High-resolution bacterial growth inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of antibacterial constituents in Chinese plants used to treat snakebites.

Bacterial infection is one of the main secondary infections caused by snakebite. The 88 plant species investigated in this study have been used as folk remedies for treatment of snakebite, and it is therefore the aim of this study to investigate whether the plants contain compounds with bacterial growth inhibition. The water and ethanol extracts of 88 plant species were screened at 200 μg/mL against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa for their antibacterial activity by micro-broth dilution assay. The most active extracts were fractionated into microplates using analytical-scale RP-HPLC, and subsequently growth inhibition was assessed for each well. The biochromatograms constructed from these assays were used to identify compounds responsible for antibacterial activity. The structures of five compounds were elucidated by HPLC-HRMS-SPE-NMR. Crude extracts of Boehmeria nivea, Colocasia esculenta, Fagopyrum cymosum, Glochidion puberum, Melastoma dodecandrum, Polygonum bistorta, Polygonum cuspidatum and Sanguisorba officinalis showed MIC values below 200 μg/mL against either Bacillus subtilis, Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa. The biochromatograms demonstrated that tannins play a main role for the bacterial growth inhibition observed for all above-mentioned plants except for Polygonum cuspidatum. Furthermore, the high-resolution bacterial growth inhibition profiling combined with HPLC-HRMS-SPE-NMR allowed fast identification of three non-tannin active compounds, i.e., piceid, resveratrol and emodin from ethanol extract of Polygonum cuspidatum. The high-resolution bacterial growth inhibition profiling allowed fast pinpointing of constituents responsible for the bioactivity, e.g., either showing tannins being the main bacterial growth inhibitors as observed for the majority of the active plants, or combined with HPLC-HRMS-SPE-NMR for fast structural identification of non-tannin constituents correlated with antibacterial activity.