Determination of antioxidant activity of phytogenic substances via a yeast-based fluorescence assay

Abstract

Plant-derived phytogenic feed additives (PFAs) are used to improve health and performance of farm animals. One proposed mode of action is an antioxidative effect. In order to evaluate the intracellular antioxidative potential of PFAs in vitro, we developed a yeast-based assay and applied it to several different substances and mixtures. Yeast cells of the strain Saccharomyces cerevisiae were starved in phosphate buffered saline (PBS) for several days before they were incubated for 30 minutes with 2′,7′‑Dichlorofluorescein diacetate (DCFH, a cell permeable, non-fluorescent substance that becomes highly fluorescent upon oxidation). After washing away extracellular DCFH, the cells were transferred to 96-well microplates containing the different test substances and were subjected to oxidative stress by addition of 400 µM H2O2 for one hour. Fluorescence (excitation: 485 nm, emission: 528 nm) was quantified using a plate-reader. Controls included cells without test substance, cells without DCFH and cells without H2O2. Ascorbic acid (4 mM) served as positive control. As expected, the positive control reduced the fluorescence signal of the H2O2-stressed cells, proving that the assay is suitable for detecting antioxidant activity. Moreover, also some of the tested PFAs led to a reduced signal, thereby underlining the antioxidant capacity of selected PFAs on eukaryotic cells.