Medical application of phloretin in preparation of klebsiella pneumoniae capsular inhibitor

Abstract

The invention provides application of phloretin in preparation of a medicine for treating klebsiella pneumoniae infection. A uronic acid experiment, a capsule staining experiment, a J774 cytotoxicity experiment and a greater wax moth larva infected klebsiella pneumonia survival experiment prove that the phloretin can inhibit klebsiella pneumonia capsule and has a relatively good protection effect on animals infected by klebsiella pneumonia. Compared with antibiotic treatment, the method for treating klebsiella pneumoniae infection by using phloretin has the advantages of no drug resistance and high cure rate. Therefore, the phloretin can be used for new drug development and has important significance on drug target confirmation.

Description

Technical Field

[0001] The invention relates to the medical use of phloretin in preparing a Klebsiella pneumoniae capsule inhibitor, and belongs to the field of medical pharmacy. Background Art

[0002] Klebsiella pneumonia is a conditionally pathogenic bacterium that can cause pneumonia, bacteremia, meningitis, liver abscess and other infectious diseases in humans, cattle, pigs, chickens and other animals. In recent years, the clinical isolation rate of Klebsiella pneumoniae has continued to increase, and its drug resistance and virulence have also been on the rise, posing a serious threat to public health and animal husbandry, and has therefore attracted widespread attention.

[0003] The capsule is an important structure formed on the surface of bacteria by polysaccharides synthesized and secreted outside the cell wall. It is critical for Klebsiella pneumoniae to establish infection. During Klebsiella pneumoniae infection, the capsule protects pathogens from being eliminated by the body through multiple mechanisms such as inhibiting immune cell phagocytosis, preventing early immune response activation, and eliminating complement. Therefore, the capsule is a potential target for the development of anti-virulence drugs for Klebsiella pneumoniae infection. In response to the national green agriculture call, the development of capsule inhibitors to fight Klebsiella pneumoniae infection under the policy of reducing and limiting antibiotics is in line with the needs of human and animal public health and market economic development.

[0004] Phloretin is a dihydrochalcone compound naturally found in fruits and vegetables such as apples, pears, and strawberries. Phloretin has a variety of physiological functions, including antibacterial, antiviral, anti-inflammatory, antioxidant, tyrosinase inhibition, hypoglycemic and anti-tumor functions, and has broad application prospects in the fields of food, medicine, and cosmetics. The present invention is the first to discover that phloretin can inhibit the capsule of Klebsiella pneumoniae and inhibit the pathogenicity of Klebsiella pneumoniae in vivo and in vitro. Summary of the invention

[0005] The molecular formula of phloretin of the present invention is: 15 H 14 O5, molecular weight: 274.27.

[0006] The present invention provides a medical use of phloretin in the preparation of a Klebsiella pneumoniae capsule inhibitor. Using a K2 serotype Klebsiella pneumoniae clinical isolate K7 as a model strain, the medical use of phloretin in inhibiting the synthesis of Klebsiella pneumoniae capsule is disclosed, providing a new target drug that can prevent and treat Klebsiella pneumoniae infection.

[0007] The present invention finds that phloretin inhibits the synthesis of Klebsiella pneumoniae capsules. The thickness of the Klebsiella pneumoniae capsules is inhibited by phloretin through optical microscope observation. The invention has a good protective effect on the infection of greater wax moth larvae mediated by Klebsiella pneumoniae. Compared with the treatment with antibiotics, the treatment with phloretin has the advantages of no drug resistance and high cure rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 : The chemical structural formula of phloretin.

[0009] Figure 2 :Phloretin has no effect on the growth of Klebsiella pneumoniae within a certain concentration range.

[0010] Figure 3 :Phloretin reduces the content of capsular polysaccharide of Klebsiella pneumoniae in a dose-dependent manner Figure 4 :Phloretin inhibits the capsule thickness of Klebsiella pneumoniae Figure 5 :Phloretin has no proliferation toxicity to A549 cells Figure 6 : Protective effect of phloretin in the Glebsiella mellonella larvae model of infection with Klebsiella pneumoniae. DETAILED DESCRIPTION

[0011] The present invention is further described by way of the following examples, which are not intended to limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change made to the present invention that is easily implemented by a person of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0012] Example 1 Phloretin can be used as a Klebsiella pneumoniae capsule inhibitor in any pharmaceutically acceptable carrier.

[0013] Example 2 Phloretin can be used as a Klebsiella pneumoniae capsule inhibitor for preparing drugs for treating infectious diseases.

[0014] Example 3 Phloretin can be used to treat infectious diseases caused by bacteria.

[0015] Test Example 1 Effects of phloretin on the growth of Klebsiella pneumoniae A single colony of Klebsiella pneumoniae was selected and cultured overnight in LB liquid medium (37°C, 200 rpm). The next day, the culture was expanded at a ratio of 1:100 until the absorbance at 600 nm (OD 600) was about 0.3, and the bacterial solution was evenly divided into five 50 ml conical flasks (about 20 ml in each flask), and different concentrations of phloretin (0, 4, 8, 16, 32, 64 μg / ml) were added respectively. The culture was continued at 37°C and 200 rpm, and the OD of each sample was measured every 1 h. 600 , until it grows to a plateau stage.

