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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.

How Does the Phenol Structure Influence the Results of the Folin-Ciocalteu Assay?