Antioxidant and Antimicrobial Potential of Romanian Propolis Extract Combinations: Linking Chemical Composition with Biological Activity

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Abstract
Background/Objectives: Propolis is a chemically complex product whose antimicrobial activity varies with geographical origin and composition. This study evaluated the chemical, antioxidant, and antimicrobial properties of five Romanian propolis ethanolic extracts (PEEs) and their combinations, focusing on antimicrobial interactions and chemical–biological relationships. Methods: Five PEEs were evaluated individually, as ten binary combinations, and as one five-extract mixture. Antioxidant activity and 20 phenolic compounds were determined. Antimicrobial activity against 16 strains was assessed using agar diffusion and broth microdilution assays. Extract interactions were evaluated using the fractional inhibitory concentration index (FICI). Principal Component Analysis (PCA), Spearman correlation, and Partial Least Squares (PLS) regression investigated chemical–biological relationships. Results: Significant differences were observed among preparations in phenolic composition, antioxidant activity, and antimicrobial responses. Total phenolic content ranged from 88.8 to 149.8 mg GAE/g, while A + S showed the highest antioxidant activity (3213.74 mg TE/g). C1 showed strong activity against several Gram-positive bacteria and Candida albicans, whereas S and S-containing combinations showed activity against several Gram-negative strains. FICI analysis showed predominantly indifferent interactions (60.8%), followed by additive (25.0%), synergistic (9.09%), and antagonistic (5.11%). The strongest synergy was observed for A + S against Pseudomonas aeruginosa (FICI = 0.195). Chemical PC1 was inversely associated with biological PC2 (ρ = −0.809, FDR-adjusted p = 0.0013). Conclusions: Antimicrobial activity depends on extract composition and the target microorganism. Combining extracts did not consistently improve activity; however, selected combinations produced additive or synergistic interactions, whereas antagonism was uncommon. Multivariate analyses further supported an association between phenolic composition and antimicrobial variability.

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