Application of licochalcone A in preparation of medicines for resisting clostridium difficile infection
A technology for Clostridium difficile infection and licochalcone, which is applied in the field of medicine, can solve the problem of no anti-Clostridium difficile activity of licochalcone A, and achieve significant inhibitory activity, relieve clinical symptoms, and reduce mortality
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Embodiment 1
[0019] Embodiment 1: In vitro bacteriostatic activity test of licochalcone A
[0020] 1. Experimental materials and equipment
[0021] Bacterial strains: Clostridium difficile ATCC43255 and BAA1803 were purchased from ATCC Company of the United States; Clostridium difficile (CICC22951) was purchased from China Industrial Microorganism Culture Collection and Management Center.
[0022] Reagents: anaerobic liquid medium, Beijing Land Bridge Technology Co., Ltd.; blood plate and agar were purchased from Guangdong Huankai Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO), metronidazole, vancomycin, nitazoxanide Purchased from sigma company; Licochalcone A was purchased from Shanghai Yuanye Biotechnology Co., Ltd.
[0023] Test equipment: ultra-clean bench, autoclave, anaerobic incubator, electronic analytical balance, cell density meter, pipette, Direct-Q 3 ultrapure water system.
[0024] 2. Test method
[0025] 2.1 Preparation of medicinal solution: Licochalcone A, metronida...
Embodiment 2
[0037] Embodiment 2: The bactericidal curve determination of licochalcone A to Clostridium difficile
[0038] 1. Test material
[0039] Bacterial strain: Clostridium difficile (ATCC43255), purchased from American ATCC Company.
[0040] Reagents: anaerobic liquid medium, Beijing Land Bridge Technology Co., Ltd.; agar was purchased from Guangdong Huankai Biotechnology Co., Ltd.; test equipment: ultra-clean bench, autoclave, anaerobic incubator, electronic analytical balance, cell density Meter, pipette, Direct-Q 3 ultrapure water system.
[0041] 2. Test method
[0042] 2.1 Preparation of medicinal solution: prepare licochalcone A with DMSO into a medicinal solution with a concentration of 1 mg / mL, and set aside.
[0043] 2.2 Bacterial solution preparation: Pick a single colony of Clostridium difficile with an inoculation loop, inoculate it into a sterilized test tube containing about 5 mL of anaerobic liquid medium, and incubate in an anaerobic incubator at 37°C for 18-24 ho...
Embodiment 3
[0047] Embodiment 3: cytotoxicity test
[0048] 1. Test material
[0049] Ccaco-2 cells, Shanghai Cell Bank, Chinese Academy of Sciences; CCK-8 cell viability detection kit, Sigma Reagent Company; carbon dioxide incubator, Memmert, Germany; microplate reader, Thermo Fisher, USA; 96-well plate, MEM medium, Fetal bovine serum and non-essential amino acids were purchased from Thermo Fisher, USA.
[0050] 2. Test method
[0051] Take Caco-2 cells in the logarithmic growth phase and adjust the cell density to 1×10 5 The density of cells / mL was seeded in a sterile 96-well plate, and 100 μL of cell suspension was added to each well, and incubated at 37 °C, 5% CO 2 Cultured in an incubator for 24 hours, discarded the original medium, then added 100 μL of different concentrations of licochalcone A solution (1.5625, 3.125, 6.25, 12.5, 25, 50 μg / mL), and set 6 for each concentration parallel replicate wells. After the cells were cultured for 12 hours, 10 μL of CCK-8 solution was add...
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