Application of an online post-column derivatization HPLC-DPPH assay to detect compounds responsible for antioxidant activity in Sonchus oleraceus L. leaf extracts

Abstract

Abstract Objectives To use an online assay to identify key antioxidants in Sonchus oleraceus leaf extracts and to investigate the effect of leaf position and extraction conditions on antioxidant concentration and activity. Methods Separation of phytochemicals and simultaneous assessment of antioxidant activity were performed online using HPLC and post-column reaction with a free-radical reagent (2, 2-diphenylpicrylhydrazyl, DPPH). Active compounds were identified using nuclear magnetic resonance spectroscopy and mass spectrometry. We applied the online HPLC-DPPH radical assay to evaluate antioxidants in leaves from different positions on the plant and to assess the effect of pre-treatment of leaves with liquid N2 before grinding, extraction time, extraction temperature and method of concentrating extracts. Key findings Key antioxidants identified in S. oleraceus leaf extracts were caftaric acid, chlorogenic acid and chicoric acid. Middle leaves contained the highest total amount of the three key antioxidant compounds, consisting mainly of chicoric acid. Pre-treatment with liquid N2, increasing the extraction temperature and time and freeze-drying the extract did not enhance the yield of the key antioxidants. Conclusion The online HPLC-DPPH radical assay was validated as a useful screening tool for investigating individual antioxidants in leaf extracts. Optimized extraction conditions were middle leaves pre-treated with liquid N2, extraction at 25°C for 0.5 h and solvent removal by rotary evaporation.