Activity-guided isolation and identification of free radical-scavenging components from an aqueous extract of Coleus aromaticus

An activity-directed fractionation and purification process was used to identify the DPPH (l,l-diphenyl-2-picrylhydrazyl) free radical-scavenging components of Coleus aromaticus Benth. Fresh leaves of C. aromaticus were extracted with water and then separated into hexane, ethyl acetate, and water fractions. Among these, only the ethyl acetate phase showed strong DPPH radical-scavenging activity in vitro, when compared with water and hexane phases. The ethyl acetate fraction was then subjected to separation and purification using Sephadex LH-20 chromatography. Three compounds showing strong DPPH radical-scavenging activity were shown, by spectral methods (1H NMR, 13C NMR, and MS) and by comparison with literature values, to be rosmarinic acid, chlorogenic acid and caffeic acid. In addition, HPLC identification and quantification of isolated compounds were also performed. Rosmarinic acid was found as a major component and principally responsible for the radical-scavenging activity of C. aromaticus.