Application of hydroquinone derivatives in inhibiting the activity of Klebsiella pneumoniae and preparing anti-Klebsiella pneumoniae drugs

Abstract

The present invention provides that the hydroquinone derivative A can significantly inhibit the activity of Klebsiella pneumoniae at a safe concentration. Specifically, in the present invention, zebrafish are used as a model animal to construct an infection model of Klebsiella pneumoniae-like bacteria, and fluorescence intensity analysis using FITC fluorescent dye proves that the hydroquinone derivative A can significantly inhibit the activity of Klebsiella pneumoniae, and it can be applied to the preparation of drugs against Klebsiella pneumoniae.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of hydroquinone derivatives in inhibiting the activity of Klebsiella pneumoniae and preparing anti-Klebsiella pneumoniae drugs. Background Art

[0002] Klebsiella pneumoniae is a Gram-negative bacterium. Klebsiella pneumoniae can colonize and reproduce in the pharynx and gastrointestinal tract of the host to form colonies. Under the conditions of reduced body immunity, endotracheal intubation or abuse of antibiotics, the pathogenic bacteria can enter the lungs through inhalation, swallowing, intubation, etc. to cause infection and form pneumonia. Klebsiella pneumoniae pneumonia can cause severe pneumonia, bronchitis, infantile meningitis, enteritis, wound infection, urinary tract infection and septicemia, etc.

[0003] Klebsiella pneumoniae (Kpn) is one of the important pathogenic bacteria isolated clinically and in hospital infections. With the widespread use of broad-spectrum antibiotics such as β-lactams and aminoglycosides, bacteria are prone to produce extended-spectrum β-lactamases (ESBLs), cephalosporinases (AmpC enzymes) and aminoglycoside modifying enzymes (AMEs), and show serious multidrug resistance to commonly used drugs including the third-generation cephalosporins and aminoglycosides. The hospital infection rate caused by Klebsiella pneumoniae has been increasing year by year recently, and the continuous increase of multi-drug resistant strains often leads to the failure of clinical antibacterial drug treatment and the prolongation of the disease course. The resistance mechanisms of Klebsiella pneumoniae mainly include the production of β-lactamase, the formation of biofilms, the deletion of outer membrane porins, the active efflux of antibacterial drugs, etc. The horizontal dissemination of antibacterial drug resistance genes is an important reason for the clinical exacerbation of multi-drug resistant strains.

[0004] Quinone / hydroquinone derivatives are an important class of marine natural products. Most natural quinones / hydroquinones are isolated from marine sponges, brown algae, and fungi, and they exhibit a wide range of biological activities, including anti-tumor, anti-viral, and antibacterial activities, especially antimicrobial activity against methicillin-resistant and multi-drug resistant strains. In most cases, quinone / hydroquinone derivatives act as important electron transfer mediators in organisms through reversible redox processes. In the earlier research of the inventors, three bioactive hydroquinone derivatives extracted from mangrove fungi in the South China Sea were synthesized using o-methylhydroquinone as a scaffold, and their structures were characterized by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. The synthesis method and characterization results are detailed in the literature "Li H, Jiang J, Liu Z, Lin S, Xia G, Xia X, Ding B, He L, Lu Y, She Z. Peniphenones A-D from the mangrove fungus Penicillium dipodomyicola HN4-3A as inhibitors of Mycobacterium tuberculosis phosphatase MptpB. J Nat Prod.

[0005] 2014; 77(4): 800-806".

[0006] Although quinone / hydroquinone derivatives have been found to have good antibacterial activity, in existing research, there has been no study on the inhibition of Klebsiella pneumoniae by hydroquinone derivatives. Therefore, in the context of the increasing infection rate and drug resistance of Klebsiella pneumoniae, studying the role of quinone / hydroquinone derivatives in the inhibitory application against Klebsiella pneumoniae has a positive impact. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the present invention provides an application of a hydroquinone derivative in inhibiting Klebsiella pneumoniae.

[0008] The present invention provides an application of a hydroquinone derivative in inhibiting the activity of Klebsiella pneumoniae, and the hydroquinone derivative is: For the convenience of description, the above hydroquinone derivative is simply referred to as hydroquinone derivative A.

