Evaluation of the ROS Inhibiting Activity and Mitochondrial Targeting of Phenolic Compounds in Fibroblast Cells Model System and Enhancement of Efficiency by Natural Deep Eutectic Solvent (NADES) Formulation

Purpose Many phenolics have already been tested for their antioxidant activities using in vitro methods. However, such assays do not consider the complexity of real cellular systems, and most of the phenolics characterized with such assays shows disappointing results when evaluated in cells. Accordingly, there is a need to develop effective screening methods. Methods Antioxidants were first evaluated by CAT assay and then, evaluated for their ability (i) to reduce the level of ROS using fluorescent probe, (ii) to cross fibroblast cell membranes using confocal microscopy, and (iii) to target mitochondria. Antioxidants were also formulated in NADES. Results Correlation was obtained when comparing CAT results with short term inhibition (2 h) in the fibroblast cells. On the contrary, it was difficult to anticipate ROS inhibiting efficiency at long term (24 h) from both the CAT assay and the short term inhibition measurements. Indeed, some molecules displayed activity rapidly but lost it over time. In contrast, other molecules were better for long term. The comparable efficiency at long term of Bis-Ethylhexyl Hydroxydimethoxy Benzylmalonate (Bis-EHBm) and decyl rosmarinate, prompted us to further investigate the potential mitochondrial targeting of the former. Using mitochondrial probes, our results confirmed its mitochondrial location. Finally, the formulation of antioxidants in NADES could greatly improve their activity. Conclusions Combinations of fast acting and slow acting molecules could be promising strategies to identify a performant antioxidant system. Bis-EHBm behaves as decyl rosmarinate with a confirmed mitochondrial location. Finally, the formulation of antioxidants in NADES could greatly improve their activity for ROS inhibition.