Application of cinnamic acid in preparation of klebsiella pneumoniae capsular inhibitor

Abstract

The invention discloses application of cinnamic acid in preparation of a klebsiella pneumoniae capsular inhibitor and belongs to the technical field of biological medicine, the CAS login number of the cinnamic acid is 140-10-3, the molecular formula of the cinnamic acid is C9H8O2, the molecular weight of the cinnamic acid is 148.16, and the cinnamic acid has an inhibiting effect on klebsiella pneumoniae capsular synthesis. The invention solves the problem that the application of cinnamic acid in preparation of klebsiella pneumoniae capsule inhibitors is not researched and reported at present. Through uronic acid determination, Anthony's capsular staining, a growth curve test, cytotoxicity, a klebsiella pneumonia infection greater wax moth protection test and other analysis, it is proved that cinnamic acid can reduce the pathogenicity of klebsiella pneumonia by inhibiting capsular synthesis, and then the effective anti-infection effect is achieved; a novel treatment means is provided for clinical drug-resistant klebsiella pneumoniae infection, and a lead compound with definite functions is provided for subsequent research.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically the use of cinnamic acid in the preparation of Klebsiella pneumoniae capsular inhibitors. Background Technology

[0002] Klebsiella pneumoniae is a significant opportunistic pathogen causing respiratory, digestive, bacteremia, and other purulent infections in livestock and poultry such as cattle, pigs, deer, and chickens. It is also one of the leading pathogens causing community and nosocomial bacterial infection-related deaths in humans, with an isolation rate second only to Escherichia coli. While long considered to pose little threat to animals, its pathogenicity and drug resistance have led to a continuous increase in its isolation rate in veterinary clinics in recent years, causing substantial economic losses to the livestock industry, particularly dairy farming.

[0003] The capsule is a crucial virulence factor for Klebsiella pneumoniae to evade the immune system and further cause pathogenicity. Recent studies have shown a correlation between capsule production levels and pathogenicity; the thicker the capsule, the stronger the pathogenicity. Therefore, the capsule has become an important target for antibiotic alternative strategies and drug development. Currently, existing strategies targeting the capsule, such as vaccines, antibodies, and phages, are still highly limited by serotype specificity. In contrast, compound inhibitors offer significant advantages such as low cost and broad spectrum. Screening for inhibitors targeting the capsule can provide effective lead compounds for the development of drugs against Klebsiella pneumoniae infection, and is of great significance for the prevention and control of multidrug-resistant Klebsiella pneumoniae infection.

[0004] Cinnamic acid, also known as cinnamic acid, is a natural phenolic acid compound derived from various plants. It is named after cinnamon (Cinnamomum cassia), from which it was initially isolated. This compound can also be obtained through chemical synthesis. Currently, cinnamic acid is widely used in food, medicine, and cosmetics, but there are no research reports on its application in the preparation of Klebsiella pneumoniae capsular inhibitors. Summary of the Invention

[0005] The purpose of this invention is to provide the use of cinnamic acid in the preparation of Klebsiella pneumoniae capsular inhibitors, providing a novel treatment for drug-resistant Klebsiella pneumoniae infections in clinical practice, and providing a functionally defined lead compound for subsequent research, thus solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The use of cinnamic acid in the preparation of Klebsiella pneumoniae capsule inhibitor, wherein the cinnamic acid has CAS Registry Number 140-10-3, molecular formula C9H8O2, and molecular weight 148.16, and the cinnamic acid has an inhibitory effect on Klebsiella pneumoniae capsule synthesis.

[0007] Preferably, the cinnamic acid inhibits capsule synthesis by suppressing the production level of Klebsiella pneumoniae capsular polysaccharides.

[0008] Preferably, the cinnamic acid reduces the pathogenicity of Klebsiella pneumoniae by inhibiting capsule synthesis, thereby achieving anti-interference.

[0009] Preferably, the Klebsiella pneumoniae strain is K7, a highly virulent K2-type Klebsiella pneumoniae strain.

[0010] Preferably, the cinnamic acid is used as a capsular inhibitor in the preparation of a drug for treating Klebsiella pneumoniae infection.

[0011] Compared with the prior art, the beneficial effects of the present invention are: This invention, through analyses such as uronic acid assay, Anthony's capsule staining, growth curve assay, cytotoxicity assay, and Klebsiella pneumoniae infection protection assay against giant wax moth, confirms that cinnamic acid can reduce the pathogenicity of Klebsiella pneumoniae by inhibiting capsule synthesis, thereby exerting an effective anti-infective effect. Cinnamic acid, as a natural compound, has a clear source and is inexpensive, providing a novel treatment for drug-resistant Klebsiella pneumoniae infection in clinical practice, and providing a lead compound with a clearly defined function for subsequent research. Attached Figure Description

