Rapid screening of natural free radical scavengers in two Polygonum herbs by fast HPLC-DAD-ESI-MSn with precolumn incubation method

Abstract

A simple and efficient method, i.e. 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) incubation followed by fast HPLC-DAD-ESI-MSⁿ based on novel core–shell particle technology, was proposed for the rapid and high-throughput screening of natural free radical scavengers directly from the crude extract of two Polygonum herbs, P. multiflorum and P. cuspidatum. All of the components in P. multiflorum or P. cuspidatum were well separated within 9 min using the core–shell column, which was much superior to previously reported HPLC methods. After incubation with DPPH, 7 compounds were demonstrated to possess potential free radical scavenging activity. They were identified as gallic acid, cis-2,3,5,4′-tetrahydroxy stilbene-2-O-glucoside and trans-2,3,5,4′-tetrahydroxystilbene-2-O-glucoside from P. multiflorum, together with resveratroloside, piceid, 3,5,4′-trihydroxystilbene-3-O-(6′′-galloyl)-glucoside, and resveratrol in P. cuspidatum. Meanwhile, 3,5,4′-trihydroxystilbene-3-O-(6′′-galloyl)-glucoside showed the strongest scavenging capability, while resveratroloside was the weakest one according to the quantitative difference in peak areas before and after incubation with DPPH. Furthermore, the free radical scavenging activity of five of the pure compounds was further evaluated by a classical DPPH colorimetric assay on a microwell plate for method validation. Collectively, the current work provides a powerful tool for the rapid discovery of natural radical scavengers directly from complex matrices like herbal medicines, and the results could also help to elucidate which components are responsible for the antioxidant activity of two Polygonum herbs.