Human serum albumin (HSA) shows the sequence homology and structural similarity with bovine serum albumin (BSA). Therefore, here, the interaction of natural phenolic antioxidants, ellagic acid (ELA), and its derivatives–urolithins A (ULA) and B (ULB)–with BSA was investigated. The results of surface plasmon resonance (SPR) indicated a high affinity of ELA, ULA, and ULB to BSA, with KD value < 1 × 10-6 M. The KD values of binding of the studied compounds to BSA increased with temperature, revealing a reduction in affinity with an increase in temperature. Fluorescence data showed that the quenching of BSA by tested compounds occurred via a static quenching. However, the affinity of ELA for BSA was higher than that of ULA and ULB, which may be because of the presence of a large number of hydroxyl groups in its structure. The assessment of the antioxidant activity of BSA and BSA–ELA/ULA/ULB complexes using the DPPH assay indicated that the DPPH scavenging activity of BSA increased after complex formation with ELA/ULA/ULB in the following order: BSA–ELA > BSA–ULA > BSA–ULB > BSA, which was due to their structural differences. The results of the docking analysis were in agreement with the experimental results.
