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

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.