significantly lower than those of the single administration group, only in the 3rd power of Log10, a reduction of 6 Log.

Claims

1. The application of aromatine in combination with an antibiotic in the preparation of a drug for treating bacterial infectious diseases, wherein the antibiotic is tigecycline or ciprofloxacin, and the bacteria is Klebsiella pneumoniae.

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