Phenolic Screening and Mixture Design Optimization for In Vitro Assessment of Antioxidant and Antimicrobial Activities of Honey, Propolis, and Bee Pollen

The current study used a design of experiments to evaluate the potential synergistic effects of three Moroccan bee products (honey (H), propolis (P), and bee pollen (BP)) on the free radical inhibition and antibacterial activity against clinical strains of Staphylococcus aureus and Escherichia coli. The phytochemical contents of these three bee products were first evaluated using HPLC-DAD with 20 identified compounds (9 in both H and P extracts and 13 in BP extract). The P extract had the highest phytochemical content, with high levels of flavanone pinocembrin, flavanol catechin, lignan pinoresinol, and simple phenolics (p-coumaric and gallic acids). Then, the optimized mixtures were determined using an augmented simplex-centroid design. The optimized formulations (H33%:P43%:BP24%) and (H21%:P47%:BP32%) presented the optimal total phenolic content and DPPH-IC₅₀ with 226.88 mgGAE/g and 10.64 µg/mL, respectively, whereas the formulations (H26%:P52%:BP22%) and (H35%:P40%:BP25%) showed the optimal antimicrobial activity against S. aureus (MIC_S.aureus equal to 4.34 µg/mL) and E. coli (MIC_E.coli equal to 5.70 µg/mL), respectively. The predicted responses from these mixture proportions were also experimentally validated. Compared to the single free radical activity and antibacterial effect of each isolated bee product, these optimized formulations demonstrated an increased biological activity, and the determination of the fractional inhibitory concentrations revealed a synergistic effect between these products. This study emphasizes the interest in optimized bee product mixtures for practical applications beyond the pharmaceutical and food industries. Their potential can be extended to nutraceuticals, cosmeceuticals, animal health, environmental sustainability, and advanced biomedical research, offering holistic solutions for diverse challenges across various sectors. Exploring these applications further can unlock new avenues for innovation and sustainable development.