[0009] The application of a hydroquinone derivative in inhibiting the activity of Klebsiella pneumoniae, and the hydroquinone derivative also includes Derivatives of hydroquinone derivative A may be compounds in which one or more hydroxyl groups of hydroquinone derivative A are halogenated, such as hydroxyl groups halogenated to -F, -Cl, -Br or -I; derivatives of hydroquinone derivative A may also be compounds in which hydroquinone derivative A is substituted with a five-membered heterocycle, such as pyrrole, furan, thiophene, oxazole, thiazole, pyrazole, imidazole, triazole and tetrazole, etc.

[0010] The present invention also provides the use of hydroquinone derivatives in the preparation of drugs against Klebsiella pneumoniae, and the hydroquinone derivatives are:

[0011] The use of hydroquinone derivatives in the preparation of drugs against Klebsiella pneumoniae, and the hydroquinone derivatives also include Derivatives of hydroquinone derivative A may be compounds in which one or more hydroxyl groups of hydroquinone derivative A are halogenated, such as hydroxyl groups halogenated to -F, -Cl, -Br or -I; derivatives of hydroquinone derivative A may also be compounds in which hydroquinone derivative A is substituted with a five-membered heterocycle, such as pyrrole, furan, thiophene, oxazole, thiazole, pyrazole, imidazole, triazole and tetrazole, etc.

[0012] The hydroquinone derivative A can be synthesized using o-methylhydroquinone as a scaffold, and the simplified reaction formula is:

[0013]

[0014] Advantages of this solution:

[0015] The present invention discloses that, at a safe concentration, hydroquinone derivative A can significantly inhibit the activity of Klebsiella pneumoniae. Specifically, in the present invention, zebrafish are used as a model animal to construct an infection model of Klebsiella pneumoniae-like bacteria, and fluorescence intensity analysis using FITC fluorescent dye proves that hydroquinone derivative A can significantly inhibit the activity of Klebsiella pneumoniae and can be applied to the preparation of drugs against Klebsiella pneumoniae. Description of the drawings

[0016] More specifically illustrated by the preferred embodiments of the present invention shown in the drawings, the above and other objects, features and advantages of the present invention will become clearer. The same reference numerals in all the drawings indicate the same parts, and the drawings are not deliberately drawn to scale in actual size, with the emphasis on showing the gist of the present invention.

[0017] Figure 1 This is the test result of the maximum safe concentration of hydroquinone derivative A provided by the present invention for zebrafish larvae;

[0018] Figure 2 This is the fluorescence test result diagram of the zebrafish model for hydroquinone derivative A provided by the present invention to inhibit the activity of Klebsiella pneumoniae;

[0019] Figure 3 This is a schematic diagram of fluorescence intensity of a zebrafish model fluorescence test for the activity of hydroquinone derivative A in inhibiting Klebsiella pneumoniae provided by the present invention. Detailed implementation mode

[0020] The following further details the technical solutions of the present invention in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the illustrated embodiments are not used as a limitation to the present invention.

[0021] Hydroquinone derivative A is a bioactive hydroquinone derivative extracted from a mangrove fungus in the South China Sea. At present, it can be artificially synthesized. However, the specific application of hydroquinone derivative A is still in the research stage. At present, there is no report on the application of hydroquinone derivative A in inhibiting the activity of Klebsiella pneumoniae and the application of hydroquinone derivative A in preparing drugs against Klebsiella pneumoniae.

[0022] The following examples are experiments to verify the safety and efficacy of hydroquinone derivative A. The experimental results are tested by statistical methods. All experiments are carried out with at least 3 independent replicates. The results are expressed as mean and standard error. All statistical analyses use P < 0.01 as the test standard for significant statistical differences.

[0023] Due to the characteristics of zebrafish such as convenient breeding, short reproductive cycle, large egg production, in vitro fertilization of embryos, in vitro development, and transparent embryos, zebrafish have become a new favorite in life science research and are widely used in studying the infection processes of mammals caused by various pathogens and viruses and the research and development of antibacterial drugs. The present invention uses zebrafish as a model animal to construct a Klebsiella pneumoniae-like infection model.

[0024] Example 1 Maximum safe concentration test of hydroquinone derivative A on zebrafish larvae

[0025] Before exploring the activity test of hydroquinone derivative A against Klebsiella pneumoniae infection, safety evaluation is required, that is, it is necessary to first determine the maximum safe concentration of hydroquinone derivative A for zebrafish larvae. Biological safety is a prerequisite for candidate drugs to exert their efficacy characteristics. First, study the safety of hydroquinone derivative A in zebrafish larvae: Hydroquinone derivative A with different concentration gradients (0.25, 0.5, 1, 2, 4 μM) is co-incubated with zebrafish larvae for 96 h, and the number of normal, deformed, and dead zebrafish embryos in each group is counted after 72 h and 96 h of cultivation. The results are shown in Table 1 and Figure 1 as follows.

