Cold ethanol extraction yields a polyphenol-rich polish propolis extract with potent anti-melanoma activity

Propolis is a complex natural product used in functional foods and nutraceuticals, and its bioactivity depends on its botanical origin and processing. In this study, we employed a cold-ethanol extraction method on Polish propolis to produce wax-free polyphenol-rich extracts while preserving heat-sensitive compounds. UHPLC-DAD-ESI-IT-MS profiling identified 72 compounds in the extracts, predominantly flavonoids and caffeic acid esters (e.g., CAPE), which is consistent with a poplar-type propolis profile. The cold-extracted propolis exhibited potent anti-melanoma activity in vitro against human melanoma cell lines (A375 and SK-MEL-3), with minimal impact on normal skin fibroblasts at similar concentrations. Both extract variants (prepared using 75% and 80% ethanol) exhibited dose-dependent cytotoxic and antiproliferative effects on melanoma cells, with the 75% ethanol extract consistently demonstrating greater potency. At 75 μg/mL, the 75% extract reduced SK-MEL-3 cell viability to ∼20%, whereas the viability remained ∼60% with the chemotherapeutic control (etoposide). In a wound-healing assay, 25 μg/mL of the 75% extract inhibited melanoma cell migration as effectively as an equal concentration of etoposide, indicating its strong anti-metastatic potential. Propidium iodide/Hoechst staining confirmed that the propolis extract induced melanoma cell death. This study is the first to correlate the region-specific chemical profile of Polish propolis with its comprehensive anti-melanoma effects. Overall, these findings demonstrate that cold-ethanol extraction yields a highly bioactive polyphenol-rich propolis extract, highlighting its potential as a valuable ingredient in functional foods and in the nutraceutical industry.