[0012] Figure 1 This is a graph showing the analytical results of the effect of cinnamic acid on the production level of capsular polysaccharides in Klebsiella pneumoniae according to the present invention. Figure 2 This is a schematic diagram illustrating the effect of cinnamic acid on the capsule thickness of Klebsiella pneumoniae according to the present invention. Figure 3 This is a schematic diagram of the growth curve of the present invention; Figure 4 This is a schematic diagram showing the drug toxicity test results of cinnamic acid on mammalian cells according to the present invention; Figure 5 This is a schematic diagram illustrating the results of the analysis of the protective effect of cinnamic acid against Klebsiella pneumoniae infection of the large wax moth. Detailed Implementation

[0013] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] To address the issue that while cinnamic acid is widely used in food, pharmaceuticals, and cosmetics, there are no studies reporting its application in the preparation of Klebsiella pneumoniae capsular inhibitors, please refer to [link to relevant documentation]. Figures 1-5 This embodiment provides the following technical solution: Cinnamic acid has the CAS Registry Number 140-10-3, the molecular formula C9H8O2, and the molecular weight 148.16. Its chemical structure is as follows: ; The Klebsiella pneumoniae strain used in this embodiment is specifically the K2 type highly virulent Klebsiella pneumoniae strain K7, which was kindly provided by Professor Gu Jingmin's team at Jilin University.

[0015] Specifically, this embodiment uses K7 strain of highly virulent Klebsiella pneumoniae (kindly provided by Professor Gu Jingmin's team at Jilin University) as the research object. Cinnamic acid, an effective capsular inhibitor, was obtained through uronic acid screening. Anthony's capsular staining further clarified that it significantly reduced the capsular thickness on the bacterial surface. Growth curve analysis indicated that cinnamic acid had no significant inhibitory effect on the survival of Klebsiella pneumoniae and was non-toxic to mammalian cells within the tested range. By inhibiting capsular-mediated pathogenicity, cinnamic acid treatment effectively improved the survival rate of Klebsiella pneumoniae infected with the large wax moth. The medical use of cinnamic acid in the preparation of capsular inhibitors is disclosed, demonstrating that cinnamic acid can reduce the pathogenicity of Klebsiella pneumoniae by inhibiting capsular synthesis, thereby exerting an effective anti-infective effect. This provides an anti-capsular lead compound for the prevention and treatment of Klebsiella pneumoniae infection.

[0016] In this embodiment, the effect of cinnamic acid on the production level of capsular polysaccharides in Klebsiella pneumoniae was analyzed. Klebsiella pneumoniae strain K7 (K2 type) was inoculated into LB liquid medium and cultured overnight. The next day, the strain was expanded at a 1:100 ratio with the addition of a specific concentration of cinnamic acid. After culturing at 180 rpm for 5 h, 500 µL of the bacterial culture was mixed with 100 µL of 1% surfactant zwitergent (w / v percentage, 100 mM citric acid). The mixture was incubated at 50 °C for 20 min and then centrifuged. 300 µL of the supernatant was mixed thoroughly with 1.2 mL of anhydrous ethanol and allowed to stand at 4 °C for 20 min to precipitate the polysaccharides. After centrifugation, the supernatant was discarded, and the precipitate was dried and dissolved in 200 µL of distilled water. Then, 1.2 mL of sodium tetraborate-concentrated sulfuric acid was added and vortexed. The mixture was boiled for 5 min and then cooled on ice for 10 min. Finally, 0.15% m-carboxybiphenyl (w / v percentage, 0.5% NaOH) was added for color development, and the result was detected using a microplate reader at 520 μL. The absorbance value at nm was used to calculate the uronic acid content of each sample based on the standard curve prepared by galactoside. The results obtained are as follows Figure 1 As shown, cinnamic acid dose-dependently reduces the production level of capsular polysaccharides in Klebsiella pneumoniae.

[0017] In this embodiment, Anthony's bacterial capsule staining test was performed; Cinnamic acid was co-cultured with Klebsiella pneumoniae strain K7 (K2 type) until OD. 600nm =0.4, take 1 mL and centrifuge to collect the bacterial pellet. Use an inoculation loop to pick up a small amount of bacteria and mix it with 10 μL of skim milk on a glass slide. After the smear is air-dried, stain it with 1% crystal violet solution for 2 minutes, and then rinse it with 20% copper sulfate solution. After drying, observe and collect images with an optical microscope. The capsule does not show color against the purple background, while the bacterial cells are stained dark purple. The results are as follows Figure 2 As shown in the figure, compared with the DMSO control group ( Figure 2 Compared to (A), cinnamic acid treatment ( Figure 2 (B) significantly reduced the thickness of bacterial surface capsules.

[0018] In this embodiment, growth curve measurement is performed; The K2 strain of Klebsiella pneumoniae K7, which had been cultured overnight, was multiplied at a ratio of 1:100 in a 250 mL Erlenmeyer flask containing 200 mL of fresh LB broth and cultured in a shaker at 37°C until OD (dose retardation). 600nm Reaching 0.3 will reduce OD 600nmThe bacterial culture with a pH of 0.3 was dispensed into 20 mL autoclaved 50 mL Erlenmeyer flasks. Simultaneously, DMSO solvent or different concentrations of cinnamic acid (4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, and 128 μg / mL) were added to each flask. The flasks were incubated at 37°C and 180 rpm, with OD values ​​measured every hour using a spectrophotometer. 600nm The values ​​were then used to plot the growth curve based on the absorbance of the bacterial culture at each time point. The results obtained are as follows Figure 3 As shown, cinnamic acid has almost no effect on the growth of Klebsiella pneumoniae within the tested range.

