DPPH assay

The antioxidant capacity (AOC) was assessed by using the DPPH assay [34]. DPPH (1,1-diphenyl-2-picryl-hydrazyl, Sigma-Aldrich, Germany) is a free radical that can be reduced by antioxidants. Upon reduction, the colour of the free radical changes, and thus, the amount of reduced DPPH can be accessed via UV-Vis spectroscopy. The DPPH assay is performed for different concentrations of the antioxidant, which enables the determination of the IC 50 that is the concentration needed to scavenge 50% of the free radical (DPPH). Consequently, low IC 50 values represent a high AOC and vice versa.

In this study, the IC 50 was determined at different time points and in different dispersion media, i.e. purified water and ethanol, respectively. Tests were performed by adding 100 µl of the samples containing different concentrations of the nanocrystals to 100 µl of a 0.3 mM ethanolic solution of DPPH. After incubation (5 min, 15 min, 20 min, 30 min and 45 min) in the dark, the absorbance was measured by a UV-VIS plate reader (Mulitskan GO, Thermo scientific, Germany) at 517 nm. The inhibition activity (inhibition %) was calculated as: inhibition%=1-Asample/A0·100, where A_sample is the absorbance of the sample and A₀ is the absorbance of the control (DPPH solution). The resulting function of inhibition against concentration was used to calculate the IC 50 value (µg/ml). Besides the determination of the IC 50 for the nanocrystals, also, the IC 50 of the bulk material and an ethanolic hesperetin solution was analysed and used as control.