There are several procedures to determine the total
antioxidant activity of substances. For example spectrophotometric
methods like DPPH assays [31], Folin-Ciolteau
assays [32], and many others. Also (bio-)amperometry and
cyclic voltammetry can be used [33]. In this study 2,2-
diphenyl-1-picrylhydrazyl (DPPH) assays were chosen,
because of the fast and easy method to investigate the total
antioxidant scavenging activity. Also the experimental
procedure can be easily and fast adjusted and customized
to the analysis experiments. It was first developed by Blois
to determine the total antioxidant activity of compounds
[34]. DPPH is a dark colored powder of stable free-radical
molecules, which is soluble in methanol or ethanol. It has
a strong absorption maximum at a wavelength around
515 nm due to the presence of an unpaired electron. As this
electron becomes paired off in the presence of an antioxidant
(hydrogen donor), the absorption strength decreases.
A color change from violet to yellow can be observed (see
Fig. 2). This change of color due to change in absorbance
can be used to determine the antioxidant activity of compounds
[35,36]. To obtain reproducible results it is important
to investigate the reaction kinetics of DPPH and the
antioxidant. The time until the reaction reaches a steady
state depends on the antioxidant compound [31].