A novel “target constituent knock-out” strategy coupled with TLC, UPLC–ELSD and microcalorimetry for preliminary screening of antibacterial constituents in Calculus bovis

Abstract A novel “target constituent knock-out” strategy was proposed and applied for preliminary screening of antibacterial constituents in Calculus bovis ( C. bovis ). This strategy was accomplished through the following steps: (1) the single constituents (A–F) in C. bovis samples were knocked out on the Silica Gel thin-layer plates by thin-layer chromatography (TLC); (2) these knocked-out constituents were identified by ultra performance liquid chromatography–evaporative light scattering detection (UPLC–ELSD); (3) the antibacterial activities of these knocked-out constituents and C. bovis samples on Staphylococcus aureus ( S. aureus ) were evaluated by microcalorimetry combined with principal component analysis (PCA); (4) the activities of these knocked-out constituents and the total extract of C. bovis , also the interaction properties between these single constituents and the total extract were elucidated. The results showed that the sum of inhibitory ratio ( I ) of constituents A–F (202.0%) was 5-fold of the I of C. bovis sample (38.01%), showing that these knocked-out constituents had strong antagonistic effects on each other in C. bovis sample and the antagonistic extent was 81.18%. And we found that the key antibacterial composition of C. bovis was not a single component, also not the high content component (cholic acid, CA), but constituent F, which was the combinatorial composition of deoxycholic acid (DCA) and hyodeoxycholic acid (HDCA). Constituent F revealed over 33-fold high activity of the sum of DCA and HDCA activity in solo-use, showing strong synergistic effect between DCA and HDCA. In addition, constituents A–E had significant antagonistic effects on constituent F. Our study indicates that this proposed “target constituent knock-out” strategy is a useful approach for screening active constituents and elucidating the multi-component interactions in C. bovis , further providing some reference for understanding the pharmacodynamic actions, controlling the quality of Chinese materia medicas (CMMs) and discovering new drugs.