[0016] Conclusion: Compared with the group without drug treatment, there was no significant difference in the bacterial growth status of the groups treated with different concentrations of phloretin, indicating that phloretin did not affect the normal growth of Klebsiella pneumoniae within the effective concentration range (see Appendix Figure 2 ).

[0017] Test Example 2 Uronate test Overnight culture of Klebsiella pneumoniae was amplified at 1:100 in 2 ml LB medium containing 0, 4, 8, 16, 32, and 64 ug / ml phloretin for 6 h. Take 1 ml of bacterial solution and add 200 ul 100 mM citric acid solution (pH 2.0, containing 1% zwittergent 3-14) and mix well, incubate at 50℃ for 20 min, centrifuge at 14000 rpm for 5 min, take 600 ul of supernatant and add to 2.4 ml of anhydrous ethanol, mix well, stand at 4℃ for 20 min, centrifuge at 14000 rpm for 5 min, discard the supernatant, and dry the precipitate at room temperature overnight. Add 200 ul of deionized water to the precipitate in the tube, shake it thoroughly to dissolve, add 1.2 ml of sodium tetraborate concentrated sulfuric acid solution and mix well, heat at 100℃ for 5 min, cool on ice for 10 min, add 20 ul, 0.15% 3-hydroxy-p-aniline, and measure the absorbance at 520 nm.

[0018] Conclusion: Adding phloretin to the culture can inhibit the content of capsular polysaccharide of Klebsiella pneumoniae. The inhibition of uronic acid by 8 μg / ml phloretin is significant. As the drug concentration increases, the inhibition is dose-dependent (see Appendix Figure 3 ).

[0019] Test Example 3 Capsule staining Overnight culture of Klebsiella pneumoniae was expanded at 1:100 to OD 600 = 0.6, wash the cells once with PBS, and adjust the OD 600 = 0.1, dilute the bacterial solution 10,000 times, take 10 ul of the bacterial solution and 10 ul of skim milk and mix them on a glass slide, use a coverslip to scrape (the bacterial solution is uniform) to dry naturally, use 1% crystal violet to stain at room temperature for 2 min, use 20% copper sulfate to rinse the dye, let it stand and dry, and then observe under a microscope.

[0020] Conclusion: Compared with the control group without drug solvent, drug treatment inhibited capsule production, making the capsule of Klebsiella pneumoniae thinner after treatment (see Appendix Figure 4 ).

[0021] Test Example 4 Toxic effect of phloretin on J774 cells The effect of benzoic acid on the cell viability of J774 cells was determined using a CCK-8 assay kit. The cells in the cell culture flask were digested with trypsin, and the cell suspension was diluted and counted. Cells were inoculated in a 96-well plate, with 5 × 10 cells per well. 4 Cells were added with 200 ul of DMEM medium containing 10% fetal bovine serum and placed in a constant temperature biochemical incubator (37°C, 5% CO2) for overnight culture to allow the cells to adhere to the wall. The culture medium in the culture wells was replaced and phloretin was added to the final mass concentrations of 0, 4, 8, 16, 32, 64, and 128 ug / ml, and the wells containing cells and the wells without cells added with the culture medium were set as controls. Three replicates were placed in each well and incubated in a 37°C constant temperature incubator for 12 h. After incubation, 20 ul of CCK-8 solution was added to each well of the 96-well plate and incubated in a 37°C constant temperature incubator for 1 h. The absorbance at 450 nm was measured using an enzyme reader, and the data was recorded and calculated.

[0022] Conclusion: Compared with the control wells without drug addition, the values ​​of cell determination after drug treatment did not change significantly, indicating that phloretin has no proliferation toxicity to J774 cells and does not affect cell growth (see Appendix Figure 5 ).

[0023] Test Example 5 The protective rate of benzoic acid against the larvae of Galleria mellonella infected with Klebsiella pneumoniae The overnight culture of Klebsiella pneumoniae was expanded into 20 ml LB medium and cultured at 37°C until OD 600 nm = 0.6~0.8, collect the cells by centrifugation, wash with sterile PBS and adjust the OD with PBS 600 =0.1. The larvae of G. mellonella were revived at 37°C for 30 min, and the right side of the last pair of ventral legs of each larva was infected with 10 μl (5 × 10 7 CFUs) were randomly divided into 2 groups (solvent control, 50 mg / kg phloretin) after infection, with 10 mice in each group. 10 μl of drug was given to the right side of the second to last pair of ventral feet. The mice were cultured at 37℃ and the results were counted every 6 hours.

[0024] Conclusion: Compared with the group without drug administration, the survival rate of G. mellonella larvae treated with 50 mg / kg phloretin increased by 40%, indicating that phloretin has a protective effect against G. mellonella larvae infected with Klebsiella pneumoniae (see Appendix Figure 6 ).

Claims

1. The medical use of phloretin in the preparation of Klebsiella pneumoniae capsule inhibitors.

2. The use according to claim 1, characterized in that The Klebsiella pneumoniae capsule refers to all type II capsule serotypes of Klebsiella pneumoniae, and phloretin can effectively inhibit the formation of its capsule.

3. The use according to claim 1, characterized in that The Klebsiella pneumoniae capsule inhibitor can effectively treat infections caused by Klebsiella pneumoniae.

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