Use of vanillin in the preparation of a klebsiella pneumoniae capsule inhibitor

Abstract

The application belongs to the technical field of medicine and pharmacy, and particularly relates to application of vanillin in preparation of a Klebsiella pneumoniae capsule inhibitor. The application relates to application of vanillin in preparation of an inhibitor for inhibiting formation, adhesion or function of a Klebsiella pneumoniae capsule, and related antibacterial or anti-infection products, wherein the vanillin reduces pathogenicity of Klebsiella pneumoniae by inhibiting capsule synthesis. It is disclosed that the vanillin can effectively inhibit formation of a Klebsiella pneumoniae capsule and reduce viscosity of bacteria, and can effectively treat mice infected with Klebsiella pneumoniae.

Description

Technical Field

[0001] This invention relates to the field of medical and pharmaceutical technology, specifically to the application of vanillin in the preparation of Klebsiella pneumoniae capsular inhibitors. Background Technology

[0002] Klebsiella pneumoniae is an important opportunistic pathogen that causes respiratory, digestive, bacteremia, and other purulent infections in animals such as cattle, pigs, deer, and poultry. It is also one of the leading causes of community and hospital-acquired bacterial infection-related deaths in humans, posing a significant threat to clinical and public health safety. With the continuous evolution of its pathogenicity and drug resistance, its detection rate in public health surveillance systems has been increasing year by year in recent years, bringing more severe challenges to disease control and clinical diagnosis and treatment, and causing significant economic losses to public safety, food safety, and even the social medical security system. Therefore, the increasing scarcity of effective treatments for Klebsiella pneumoniae (especially multidrug-resistant strains) makes the development of novel anti-infection strategies (such as antibiotic alternative therapies) a critical scientific issue that urgently needs to be addressed.

[0003] The capsule, a key virulence factor in Klebsiella pneumoniae's immune evasion and infection initiation, has a structure and biosynthetic level closely related to the bacterium's pathogenicity. Therefore, intervention strategies targeting the capsule have become an important research direction for developing novel antibiotic alternatives and a potential target for novel anti-infective drugs. Currently, existing methods targeting the capsule, such as vaccines, antibodies, and phage-derived enzymes, still have significant serotype-specific limitations, restricting their application. In contrast, small molecule inhibitors have significant advantages, including well-defined structures, ease of synthesis, low production costs, and potential broad-spectrum antibacterial activity. Screening for inhibitors targeting the capsule's synthesis or assembly pathways is an effective way to discover novel lead compounds against Klebsiella pneumoniae, and it has important practical significance for addressing the increasingly severe challenge of controlling multidrug-resistant strains in clinical practice.

[0004] Vanillin, also known as vanillin aldehyde, is an aromatic aldehyde compound widely found in natural plants, initially isolated from vanilla beans. Furthermore, with industrial development, vanillin can now be produced on a large scale through various methods, including chemical synthesis and biotransformation. Currently, vanillin is one of the world's most widely used food flavorings, not only in the food, beverage, and baking industries, but also in cosmetics, daily chemical fragrances, and pharmaceutical excipients. However, there are no research reports on the application of vanillin in the preparation of Klebsiella pneumoniae capsular inhibitors. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution:

[0007] Application of vanillin in the preparation of Klebsiella pneumoniae capsular inhibitor.

[0008] As a preferred embodiment of the application of vanillin in the preparation of Klebsiella pneumoniae capsular inhibitors according to the present invention, the vanillin has a CAS Registry Number of 121-33-5.

[0009] As a preferred embodiment of the application of vanillin in the preparation of Klebsiella pneumoniae capsule inhibitors according to the present invention, the vanillin is used in the preparation of inhibitors for inhibiting the formation, adhesion or function of Klebsiella pneumoniae capsules, as well as related antibacterial or anti-infective products.

[0010] As a preferred embodiment of the application of vanillin in the preparation of Klebsiella pneumoniae capsular inhibitors according to the present invention, the vanillin reduces the pathogenicity of Klebsiella pneumoniae by inhibiting capsular synthesis.

