An integrated chromatographic strategy to discover free radical inhibitors from trace fraction of Ribes himalense Royle ex Decne

Abstract

Efficient and rapid chromatographic strategies are indispensable for analyzing the chemical components of natural products as well as their biological functions. The current study developed an integrated chromatography-based strategy to recognize and separate trace free radical inhibitors from the traditional Tibetan medicine Ribes himalense Royle ex Decne (R. himalense). First, R. himalense was processed using medium pressure liquid chromatography (MPLC) involving polyamide as a stationary phase. Then the free radical inhibitors in target fraction (Fr4) were purified using a two-dimensional reversed-phase/reversed-phase interaction liquid chromatography (2D RP/RPLC) mode, with the procedure being directed by an online HPLC-1,1-diphenyl-2-picrylhydrazyl (HPLC-DPPH) activity screening method. Finally, three free radical inhibitors (3-caffeoylquinic acid methyl ester, rutin and myricetin-3'-α-L-rhamnopyranoside) were successfully isolated with a percentage purity above 95%, with their IC50 values being 22.5 µg/mL, 9.5 µg/mL and 15.4 µg/mL respectively. To the best of our knowledge, these free radical inhibitors are being recorded for the first time in R. himalense. Furthermore, the integrated chromatography-based strategy developed in this work was shown to be reliable and efficient, which further supplemented the chemical components of R. himalense, as well as provided methodological support for the analysis of active substances in other natural products.