Medical application of myricetin in preparation of Listeria monocytogenes sortase inhibitor
By using bayerine to inhibit the catalytic activity of Listeria sorting enzyme A, the drug resistance problem of Listeria monocytogenes was solved, and a new treatment method for Listeria monocytogenes infection was provided, and effective inhibition of bacterial virulence factors and protection of host cells were achieved.
Patent Information
- Application Number
- CN202510823735.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-08-29
AI Technical Summary
In the prior art, Listeria monocytogenes has become highly resistant to commonly used antibiotics and lacks effective treatment methods, especially the method of inhibiting the activity of sorting enzyme A to prevent Listeria infection.
Using bayberry as an inhibitor, by inhibiting the catalytic activity of Listeria sorting enzyme A, the effect of bacterial virulence factors is blocked, thereby inhibiting bacterial growth and biological membrane formation, and reducing the ability to internalize into host cells.
While not affecting bacterial growth, bayberry significantly inhibits the activity of sorting enzyme A, reduces the ability of biological membrane formation and internalization into host cells, providing a new therapeutic option for Listeria monocytogenes infection, and has no obvious cytotoxicity.
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Figure CN120549908A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to use of myricetin in preparing medicine for inhibiting the activity of Listeria sortase A and thus resisting Listeria monocytogenes infection, and belongs to the technical field of medical pharmaceuticals. Background Art
[0002] Listeria monocytogenes is a major zoonotic pathogen that causes listeriosis. It has a wide host range and has been successfully isolated from a variety of animals, including cattle, sheep, poultry, and gerbils. Infecting animals, it primarily causes invasive disease, with clinical symptoms including encephalitis, meningitis, sepsis, and abortion. Furthermore, L. monocytogenes is highly adaptable and can survive in a wide range of conditions, making it highly susceptible to contaminating farm environments and feed, leading to listeriosis in humans and animals. This not only causes significant economic losses to the livestock industry but also poses a serious threat to human health. Antibiotic therapy is the primary treatment strategy for L. monocytogenes, but reports indicate that L. monocytogenes has developed significant resistance to tetracyclines, cephalosporins, erythromycin, and ciprofloxacin. Natural compounds are widely distributed and generally have low toxicity. Antibiotic-independent strategies that target virulence factors present unique advantages, do not impose stress on the bacteria, and are less likely to develop resistance. These strategies offer new therapeutic options for the prevention and treatment of L. monocytogenes.
[0003] The virulence factors of Listeria monocytogenes play a crucial role in host invasion, proliferation, and disease-causing processes. Sortase A, a virulence factor-anchoring enzyme, mediates the covalent attachment of bacterial surface proteins to cell wall peptidoglycan, and is closely involved in host adhesion and immune evasion. Therefore, the crucial role of sortase A in the pathogenicity of Listeria monocytogenes makes it an important target for the development of anti-virulence drugs.
[0004] Myricetin is a natural flavonol compound primarily found in the bark and leaves of the Myrica rubra (Myricetin), a plant of the Myricaceae family. It is also found in onions, berries, tea, and other plants, and is a key flavonol component of red wine. Modern pharmacological research has confirmed that myricetin exhibits a wide range of pharmacological activities, including anti-inflammatory, analgesic, anti-tumor, hypoglycemic, and hepatoprotective properties, with particular significance in the prevention and treatment of cardiovascular disease. This substance has significant development potential, thanks to its significant antioxidant and cholesterol-lowering properties. Currently, health foods containing myricetin are marketed in Europe, particularly in Mediterranean countries, but no pharmaceutical products have been approved. Therefore, myricetin holds broad potential for application in the pharmaceutical field. Currently, there are no reports, domestically or internationally, demonstrating that myricetin inhibits Listeria monocytogenes infection by inhibiting the catalytic activity of Listeria sortase A. Summary of the Invention
[0005] The present invention proposes the medical use of myricetin in the preparation of a Listeria sortase A inhibitor, providing a new option for drugs for treating Listeria monocytogenes infection.
[0006] The present invention uses Listeria monocytogenes as a research model bacteria, and verifies through tests such as sortase activity inhibition test, growth curve, biofilm formation inhibition test, and bacterial internalization analysis test that myricetin can effectively inhibit the activity of sortase A without affecting bacterial growth, and has a certain inhibitory effect on the pathogenicity of Listeria monocytogenes.
[0007] The molecular formula of myricetin of the present invention is: 15 H 10 O8, molecular weight: 318.24. The molecular structure of myricetin is as follows: BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 : Myricetin inhibits the catalytic activity of Listeria sortase A; Figure 2 : Myricetin does not inhibit the growth of Listeria monocytogenes within the effective concentration range; Figure 3 : Myricetin has no toxic effect on Caco-2 cells within the effective concentration range; Figure 4 : Myricetin inhibits the formation of Listeria monocytogenes biofilm; Figure 5 : Myricetin inhibits the internalization of Listeria monocytogenes. DETAILED DESCRIPTION
[0009] The present invention is further described by way of examples through the following embodiments, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any changes or modifications to the present invention that are easily implemented by a person of ordinary skill in the art will fall within the scope of the claims of the present invention.
