Thunb. as a promising antibacterial agent for ATCC14579 based on network pharmacology, metabolomics, and experiments.

Thunb. has attracted much attention for its treatment of bacterial and viral infectious diseases, while its active ingredients and potential mechanisms of action have not been fully elucidated. Here, we combined metabolomics, and network pharmacology to explore the molecular mechanism of ATCC14579 inhibition by Thunb. inhibition experiments showed that the Thunb.'s water extracts, ethanolic extract, luteolin, quercetin, and kaempferol strongly inhibited ATCC14579. In contrast, chlorogenic acid and macranthoidin B had no inhibitory effect on ATCC14579. Meanwhile, the minimum inhibitory concentrations of luteolin, quercetin, and kaempferol against ATCC14579 were 15.625 μg mL, 31.25 μg mL, and 15.625 μg mL. Based on the previous experimental basis, the metabolomic analysis showed the presence of 16 active ingredients in Thunb.'s water extracts and ethanol extracts, with differences in the luteolin, quercetin, and kaempferol contents between the water extracts and ethanol extracts. Network pharmacology studies indicated that fabZ, tig, glmU, secA, deoD, nagB, pgi, rpmB, recA, and upp were potential key targets. Active ingredients of Thunb. may exert their inhibitory effects by inhibiting ribosome assembly, the peptidoglycan biosynthesis process, and the phospholipid biosynthesis process of ATCC14579. An alkaline phosphatase activity assay, peptidoglycan concentration assay, and protein concentration assay showed that luteolin, quercetin, and kaempferol disrupted the ATCC14579 cell wall and cell membrane integrity. Transmission electron microscopy results showed significant changes in the morphology and ultrastructure of the cell wall and cell membrane of ATCC14579, further confirming the disruption of the cell wall and cell membrane integrity of ATCC14579 by luteolin, quercetin, and kaempferol. In conclusion, Thunb. can be used as a potential antibacterial agent for ATCC14579, which may exert its antibacterial activity by destroying the integrity of the cell wall and membrane.