A selective knockout method for discovery of minor active components from plant extracts: Feasibility and challenges as illustrated by an application to Salvia miltiorrhiza

Natural products have been recognized to play an invaluable role in drug discovery. However, efficient discovery of minor active constituents from natural sources is challenging due to the low abundance and complex matrices. In this study, we developed a selective knockout method to discover minor bioactive components from complex phytochemical mixtures, using a Chinese medicine as an example. Based on the chromatographic fingerprint, six major components in the ethyl acetate extract of the root of Salvia miltiorrhiza (EASM) were selectively knocked out via high-resolution peak fraction (HRPF) approach. The remaining extract was automatically enriched and fractionated to generate a chemical library consisting of 62 minor components with contents less than 3‰. Simultaneously, a parallel mass-spectrometry (MS) analysis was performed to ensure purity and to characterize the structure of the compound in each fraction. Via an antioxidant response element (ARE)-driven luciferase reporter system, 33 minor components were screened out as nuclear factor erythroid 2-related factor 2 (Nrf2) activators and 30 components were identified. Here, the Nrf2 activation activities of 21 components have been reported for the first time. Different from the existing methods for discovery of active compounds from natural products, in the developed method of this manuscript, the major components are selectively removed, and the fractions of the minor components are prepared after several times of preparative HPLC enrichment by high-resolution peak fraction approach. It improves the prospective discovery of minor active components from complex medicinal herbs.