[0019] In this embodiment, the drug toxicity of cinnamic acid to mammalian cells was tested; A549 cells (purchased from the American Culture Collection) were seeded into 96-well plates (2 × 10⁶ cells per well). 4 Cells were cultured overnight (cells / well). The next day, cells were treated with DMSO solvent (solvent control group) or a specific concentration of cinnamic acid. The cell-free control group served as a blank control, and the 0.1% Triton X-100 treatment group served as a positive control group. Each group was set up in triplicate. After incubation at 37°C for 8 h, the cells were detected according to the LDH kit instructions. The detection results were calculated using the following formula: Cytotoxicity (%) = (Test group - Cell-free control group) / (Positive treatment group - Cell-free control group) × 100%; The results obtained are as follows Figure 4 As shown, cinnamic acid is almost non-toxic to A549 cells in the concentration range of 4-256 μg / mL compared with the solvent control group.

[0020] In this embodiment, the protective effect of cinnamic acid against Klebsiella pneumoniae infection of the large wax moth was analyzed; The K2 strain of Klebsiella pneumoniae K7, which had been cultured overnight, was multiplied at a ratio of 1:100 into 20 mL of LB medium and cultured at 37°C and 180 rpm until OD200 was reached. 600nm =0.6-0.8, collect bacterial cells and wash three times with sterile PBS for later use; larvae of the large wax moth (purchased from Tianjin Huiyude Biotechnology Co., Ltd.) were pre-warmed to room temperature for 30 minutes, and 10 μL (2×10) was injected into the right side of the first pair of abdominal legs of each larva. 4 CFU bacterial suspension was randomly divided into two groups after infection (biological solvent control group, 50 mg / kg cinnamic acid group), with 8 animals in each group. 10 μL of the drug was given to the right side of the second to last pair of abdominal feet. After administration, the animals were incubated at 37℃ and the results were recorded every 6 h. The results are as follows Figure 5As shown, treatment with 50 mg / kg cinnamic acid can increase the survival rate of the large wax moth from 12.5% ​​to 50%.

[0021] It should be noted that, Figure 1-5 All statistical analyses were performed using GraphPad Prism 9.1. Unless otherwise stated, all quantitative data were derived from at least three independent experiments and are expressed as mean ± standard error of mean (mean ± SEM). One-way ANOVA was used for comparisons between groups. ns indicates no significant difference. *P < 0.05, **P < 0.01.

[0022] In summary, this invention demonstrates that cinnamic acid can effectively inhibit capsule synthesis, significantly reduce the capsule thickness on the bacterial surface, and has no significant inhibitory effect on the survival of Klebsiella pneumoniae. Furthermore, cinnamic acid is non-toxic to mammalian cells within the tested range. Cinnamic acid effectively improves the survival rate of Klebsiella pneumoniae infected with the large wax moth. Based on this, cinnamic acid can be used as a capsule inhibitor of Klebsiella pneumoniae and as a pharmaceutically acceptable carrier in the preparation of drugs for treating Klebsiella pneumoniae infections. Cinnamic acid can also be used as a capsule inhibitor in the preparation of potential drugs for Klebsiella pneumoniae infectious diseases.

[0023] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0024] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The use of cinnamic acid in the preparation of Klebsiella pneumoniae capsular inhibitors, characterized in that, The cinnamic acid has a CAS registry number of 140-10-3, a molecular formula of C9H8O2, and a molecular weight of 148.

16. The cinnamic acid has an inhibitory effect on the capsule synthesis of Klebsiella pneumoniae.

2. The use of cinnamic acid according to claim 1 in the preparation of Klebsiella pneumoniae capsular inhibitors, characterized in that, The cinnamic acid inhibits capsule synthesis by suppressing the production level of Klebsiella pneumoniae capsular polysaccharides.

3. The use of cinnamic acid according to claim 1 in the preparation of Klebsiella pneumoniae capsular inhibitors, characterized in that, The cinnamic acid reduces the pathogenicity of Klebsiella pneumoniae by inhibiting capsule synthesis, thus achieving anti-interference.

4. The use of cinnamic acid according to claim 1 in the preparation of Klebsiella pneumoniae capsular inhibitors, characterized in that, The Klebsiella pneumoniae strain mentioned is K7, a highly virulent strain of K2 Klebsiella pneumoniae.

5. The use of cinnamic acid according to claim 1 in the preparation of Klebsiella pneumoniae capsular inhibitors, characterized in that, The cinnamic acid is used as a capsular inhibitor in the preparation of drugs for treating Klebsiella pneumoniae infection.

Images