title: Free radical scavenging activities of polyphenolic compounds isolated from Medicago sativa and Medicago truncatula assessed by means of thin-layer chromatography DPPH˙ rapid test.
abstract: BACKGROUND
  The structure of polyphenolic compounds influences their anti-oxidant potential. Finding a simple, rapid and reliable analytical method to study the structure-activity relationships for numerous samples is challenging.
  
  
  OBJECTIVE
  To develop a simple thin-layer chromatography-2,2-diphenyl-1-picrylhdrazyl (TLC-DPPH˙) protocol with image processing to study the influence of the structure of polyphenols on observed direct anti-oxidant properties.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  A simple, rapid and reliable TLC-DPPH˙ 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.
authors: Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation-State Research Institute, 8 Czartoryskich Street, 24-100 Puławy, Poland. lukecarpenter@poczta.onet.pl
authors: Łukasz
authors: Ł
authors: Cieśla
authors: Iwona
authors: I
authors: Kowalska
authors: Wiesław
authors: W
authors: Oleszek
authors: Anna
authors: A
authors: Stochmal
mesh_terms: D000215:Acylation; D001713:Biphenyl Compounds / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods*; D016166:Free Radical Scavengers / Q000737:chemistry* / Q000494:pharmacology*; D007091:Image Processing, Computer-Assisted; D000455:Medicago sativa / Q000737:chemistry*; D046913:Medicago truncatula / Q000737:chemistry*; D010851:Picrates / Q000378:metabolism; D059808:Polyphenols / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D001713:Biphenyl Compounds; D016166:Free Radical Scavengers; D010851:Picrates; D059808:Polyphenols; C004931:1,1-diphenyl-2-picrylhydrazyl