[0026] Table 1 Maximum safe concentration test of hydroquinone derivative A on zebrafish larvae

[0027]

[0028] From Table 1 and Figure 1 it can be seen that when hydroquinone derivative A with concentrations of 0.25 μM, 0.5 μM, and 1 μM was co-cultured with zebrafish larvae, it did not cause malformation and death of zebrafish embryos, while hydroquinone derivative A with concentrations of 2 μM and 4 μM could cause malformation and death of zebrafish embryos. In summary, at 96 h of co-culture, the maximum safe concentration of hydroquinone derivative A for zebrafish was 1 μM. Therefore, in the following experiments of the present invention, the application of hydroquinone derivative A against Klebsiella pneumoniae activity was explored at the 1 μM safe concentration of hydroquinone derivative A.

[0029] Example 2 Experiment on the effect of hydroquinone derivative A on inhibiting the activity of Klebsiella pneumoniae

[0030] 1. Construction of a zebrafish model infected with Klebsiella pneumoniae

[0031] Klebsiella pneumoniae was stained with FITC (fluorescein isothiocyanate) fluorescent dye under light-proof conditions. After incubating in the dark for 1 h, the staining working solution was centrifuged and aspirated clean. After rinsing three times with PBS (polybutylene succinate), a bacterial suspension (1×10 9 CFU / mL) was prepared with PBS for standby. Zebrafish embryos at 24 hpf (hours-post fertilization) were dechorionated with 0.25% trypsin and incubated in a 28 °C biochemical incubator until 48 hpf; then the zebrafish larvae were anesthetized with 0.02% tricaine and placed on an agar plate, and 2 nL of the above-stained bacteria were injected into the yolk sac of the zebrafish larvae using a microinjector (PV850, Shenzhen Decheng Instrument Technology Co., Ltd.). After injection, the larvae were rinsed at least twice with embryo medium without methylene blue to remove tricaine and free bacteria.

[0032] The zebrafish model infected with Klebsiella pneumoniae was placed on a glass slide, and the distribution of bacteria was observed one by one under a fluorescence microscope (NIB950-FL, Ningbo Yongxin Optics Co., Ltd.). Zebrafish models with uniform distribution and similar numbers of Klebsiella pneumoniae were selected and randomly divided into four groups, namely a blank control group, experimental group 1, experimental group 2, and experimental group 3. The above zebrafish models of each group were placed in a 48-well plate, with 12 replicates in each group and 1 fish in each well.

[0033] 2. Test method

[0034] (1) Set up groups

[0035] Zebrafish models with uniform distribution and similar numbers of Klebsiella pneumoniae were selected and randomly divided into four groups, namely a blank control group, experimental group 1, experimental group 2, and experimental group 3.

[0036] Prepare hydroquinone derivative A solutions with different concentrations: Dissolve hydroquinone derivative A in 0.1% DMSO (dimethyl sulfoxide) to form hydroquinone derivative A solutions with concentrations of 0.25 μM, 0.5 μM, and 1 μM, corresponding to experimental group 1, experimental group 2, and experimental group 3, respectively.

[0037] Add an equal amount of 0.1% DMSO to each well in the blank control group, add an equal amount of 0.25 μM hydroquinone derivative A to each well in experimental group 1, add an equal amount of 0.5 μM hydroquinone derivative A to each well in experimental group 2, and add an equal amount of 1 μM hydroquinone derivative A to each well in experimental group 3. Incubate the additives with the zebrafish model for 48 h.

[0038] (2) Result statistics

[0039] After 48 h of intervention with DMSO and hydroquinone derivative A, place the Klebsiella pneumoniae-infected zebrafish model one by one under a fluorescence microscope to record the distribution of Klebsiella pneumoniae in the zebrafish and count the fluorescence intensity. The results are shown in Table 2, Figure 2 and Figure 3 as follows.