[0011] Compared with the prior art, the beneficial effects of the present invention are: the present invention discloses that vanillin can effectively inhibit the formation of Klebsiella pneumoniae capsule and reduce bacterial viscosity, while effectively treating mice infected with Klebsiella pneumoniae. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0013] Figure 1 The graph shows the effect of vanillin at concentrations of 8 μg / mL and 16 μg / mL on the uronic acid content of Klebsiella pneumoniae.

[0014] Figure 2 This is a graph showing the effect of vanillin concentrations of 8 μg / mL and 16 μg / mL on the viscosity of Klebsiella pneumoniae.

[0015] Figure 3This is a graph showing the effect of vanillin on the growth curve of Klebsiella pneumoniae according to the present invention;

[0016] Figure 4 This is a graph showing the effect of vanillin on the viability of A549 cells according to the present invention;

[0017] Figure 5 The figure shows the effect of vanillin of the present invention on the survival rate of mice infected with Klebsiella pneumoniae. Detailed Implementation

[0018] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0019] Secondly, the present invention is described in detail with reference to the schematic diagrams. When describing the embodiments of the present invention, for ease of explanation, the cross-sectional views illustrating the device structure may be partially enlarged, not according to the usual scale. Furthermore, the schematic diagrams are merely examples and should not limit the scope of protection of the present invention. In addition, actual fabrication should include the three-dimensional spatial dimensions of length, width, and depth.

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0021] This invention discloses the medical use of vanillin in the preparation of capsular inhibitors, and discloses that vanillin can reduce the pathogenicity of Klebsiella pneumoniae by inhibiting capsular synthesis, thereby exerting an effective anti-infective effect, and provides an anti-capsular lead compound that can prevent and treat Klebsiella pneumoniae infection.

[0022] In this embodiment, the highly virulent Klebsiella pneumoniae strain K7 (K2 type) was used as the research object. Vanillin, an effective capsule inhibitor, was screened using a uronic acid test. Viscosity centrifugation tests demonstrated that vanillin effectively reduced the high-viscosity phenotype of Klebsiella pneumoniae mediated by the bacterial capsule. Growth curve and cytotoxicity assays indicated that vanillin did not show significant inhibitory effects on the growth of Klebsiella pneumoniae and had no toxic effects on mammalian cells within the tested range. By inhibiting capsule-mediated pathogenicity, vanillin treatment effectively improved the survival rate of Klebsiella pneumoniae infected with the large wax moth.

[0023] Example 1

[0024] Vanillin can be used as a capsular inhibitor of Klebsiella pneumoniae and as a pharmaceutically acceptable carrier in the preparation of drugs for treating Klebsiella pneumoniae infection.

[0025] Example 2

[0026] Vanillin can be used as a capsular inhibitor in the preparation of potential drugs for Klebsiella pneumoniae infectious diseases.

[0027] Example 3

[0028] Vanillin, as a capsular inhibitor, can be used to treat infectious diseases caused by Klebsiella pneumoniae.

[0029] Experimental Example 1

[0030] Effects of vanillin on capsular polysaccharide levels in Klebsiella pneumoniae:

[0031] K2 type Klebsiella pneumoniae strain K7 was cultured overnight in LB liquid medium and then transferred at a ratio of 1:100 to a medium containing a specific concentration of vanillin. After shaking and culturing at 180 rpm, 500 µL of the bacterial culture was mixed with 100 µL of 1% zwitergent (w / v, 100 mM citric acid), incubated at 50 °C, and centrifuged. The supernatant was collected, thoroughly mixed with anhydrous ethanol, and allowed to stand at 4 °C to precipitate the polysaccharides. After centrifugation again and discarding the supernatant, the precipitate was dried, dissolved in 200 µL of distilled water, and 1.2 mL of sodium tetraborate-concentrated sulfuric acid was added. The mixture was boiled for 5 minutes, cooled on ice, and then 0.15% m-carboxybiphenyl (w / v, 0.5% NaOH) was added for color development. The uronic acid content in each sample was calculated by measuring the absorbance at 600 nm and using a standard curve plotted based on galactosides.

[0032] Conclusion: Vanillin dose-dependently reduces the capsular polysaccharide level of Klebsiella pneumoniae (see Appendix). Figure 1 .

