Differential Biological Effects of Trifolium pratense Extracts—In Vitro Studies on Breast Cancer Models

The increasing popularity of herbal supplements emphasizes the need of scientific data regarding their health benefits and possible toxicological concerns. The complexity of botanical extracts, which include thousands of distinct compounds, contributes to the challenging nature of this endeavor. In this study, we explored the hormetic effects of two <i>Trifolium pratense</i> extracts on breast cell lines. Using a wide range of concentrations (0.1 to 3.33 mg/mL), we analyzed how extracts modulate cellular processes such as viability, proliferation, and oxidative stress on breast adenocarcinoma highly invasive estrogen receptor negative (ER-) and noninvasive ER+ cells, as well as on non-tumorigenic ER- normal cells. The cytotoxicity and real-time cell analysis (RTCA) assays showed that both extracts exercised a biphasic dose effect on adenocarcinoma ER+ and normal ER- cell proliferation and oxidative stress. We report a monotonic dose-dependent cytotoxicity on highly invasive adenocarcinoma ER- cells; the induced apoptosis was based on the pro-oxidant activity of extracts. The reactive oxygen species (ROS) generation by high-dose ethanolic extract was observed in all cells, followed by mitochondria dysfunction. Oxidative stress parameters, such as malondialdehyde (MDA) and reduced glutathione (GSH) levels, and superoxide dismutase (SOD) activity were affected. Our study demonstrates that <i>T. pratense</i> extracts have chemoprevention potential in normal and tumorigenic breast cells by modulating cellular proliferation and oxidative stress.