[0010] 1. Effect of myricetin on the catalytic activity of Listeria monocytogenes sortase A In this study, the inhibitory effect of myricetin on the catalytic activity of Listeria monocytogenes SrtA was detected by fluorescence resonance energy transfer (FRET). Purified SrtA protein was incubated with different concentrations of myricetin (0, 4, 8, 16, and 32 μg / mL) in a black 96-well plate at 37°C for 30 minutes. The fluorescent peptide substrate (Dabcyl-QALPNTGEE-Edans) was added to the reaction system and incubated in the dark for 1 hour. The fluorescence intensity of each sample was measured using a microplate reader at an excitation wavelength of 350 nm and an emission wavelength of 520 nm. The results are shown in the attached figure. Figure 1 .
[0011] Conclusion: This study used fluorescence resonance energy transfer to detect the inhibitory effect of myricetin on SrtA enzyme activity. The results showed that myricetin could inhibit SrtA enzyme activity in a dose-dependent manner, with the inhibition rate exceeding 50% at 32 μg / mL myricetin.
[0012] 2. Effect of myricetin on the growth of Listeria monocytogenes To evaluate the effect of myricetin on the growth of Listeria monocytogenes, a growth curve was drawn. Listeria monocytogenes strains were cultured in TSB broth with an initial OD600nm of 0.3. Myricetin at various concentrations (0, 8, 16, 32, and 64 µg / mL) was then added. The absorbance at 600 nm was measured every 30 minutes using a UV spectrophotometer to assess bacterial growth at 37°C. The results are shown in the attached figure. Figure 2 . Conclusion: Within the effective concentration range (8-64µg / mL), myricetin did not affect the growth of Listeria monocytogenes.
[0013] 3. Toxicity of Myricetin to Caco-2 Cells Caco-2 cells were plated into 96-well culture plates (2 × 10 4 100 μL of DMEM (containing 10% fetal bovine serum) containing different concentrations of myricetin was added and incubated in a 37°C constant temperature incubator for 5 h. The OD value was measured at a wavelength of 490 nm using a CCK8 cytotoxicity detection kit and a microplate spectrophotometer to evaluate the cytotoxicity of myricetin. The results are shown in the attached Figure 3 .
[0014] Conclusion: Myricetin has no obvious cytotoxic effect on Caco-2 cells within the effective concentration range (4-64µg / mL).
[0015] 4. Effect of Myricetin on Listeria monocytogenes Biofilm Formation In this study, crystal violet staining was used to stain the biofilm formed by Listeria monocytogenes. The effects of different concentrations of myricetin on the biofilm formation of Listeria monocytogenes were analyzed based on the degree of crystal violet staining. The BUG1600 strain of Listeria monocytogenes was cultured overnight, and the bacterial suspension in the middle logarithmic growth phase was diluted to 1×10 8CFU / mL, expanded with 1mL of fresh sterile TSB medium at a ratio of 1:100, added to 24-well culture plates containing different concentrations of myricetin (0, 4, 8, 16, 32 and 64µg / mL), and cultured at 30°C and 5% CO2 for 72h. After incubation, the culture medium was aspirated and the plate was gently washed 3 times with 1mL of sterile PBS. The plate was then air-dried and stained with 400µL of 0.1% crystal violet for 1 hour. Excess stain was aspirated and the plate was washed 3 times with sterile distilled water. After the 24-well plate was dried, 500µL of 33% glacial acetic acid was added to each well. The absorbance at OD570nm wavelength was measured using a microplate reader, and the results were averaged over three replicates. The results are shown in the Appendix. Figure 4 .
[0016] Conclusion: The absorbance results of crystal violet staining showed that myricetin inhibited the formation of Listeria monocytogenes biofilm in a concentration-dependent manner.
[0017] 5. Effect of Myricetin on the Internalization of Listeria monocytogenes into Caco-2 Cells In this study, the effect of different concentrations of myricetin on the internalization of Listeria monocytogenes was detected by using a Caco-2 cell test. Caco-2 cells were cultured in DMEM medium containing 10% fetal bovine serum without antibiotics, seeded in 24-well culture plates at a density of 2×105 cells per well, and cultured overnight at 37°C and 5% CO2. Caco-2 cells were infected with overnight cultured Listeria monocytogenes BUG1600 at an MOI of 50 and co-cultured with different concentrations of myricetin (0, 4, 16, 32, and 64µg / mL). After 3 hours of culture, fresh culture medium containing 50µg / mL gentamicin was replaced and incubated for 30 minutes, and washed three times with sterile PBS. Sterile water was added to lyse the cells, and the cells were diluted and inoculated for counting. The results are shown in the attached figure. Figure 5 .
[0018] Conclusion: The effect of myricetin on the internalization of Listeria monocytogenes was evaluated by the Listeria monocytogenes internalization assay. The results showed that within 3 h after exposure to different concentrations of myricetin, the degree of internalization of Listeria monocytogenes into Caco-2 cells showed a decreasing trend with increasing myricetin concentration.
Claims
1. The medical use of myricetin in the preparation of Listeria monocytogenes sortase A inhibitor.
2. The use according to claim 1, characterized in that The Listeria monocytogenes sortase A inhibitor myricetin can effectively inhibit the activity of sortase A, thereby reducing the pathogenicity of Listeria monocytogenes.
3. The use according to claim 1, characterized in that The invention relates to an application of myricetin, a Listeria monocytogenes sortase A inhibitor, in the preparation of a medicine for treating Listeria monocytogenes infection.