Comparative Chromatographic Separation of Free Radicals From Natural Products Using Green Organic Solvents

A well-designed liquid chromatographic strategy is essential for the reliable characterization and preparative isolation of natural products (NPs). In this study, an integrated "medium-pressure pretreatment followed by 0.2% formic acid (FA) H₂O-EtOH high-pressure purification" strategy was employed to achieve the efficient separation of active compounds in Ribes himalense. Guided by the online high-performance liquid chromatography-DPPH assay, Fr111 and Fr112 were recovered from the R. himalense extract by polyamide- and Spherical C18-medium-pressure liquid chromatography. The chromatographic behavior of these fractions was comparatively examined under hydrophilic and reversed-phase conditions using three commonly employed aqueous-organic mobile phases, namely 0.2% FA H₂O-ACN, 0.2% FA H₂O-MeOH, and 0.2% FA H₂O-EtOH. Under the investigated experimental conditions, the 0.2% FA H₂O-EtOH system exhibited improved peak resolution and separation selectivity for both fractions relative to the other mobile phases. Ultimately, four free radical inhibitors (>95% purity) with flavonoids as the parent nucleus were obtained from Fr111 and Fr112. Their significant in vitro free radical scavenging activity was validated using the DPPH and ABTS assays. In conclusion, the 0.2% FA H₂O-EtOH system delivered favorable chromatographic performance while reducing solvent toxicity, demonstrating promise for efficient and greener preparative workflows. The proposed strategy offers a practical and scalable approach for the targeted isolation of structurally similar bioactive compounds from NPs in the foreseeable future.