Apoptotic and Cytotoxic Potential of Carnosic Acid- and Carnosol-Rich Fractions From Salvia dorystaechas on Human Hepatoma (HepG2) Cells

Plant isolates from Salvia dorystaechas (Lamiaceae) were investigated for their apoptotic and cytotoxic effects on human hepatocellular carcinoma (HepG2) cells under various treatment conditions. Chromatographic isolation yielded fractions (Fr C, D, F, and H) rich in carnosic acid (CA) and carnosol (CL), with significantly higher concentrations (26.3–93.5 g/kg) compared to the crude extract (10.0 g/kg). Other phenolic compounds, including caftaric acid, epicatechin, quercetin, and rutin, were also detected in notable amounts via ultra-high-performance liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS). CA-/CL-rich fractions effectively induced caspase-9 and caspase-3 activities, decreased cell viability, and increased lactate dehydrogenase (LDH) release. These effects were significant compared to those induced by 0.1 mM hydrogen peroxide (H₂O₂). Notably, Fr D and Fr H, which contained the highest CA/CL concentrations, showed caspase-9/caspase-3 activities of 1.60/1.83 and 1.63/1.94, respectively, at 25 μg/mL after 24 h of treatment, whereas the crude plant extract showed corresponding activity values of 1.33/1.36 under the same conditions. In contrast, the crude extract exhibited its strongest apoptotic and cytotoxic effects under harsher conditions (50 μg/mL and 48 h), with caspase-9/caspase-3 activities of 1.70/2.59, 12.0% cell viability, and 65.4% LDH release. Apoptosis was further supported by superoxide (O₂•⁻) production mediated by NADPH oxidases (NOXs), as the NOX inhibitor diphenyleneiodonium (DPI) reduced O₂•⁻ levels by 28.6%–48.6%. Additionally, nuclei-stained cells revealed morphological changes, including nuclear fragmentation, apoptotic body formation, and loss of adhesion. In conclusion, CA-/CL-rich S. dorystaechas isolates demonstrated strong apoptotic and cytotoxic effects on HepG2 cells, with contributions from other phenolic compounds. These findings highlight the potential therapeutic value of S. dorystaechas isolates in liver cancer treatment.