2.5.1. In Vitro Anti-Inflammatory Assay: Inhibition of BSA Denaturation

The in vitro anti-inflammatory potential of propolis from Gerês was determined using the inhibition of heat-induced bovine serum albumin (BSA) denaturation technique [54,55], with some minor modifications. Briefly, the propolis samples, namely G.EEs and the mG.EE, were dissolved in phosphate buffer 0.066 M, pH 5.3, at 27 °C and mixed with equal amounts of BSA (Sigma-Aldrich Portugal, Lisbon, Portugal) 0.16% (w/v) in the same buffer. Then, the mixture was incubated at 70 °C for 35–40 min and the absorbance was measured at 594 nm. Negative and positive controls were made with phosphate buffer and acetylsalicylic acid (ASA) or aspirin, a known non-steroid anti-inflammatory drug (NSAID), respectively. The percentage of inhibition of precipitation (albumin denaturation) by each propolis sample was determined and expressed as the mean value of three independent assays with triplicates each.