[0033] Experimental Example 2

[0034] Effects of vanillin on the capsular-mediated high-viscosity phenotype of Klebsiella pneumoniae:

[0035] K2 type Klebsiella pneumoniae strain K7 was inoculated into LB liquid medium and cultured overnight. The next day, the strain was expanded at a ratio of 1:100 and a specific concentration of vanillin was added. After shaking and culturing for 5 h at 180 rpm, 500 µL of bacterial solution was centrifuged at 1000g for 5 minutes. The absorbance of the supernatant was measured at 600 nm. The effect of vanillin at each concentration on the capsule-mediated high-viscosity phenotype of Klebsiella pneumoniae was calculated by comparing the absorbance with that of the initial bacterial solution.

[0036] Conclusion: Compared with the solvent control group, vanillin treatment significantly weakened the bacterial capsule-mediated high-viscosity phenotype (see Appendix). Figure 2 .

[0037] Experimental Example 3

[0038] Growth curve determination:

[0039] The K2 strain of Klebsiella pneumoniae K7, cultured overnight, was multiplied by 1:100 and cultured in a constant temperature shaker at 37°C until OD (dose dissipation) was reached. 600nmThe concentration was 0.3. Dispenses were prepared and DMSO solvent or different concentrations of vanillin were added (final concentrations of 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, and 128 μg / mL). The mixture was incubated at 37°C and 180 rpm, with OD measured every hour. 600 Numerical values ​​are used to plot growth curves based on absorbance values.

[0040] Conclusion: Vanillin did not affect the growth viability of Klebsiella pneumoniae within the tested range (see Appendix). Figure 3 .

[0041] Test Example 4

[0042] Cytotoxicity assay of vanillin:

[0043] A549 cells were seeded in 96-well plates (2 × 10⁶ cells / well). 4 Cells were cultured overnight (number of cells / well). The following day, cells were treated with either DMSO solvent or a specific concentration of vanillin. A cell-free control group served as a blank control, and a 0.1% Triton X-100 treatment group served as a positive control group. Each group was divided into three replicates. Cells were incubated at 37°C for 8 hours and then analyzed according to the CCK8 reagent kit instructions. The results were calculated using the following formula: Cell viability (%) = (Test group - Cell-free control group) / (Blank treatment group - Cell-free control group) × 100%.

[0044] Conclusion: Compared with the solvent control group, vanillin showed almost no toxicity to A549 cells in the concentration range of 4-128 μg / ml (see appendix). Figure 4 .

[0045] Experimental Example 5

[0046] Analysis of the protective effect of vanillin against Klebsiella pneumoniae infection in the large wax moth:

[0047] Overnight cultured Klebsiella pneumoniae was diluted 1:100 and cultured in 20 mL LB medium at 37°C and 180 rpm until OD reached. 600nm =0.6-0.8, collect bacterial cells and wash three times with sterile PBS for later use. Large wax moth larvae were pre-warmed to room temperature for 30 minutes, and each larva was infected with 2 × 10⁻⁶ bacteria on the right side of the first pair of abdominal legs. 4 CFU bacterial culture was used to inoculate animals into two groups (infection group and 50 mg / kg vanillin treatment group), with 10 animals in each group. 10 μL of the drug (final concentration of 50 mg / kg) was administered to the right side of the second to last pair of abdominal paws. The animals were incubated at 37°C after administration, and results were recorded every 8 hours.

[0048] Conclusion: Treatment with 50 mg / kg vanillin increased the survival rate of the large wax moth from 0% to 20%. (See appendix) Figure 5 .

[0049] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. The application of vanillin in the preparation of Klebsiella pneumoniae capsular inhibitors.

2. The application of vanillin according to claim 1 in the preparation of Klebsiella pneumoniae capsular inhibitor, characterized in that, The CAS Registry Number for vanillin is 121-33-5.

3. The application of vanillin according to claim 1 in the preparation of Klebsiella pneumoniae capsular inhibitor, characterized in that, The vanillin is used in the preparation of inhibitors for inhibiting the formation, adhesion, or function of Klebsiella pneumoniae capsules, as well as in related antibacterial or anti-infective products.

4. The application of vanillin according to claim 1 in the preparation of Klebsiella pneumoniae capsular inhibitor, characterized in that, The vanillin reduces the pathogenicity of Klebsiella pneumoniae by inhibiting capsule synthesis.

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