Free Radical Scavenging Activities of Polyphenolic Compounds Isolated from Medicago sativa and Medicago truncatula Assessed by Means of Thin-layer Chromatography DPPH? Rapid Test

Introduction The structure of polyphenolic compounds influences their anti-oxidant potential. Finding a simple, rapid and reliable analytical method to study the structureactivity relationships for numerous samples is challenging. Objective To develop a simple thin-layer chromotography2,2-diphenyl-1-picrylhdrazyl (TLCDPPH?) protocol with image processing to study the influence of the structure of polyphenols on observed direct anti-oxidant properties. Methodology First, compounds exhibiting free radical scavenging activities were chosen from among the isolated compounds with the application of a rapid TLC dot-blot test. The active ones were further chromatographed on silica gel plates using the mobile phase: acetonitrile:water:chloroform:formic acid (60:15:10:5, v/v/v/v). Subsequently the plates were stained with DPPH? methanolic solution. An improved image processing protocol was used to quantitatively measure the polyphenols activity. Results The application of a properly optimised chromatographic system enabled separation of the investigated compounds from dimethyl sulphoxide (DMSO) that influences the results of an anti-oxidant test. New solutions enabling better data processing are proposed. It has been discovered that acylation of flavonoid glycosides with hydroxycinnamic acids increases their direct anti-oxidant properties. Some of the analysed glycosides acylated with ferulic acid molecule were found to be the most potent free radical scavengers from among those analysed. The amount of sugar moieties as well as their type also influenced the observed activity. Conclusion A simple, rapid and reliable TLCDPPH? test with image processing has been developed enabling comparison of free radical scavenging activity of plant polyphenols. The influence of different structural features on the observed activity was measured successfully. Copyright (c) 2012 John Wiley & Sons, Ltd.