Comparative Methods to Evaluate the Antioxidant Capacity of Propolis: An Attempt to Explain the Differences

Propolis is a natural product produced by bees that contains a complex mixture of compounds, including phenolic compounds and flavonoids. These compounds contribute to its biological activities, such as antioxidant capacity. This study analysed the pollen profile, total phenolic content (TPC), antioxidant properties, and phenolic compound profile of four propolis samples from Portugal. The total phenolic compounds in the samples were determined by six different techniques: four different Folin–Ciocalteu (F-C) methods, spectrophotometry (SPECT), and voltammetry (SWV). Of the six methods, SPECT allowed the highest quantification, while SWV achieved the lowest. The mean TPC values for these methods were 422 ± 98 and 47 ± 11 mg GAE/g sample, respectively. Antioxidant capacity was determined by four different methods: DPPH, FRAP, original ferrocyanide (OFec), and modified ferrocyanide (MFec). The MFec method gave the highest antioxidant capacity for all samples, followed by the DPPH method. The study also investigated the correlation between TPC and antioxidant capacity with the presence of hydroxybenzoic acid (HBA), hydroxycinnamic acid (HCA), and flavonoids (FLAV) in propolis samples. The results showed that the concentrations of specific compounds in propolis samples can significantly impact their antioxidant capacity and TPC quantification. Analysis of the profile of phenolic compounds by the UHPLC-DAD-ESI-MS technique identified chrysin, caffeic acid isoprenyl ester, pinocembrin, galangin, pinobanksin-3-O-acetate, and caffeic acid phenyl ester as the major compounds in the four propolis samples. In conclusion, this study shows the importance of the choice of method for determining TPC and antioxidant activity in samples and the contribution of HBA and HCA content to their quantification.