Numerous assays were developed to measure the antioxidant activity, but each has limitations and the results obtained by different methods are not always comparable. Popular examples are the DPPH and ABTS assay. Our aim was to study similarities and differences of these two assay regarding the measured antioxidant potentials of 24 phenolic compounds using the same measurement and evaluation methods. This should allow conclusions to be drawn as to whether one of the assays is more suitable for measuring specific subgroups like phenolic acids, flavonols, flavanones, dihydrochalcones or flavanols. The assays showed common trends for the mean values of most of the subgroups. Some dihydrochalcones and flavanones did not react with the DPPH radical in contrast to the ABTS radical, leading to significant differences. Therefore, to determine the antioxidant potential of dihydrochalcone or flavanone-rich extracts, the ABTS assay should be preferred. We found that the results of the flavonoids in the DPPH assay were dependent on the Bors criteria, whereas the structure–activity relationship in the ABTS assay was not clear. For the phenolic acids, the results in the ABTS assay were only high for pyrogallol structures, while the DPPH assay was mainly determined by the number of OH groups.

Plants produce a diverse array of secondary metabolites that are generally nonessential but facilitate ecological interactions. Fruits, vegetables, seeds and nuts can accumulate bioactive secondary metabolites with health-promoting properties, including the potent antioxidant activities of phenolic compounds. Several in vitro assays have been developed to measure the polyphenol content and antioxidant activity of plant extracts, e.g., the simple and highly popular Folin-Ciocalteu (FC) assay. However, the literature contains a number of different descriptions of the assay and it is unclear whether the assay measures the polyphenol content or reducing capacity of the sample. To determine the influence of phenolic structures on the outcome of the FC assay, we tested phenols representing different subgroups (phenolic acids, flavonols, flavanols, dihydrochalcones and flavanones). We observed different results for each reference substance and subgroup. Accordingly, we concluded that the FC assay does not measure the polyphenol content of a sample but determines its reducing capacity instead. Assigning the substances to five structural classes showed that the FC results depend on the number of fulfilled Bors criteria. If a molecule fulfills none of the Bors criteria, the FC results depend on the number of OH groups. We did not find a correlation with other single electron transfer assays (e.g., ABTS and DPPH assays). Furthermore, the FC assay was compatible with all five subgroups and should be preferred over the DPPH assay, which is specific for extracts rich in dihydrochalcones or flavanones.

Due to their antioxidant properties, secondary plant metabolites can scavenge free radicals such as reactive oxygen species and protect foods from oxidation processes. Our aim was to study structural influences, like basic structure, number of hydroxyl groups and number of Bors criteria on the outcome of the oxygen radical absorbance capacity (ORAC) assay. Furthermore, similarities and differences to other in vitro antioxidant assays were analyzed by principal component analysis. Our studies confirmed that the antioxidant behavior in the ORAC assay is dominated by the number and types of substituents and not by the Bors criteria, as long as no steric hindrance occurs. For example, morin (MOR) with five hydroxyl groups and two Bors criteria reached an area under the curve of (3.64 ± 0.08) × 105, which was significantly higher than quercetin-7-D-glucoside (QGU7) (P < 0.001), and thus the highest result. Principal component analysis showed different dependencies regarding structural properties of Folin-Ciocalteu (FC)- and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-assays or 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)- and ORAC-assays, respectively. Therefore, we conclude that they are based on different reaction mechanisms. The number of hydroxyl groups showed a stronger influence on the antioxidant activity than the Bors criteria. Due to these differences, the correlation of these rapid tests to specific applications should be validated.

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