Application of luteolin in preparation of medicine for inhibiting functional activity of pyogenic streptococcus hemolysin
A technology of streptolysin and luteolin, applied in the field of medicine, can solve the problems of immune system dysfunction, exacerbation of drug resistance of pathogenic bacteria, and reduction of clinical treatment effect
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Embodiment 1
[0026] Embodiment 1, antibacterial activity and time killing test
[0027] GAS (ATCC, USA; SF370) was cultured at 37°C until OD 600=0.8, and the bacterial solution was diluted to 5×10 5 Density in CFU / mL. The minimum inhibitory concentration (MIC) test was performed using the broth microdilution method according to the procedures in the CLSI guidelines (M31-A2). MIC is recorded as the lowest drug concentration at which the culture system becomes turbid after 24 hours.
[0028] Another 18 15mL glass test tubes were taken and divided into 3 groups, 6 in each group. Prepare luteolin (Shanghai Yuanye Biotechnology Co., Ltd.; B20888) into 5 concentrations of 64, 128, 256, 512, and 1024 μg / mL, with a total of 6 mL for each concentration, and add them to the test tubes in sequence, and add 2mL, add THY medium without luteolin to the last test tube, and dilute the bacterial solution to 2×10 5 The density of CFU / mL was added to 2.5 mL of the above test tubes respectively. The fin...
Embodiment 2
[0032] Embodiment 2, hemolytic activity test
[0033] Refer to the previous experimental protocol of red blood cell hemolysis [Dong J, Liu Y, Xu N, Yang Q, Ai X. MorinProtects Channel Catfish From Aeromonas hydrophila Infection by Blocking Aerolysin Activity. Front Microbiol. 2018; 9:2828, Dong J, Zhang L, Liu Y, Xu N, Zhou S, Yang Y, et al. Luteolin decreases the pathogenicity of Aeromonas hydrophilavia inhibiting the activity of aerolysin. Virulence. 2021; 12:165-76] with modifications. Briefly, recombinant SLO (0.1 g / mL) was added to the reaction system with or without luteolin, and sheep red blood cells (RBCs) were collected and added to the above reaction system to the final concentration 2% (v / v). The entire mixed system was incubated at 37°C for 30 minutes, then centrifuged at 5000 rpm for 5 minutes, and the OD of the supernatant was obtained 540 . The completely hemolyzed system treated with 0.1% Triton-X100 was used as a positive control. Final hemolysis percentag...
Embodiment 3
[0035] Embodiment 3, surface plasmon resonance analysis (Surface plasmon resonance, SPR)
[0036] The affinity and kinetics of luteolin for SLO were measured by SPR at 25 °C on a REICHERT 4SPR using a carboxymethyl dextran hydrogel surface sensor chip. Firstly, to measure the binding of protein to small molecules, the protein was dissolved in 10 mM sodium acetate (pH 4.5) and immobilized on a chip with 6000 response units (RU) at a flow rate of 10 μL / min. Luteolin was serially diluted in PBST buffer (PBS containing 0.005% Tween 20) to a concentration of 0.125g / mL, 0.25g / mL, 0.5g / mL, 1g / mL, 2g / mL, 4g / mL, The infusion was performed at a flow rate of 25 μL / min for 3 min; for dissociation, the flow rate was set at 25 μL / min for 5 min. Reference values and signals from buffer-injected controls were subtracted and sensor traces were fitted to a 1:1 Langmuir binding model using the data analysis program Scriber 2.0 to calculate ka, kd and KD values.
[0037] In order to verify th...
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