[0040] Table 2 Statistical results of the fluorescence intensity of Klebsiella pneumoniae in zebrafish

[0041]

[0042] Figure 2 (a)-(d) are the control charts of the green fluorescence intensity of the yolk sac of the Klebsiella pneumoniae-infected zebrafish model in four groups at 0 h and 48 h of the experiment.

[0043] Figure (a) is the control chart of the green fluorescence intensity of the yolk sac of the Klebsiella pneumoniae-infected zebrafish model in the blank control group at 0 h and 48 h of the experiment. It can be seen that after 48 h of the experiment, the green fluorescence intensity of the yolk sac of the zebrafish is significantly stronger than that at 0 h of the experiment, indicating that in the case of the blank control, that is, without drug intervention, Klebsiella pneumoniae further infects the zebrafish after 48 h. From Figure 3 it can also be seen that at 0 h of the experiment, the green fluorescence intensity of the zebrafish model is 809 au, while after 48 h of the experiment, the green fluorescence intensity of the zebrafish model is 1090 au, and the green fluorescence intensity increases significantly.

[0044] Figure (b) is a comparison chart of the green fluorescence intensity of the yolk sac of the Klebsiella pneumoniae-infected zebrafish model in experimental group 1 at 0 h and 48 h of the experiment. It can be seen that after 48 h of the experiment, the green fluorescence intensity at the yolk sac of the zebrafish is close to that at 0 h of the experiment, indicating that in the case of experimental group 1 (the concentration of hydroquinone derivative A is 0.25 μM), that is, in the case of intervention with 0.25 μM hydroquinone derivative A, Klebsiella pneumoniae did not further infect the zebrafish after 48 h. Compared with the blank experimental group, it has a certain inhibitory effect on Klebsiella pneumoniae. From Figure 3 It can also be seen that at 0 h of the experiment, the green fluorescence intensity of the zebrafish model is 891 au, while after 48 h of the experiment, the green fluorescence intensity of the zebrafish model is 922 au, and the green fluorescence intensity is basically the same.

[0045] Figure (c) is a comparison chart of the green fluorescence intensity of the yolk sac of the Klebsiella pneumoniae-infected zebrafish model in experimental group 2 at 0 h and 48 h of the experiment. It can be seen that after 48 h of the experiment, the green fluorescence intensity at the yolk sac of the zebrafish is lower than that at 0 h of the experiment, indicating that in the case of experimental group 1 (the concentration of hydroquinone derivative A is 0.5 μM), that is, in the case of intervention with 0.5 μM hydroquinone derivative A, compared with 0 h of the experiment, Klebsiella pneumoniae was effectively inhibited after 48 h; and compared with the blank experimental group, there has been an obvious inhibitory effect on Klebsiella pneumoniae. From Figure 3 It can also be seen that at 0 h of the experiment, the green fluorescence intensity of the zebrafish model is 823 au, while after 48 h of the experiment, the green fluorescence intensity of the zebrafish model is 781 au, and the green fluorescence intensity decreased (P < 0.01).

[0046] Figure (d) is a comparison chart of the green fluorescence intensity of the yolk sac of the Klebsiella pneumoniae-infected zebrafish model in experimental group 3 at 0 h and 48 h of the experiment. It can be seen that after 48 h of the experiment, the green fluorescence intensity at the yolk sac of the zebrafish is significantly lower than that at 0 h of the experiment, indicating that in the case of experimental group 1 (the concentration of hydroquinone derivative A is 1 μM), that is, in the case of intervention with 1 μM hydroquinone derivative A, compared with 0 h of the experiment, Klebsiella pneumoniae was significantly inhibited after 48 h; and compared with the blank experimental group, there has been a significant inhibitory effect on Klebsiella pneumoniae. From Figure 3 It can also be seen that at 0 h of the experiment, the green fluorescence intensity of the zebrafish model is 816 au, while after 48 h of the experiment, the green fluorescence intensity of the zebrafish model is 419 au, and the green fluorescence intensity decreased significantly (P < 0.001).

[0047] In summary, the statistical analysis of fluorescence intensity showed that when the treatment concentration of hydroquinone derivative A was ≥ 0.5 μM, the growth of Klebsiella pneumoniae in zebrafish was significantly inhibited (P < 0.01), and it could be used to prepare drugs against Klebsiella pneumoniae.

[0048] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present invention.

Claims

1. Use of hydroquinone derivatives in the preparation of drugs against Klebsiella pneumoniae, wherein the hydroquinone derivatives are:

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