Extraction Strategy and C18 Solid-Phase Fractionation Shape Phenolic Profiles, Antioxidant Capacity, and Cancer Cell...

Medicinal plants are a rich source of biologically active compounds, including phenolic acids, flavonoids, ellagitannins and other secondary metabolites. However, the contribution of specific groups of phenolic compounds to antiproliferative activity remains insufficiently clarified. This study extends our previous crude-extract screening by evaluating whether C18 solid-phase extraction (SPE) fractions with different phenolic profiles are associated with different antiproliferative responses. In parallel, extraction strategies were compared to assess method-dependent changes in phenolic recovery and antioxidant capacity, and an additional single-species vegetation-stage analysis of Chamaenerion angustifolium L. Holub was performed to evaluate harvest-stage effects. Phytochemical characterisation was performed using spectrophotometric assays and high-performance liquid chromatography (HPLC) analyses. Among the tested extraction methods, 75% (v/v) methanol in water was the most effective conventional solvent, and ultrasound-assisted extraction yielded the highest overall TPC (total phenolic content), TFC (total flavonoid content), and RSA (radical scavenging activity) values. Vegetation stage analysis of C. angustifolium L. Holub revealed significant variation in phenolic content and antioxidant activity, with the highest levels observed at the beginning of the flowering. Antiproliferative activity was assessed against five cancer cell lines (4T1, A549, Caki-1, HCT116 and MCF7), while HEK-293 cells were used as an immortalised non-cancerous reference model for general cytotoxicity evaluation. Linear mixed-model analysis confirmed a significant incubation-time effect in all tested cancer cell lines, with IC₅₀ values generally decreasing after prolonged exposure. Statistically significant F2-F3 differences were plant-dependent. The 30% (v/v) methanol in water fraction (F2), enriched in oenothein B in C. angustifolium L., showed stronger antiproliferative activity, whereas the 60% (v/v) methanol in water fraction (F3) showed stronger activity in Quercus robur L., Juglans nigra L., Juglans regia L., and Solidago canadensis L. These findings indicate that antiproliferative activity was associated with the qualitative and quantitative composition of the selected phenolic-rich SPE fractions rather than with a single universal fraction effect. All tested fractions exhibited lower cytotoxicity toward HEK-293 cells under the applied conditions; however, claims of selectivity should be confirmed using additional normal or primary cell models. Overall, the findings clarify the role of extraction strategy, harvest stage and targeted fractionation in linking phenolic composition with biological activity.