China Military Institute of Chinese Materia Medica, 302 Military Hospital of China, Beijing, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Nov 15;879(30):3565-73. doi: 10.1016/j.jchromb.2011.09.045. Epub 2011 Oct 1.
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.
提出并应用了一种新的“靶成分敲除”策略，用于初步筛选牛胆（C. bovis）中的抗菌成分。该策略通过以下步骤完成：（1）通过薄层层析（TLC）在 C. bovis 样品的硅胶薄层板上敲除单个成分（A-F）；（2）通过超高效液相色谱-蒸发光散射检测（UPLC-ELSD）鉴定敲除的成分；（3）通过微量热法结合主成分分析（PCA）评估这些敲除的成分和 C. bovis 样品对金黄色葡萄球菌（S. aureus）的抗菌活性；（4）阐明这些敲除的成分和 C. bovis 总提取物的活性，以及这些单个成分与总提取物之间的相互作用性质。结果表明，成分 A-F 的抑制率（I）总和（202.0%）是 C. bovis 样品 I 的 5 倍（38.01%），表明这些敲除的成分在 C. bovis 样品中彼此之间具有强烈的拮抗作用，拮抗程度为 81.18%。我们发现，C. bovis 的关键抗菌成分不是单一成分，也不是高含量成分（胆酸，CA），而是成分 F，它是脱氧胆酸（DCA）和去氧胆酸（HDCA）的组合成分。成分 F 在单独使用时表现出超过 DCA 和 HDCA 活性总和的 33 倍高活性，显示出 DCA 和 HDCA 之间的强烈协同作用。此外，成分 A-E 对成分 F 有明显的拮抗作用。我们的研究表明，这种新提出的“靶成分敲除”策略是筛选活性成分和阐明 C. bovis 中多成分相互作用的有用方法，为进一步了解药效作用、控制中药材质量和发现新药提供了一些参考。
