Apoptotic and Antiproliferative Effects of Ajuga reptans L. Ethanol Extract via Inhibition of NF-κB and eNOS Activity in HCT116 Colon Cancer Cells

Objective: This study aimed to investigate the ethanol extract of Ajuga reptans L. (Lamiaceae) on cell proliferation, apoptotic response, expression levels of Caspase-3, NF-κB and eNOS proteins on HCT116 human colorectal carcinoma cells. Materials and Methods: A. reptans was extracted using a Soxhlet apparatus with ethanol, and the obtained extract was applied to HCT116 and HUVEC cells (as healthy controls) in increasing concentrations (0–800 µg/mL). Cell viability was analysed by the WST-1 assay at 24 h and 48 h. Morphological changes were observed via inverted microscopy. ELISA was performed to quantify Caspase-3, NF-κB, and eNOS protein levels. Results: A. reptans ethanol extract showed concentration and duration-dependent cytotoxic effects on HCT116 cells, with IC₅₀ values of 206.9 µg/mL (24 h) and 147.5 µg/mL (48 h). Minimal cytotoxicity was observed in HUVEC cells. Microscopy confirmed morphological signs of apoptosis in HCT116 cells. ELISA results demonstrated increased Caspase-3 and decreased NF-κB and eNOS levels, indicating induction of apoptosis and suppression of pro-survival pathways. Conclusions: The ethanol extract of Ajuga reptans selectively inhibited the proliferation of colorectal cancer cells by apoptotic induction and modulation of NF-κB and eNOS signaling, suggesting its potential as a plant-based adjunctive agent in colorectal cancer therapy.