Cytotoxic Effects of Bifora testiculata (L.) Spreng. Essential Oil and Its Main Component on Cancer Cell Lines

<i>Bifora testiculata</i> (L.) Spreng. (Apiaceae), an understudied species endemic to the Mediterranean and the only representative species of the genus <i>Bifora</i> in Sicily, was investigated for the first time for its essential oil (EO) chemical composition and cytotoxic properties. The EO was obtained via hydrodistillation and analyzed using GC-MS, revealing an aldehyde-rich profile (86.10%), dominated by <i>trans</i>-2-dodecenal (67.49%). Comparative analysis with previous studies on <i>B. testiculata</i> from Greece confirmed a similar aldehyde-rich profile, although minor compositional differences suggest potential chemotype variation. Given the biological relevance of <i>trans</i>-2-dodecenal and related aldehydes, further investigations into the cytotoxic properties of the EO of <i>B. testiculata</i> (<b>Bt</b>) and its main constituent against cancer cell lines were undertaken. Three human tumor cell lines (MDA-MB 231, A375, and CaCo2) and a human non-tumor cell line (HEK293) were subjected to viability tests using the MTT assay. The EO and <i>trans</i>-2-dodecenal exhibited remarkable cytotoxic activity against all cell lines, with IC₅₀ values ranging between 7.93 and 14.41 µg/mL for <b>Bt</b> and between 1.88 and 5.29 µg/mL for <i>trans</i>-2-dodecenal. AO/BE fluorescent staining and Hoechst nuclear staining showed the presence of apoptotic bodies in the treated cells. <i>N</i>-acetyl-L-cysteine was able to invert the effects of <b>Bt</b> and <i>trans</i>-2-dodecenal on cell lines, suggesting ROS involvement in cytotoxic activity. The results demonstrated that the <b>Bt</b> cytotoxic activity was mainly due to the presence of <i>trans</i>-2-dodecenal.