Development of fast and accurate methods to discover lead compounds for drug candidates is highly important. In this study, a reliable and effective post-column on-flow biochemical assay (POBA) was established to screen potent peroxidase inhibitors from complex chemical mixtures (e.g., natural product extracts). Multiple factors such as flow rate, organic phase, detection wavelength, and reaction coil were carefully investigated. To better understand the features of POBA, another emerging technology of ultrafiltration LC-MS was used for comparison. The result showed that POBA had advantages in saving time, avoiding false positives, and improving the accuracy. To illustrate the practicality of the method, Radix Salvia Miltiorrhizae, a traditional herb for cardiovascular disease treatment, was applied as the research objective. Finally, six compounds including tanshinol, protocatechuic aldehyde, salvianolic acid D, rosmarinic acid, lithospermic acid, and salvianolic acid B were determined as novel peroxidase inhibitors. Their bioactivities were validated by microplate-based assay, molecular docking, and pharmacophore modeling. This study demonstrates a great potential of POBA in the efficient and accurate discovery of drug candidates.

Open image in new window

开发快速准确的方法以发现候选药物的前导化合物非常重要。在这项研究中，建立了一种可靠而有效的柱后流动生化测定法（POBA），以从复杂的化学混合物（例如天然产物提取物）中筛选有效的过氧化物酶抑制剂。仔细研究了流速，有机相，检测波长和反应线圈等多种因素。为了更好地了解POBA的特性，使用了另一种新兴的超滤LC-MS技术进行比较。结果表明，POBA具有节省时间，避免误报，提高准确性的优点。为了说明该方法的实用性，将丹参丹参（一种用于治疗心血管疾病的传统草药）用作研究目标。最后，确定了包括丹参酚，原儿茶醛，丹酚酸D，迷迭香酸，紫草酸和丹酚酸B在内的6种化合物作为新型过氧化物酶抑制剂。通过基于微孔板的测定，分子对接和药效团模型验证了它们的生物活性。这项研究证明了POBA在高效，准确发现候选药物方面的巨大潜力。

在新窗口中打开图像