Yichao Ma1, Yuan Wang1, Hua Zhang1, Weidong Sun1, Zhenzhen Li1, Fengyichi Zhang1, Haibin Zhang1, Fuxin Chen2, Hang Zhang3, Jun An4, Chenghua He5. 1. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China. 2. School of Chemistry and Chemical Engineering, Xi'an University of Science and Technology, Xi'an, 710054, China. Electronic address: chenfuxin1981@163.com. 3. Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, Co-innovation Center of Henan Province for New Drug R&D and Preclinical Safety, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, 450001, China. 4. Qinnan Institute for Food and Drug Control, Duyun, 558000, China. 5. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China. Electronic address: hechenghua@njau.edu.cn.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: The root of Rosa roxburghii Tratt (Ci Li Gen) is a kind of Chinese ethnomedicine in Gui Zhou province, used for the treatment of abdominal pain, acute bacillary dysentery, gastroenteritis and other diseases in human and livestock. AIM OF THE STUDY: The aim of this study was to isolate and identify the effective antimicrobial components from the ethyl acetate extract of the Ci Li Gen and to investigate its antimicrobial mechanism afterwards. MATERIALS AND METHODS: The effective antimicrobial components in the ethyl acetate extract from the Ci Li Gen were isolated by high-performance liquid chromatography (HPLC) and identified by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). The antibacterial activity was evaluated by the minimum inhibition concentration (MIC) measured by microdilution technique. The antibacterial mechanism was investigated by the time-kill curve, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with NanoLC-ESI-MS/MS, intracellular esterase activity detected by Flow cytometry, and the ultrastructural changes of the Escherichia coli ATCC 25922 observed by scanning electron microscope (SEM). RESULTS: The effective antimicrobial component (peak 4) was identified as strictinin isomers by HRMS and NMR. The MIC of strictinin isomers against E. coli was 0.125 mg/mL. With respect to the negative control group, the results of SDS-PAGE and NanoLC-ESI-MS/MS showed that the up-regulated proteins of the strictinin isomers treated group were Metal-binding protein ZinT, 30S ribosomal protein S4 and 50S ribosomal protein L4, while the down-regulated protein was hydroperoxide reductase subunit C. Moreover, in the strictinin isomers treated group, the esterase activity in the E. coli cells was reduced and the bacteria E. coli became atrophied, pitted and contorted, and the surface of E. coli was rough and blurred. CONCLUSIONS: According to the above results, the antimicrobial mechanism of strictinin isomers against E. coli were oxidative stress and protein synthesis disorder, which inhibited the activity of the enzymes required for bacterial growth and metabolism. These findings reflected the pleiotropic effects of strictinin isomers, making it a promising antimicrobial agent for pharmaceutical research.
ETHNOPHARMACOLOGICAL RELEVANCE: The root of Rosa roxburghii Tratt (Ci LiGen) is a kind of Chinese ethnomedicine in Gui Zhou province, used for the treatment of abdominal pain, acute bacillary dysentery, gastroenteritis and other diseases in human and livestock. AIM OF THE STUDY: The aim of this study was to isolate and identify the effective antimicrobial components from the ethyl acetate extract of the Ci LiGen and to investigate its antimicrobial mechanism afterwards. MATERIALS AND METHODS: The effective antimicrobial components in the ethyl acetate extract from the Ci LiGen were isolated by high-performance liquid chromatography (HPLC) and identified by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). The antibacterial activity was evaluated by the minimum inhibition concentration (MIC) measured by microdilution technique. The antibacterial mechanism was investigated by the time-kill curve, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with NanoLC-ESI-MS/MS, intracellular esterase activity detected by Flow cytometry, and the ultrastructural changes of the Escherichia coli ATCC 25922 observed by scanning electron microscope (SEM). RESULTS: The effective antimicrobial component (peak 4) was identified as strictinin isomers by HRMS and NMR. The MIC of strictinin isomers against E. coli was 0.125 mg/mL. With respect to the negative control group, the results of SDS-PAGE and NanoLC-ESI-MS/MS showed that the up-regulated proteins of the strictinin isomers treated group were Metal-binding protein ZinT, 30S ribosomal protein S4 and 50S ribosomal protein L4, while the down-regulated protein was hydroperoxide reductase subunit C. Moreover, in the strictinin isomers treated group, the esterase activity in the E. coli cells was reduced and the bacteria E. coli became atrophied, pitted and contorted, and the surface of E. coli was rough and blurred. CONCLUSIONS: According to the above results, the antimicrobial mechanism of strictinin isomers against E. coli were oxidative stress and protein synthesis disorder, which inhibited the activity of the enzymes required for bacterial growth and metabolism. These findings reflected the pleiotropic effects of strictinin isomers, making it a promising antimicrobial agent for pharmaceutical research.
Keywords:
Antibacterial activity; Antimicrobial mechanism; Escherichia coli ATCC 25922; Inhibition of protein expression; The root of Rosa roxburghii Tratt (Ci Li Gen)