Therapeutic Potential of Salvia rosmarinus: Seasonal and Geographical Variation in Phytochemical Composition, Bioactivity, and Synergistic Effects of Rosmarinic Acid with 5-FU

<i>Salvia rosmarinus</i> Spenn. (rosemary) is a medicinal and aromatic plant of notable pharmacological value. This study evaluated the therapeutic properties of rosemary leaves collected from two Serbian continental (L1, L2) and one Montenegrin Mediterranean (L3) locations, harvested in November (N), March (M), and July (J). Extracts prepared with 70% methanol, 70% ethanol, and water were analyzed for chemical composition and biological activity. L3 extracts exhibited the highest polyphenolic content, with L3M methanolic extract showing the greatest total phenolic (134.60 mg GAE/g) and phenolic acid levels (211.96 mg CAE/g), and L3M ethanolic extract the highest flavonoid content (25.54 mg QE/g). LC/MS analysis identified 28 previously unreported compounds in <i>Rosmarinus</i> sp. extracts, revealing hydroxycinnamic acid derivatives and flavonoid <i>O</i>-glycosides as the main constituents in <i>S. rosmarinus</i>. The alcoholic extracts were rich in 1,8-cineole, camphor, borneol, terpinen-4-ol, and verbenone. L3 extracts demonstrated the strongest antioxidant and enzyme-inhibitory activities, often surpassing positive controls. L3J showed pronounced cytotoxicity against HCT-116 colorectal cancer cells (IC₅₀ = 13.08 µg/mL after 24 h incubation), while showing non-cytotoxic effects on normal human keratinocytes (IC₅₀ > 500 µg/mL). Finally, rosmarinic acid alone synergistically enhanced the cytotoxic effect of 5-fluorouracil (combination index < 0.8). This comprehensive study highlights the influence of geography, season, and solvent on phytochemical profile and bioactivity of rosemary extracts, emphasizing the therapeutic potential of distinct rosemary populations.