Extraction and Quantification of Formononetin from RedClover (Trifolium pratense L.) and its Anticancer Effects on Hepatocellular Carcinoma Cells

Formononetin, a major isoflavone derived from Trifolium pratense L., exhibits promising anticancer effects in various tumor models; however, its activity against hepatocellular carcinoma (HCC) remains underexplored. In the current report, formononetin was extracted and purified from the aerial parts of T. pratense L., and UPLC-ESI-MS/MS analysis confirmed a concentration of 800 ± 15 ppm in the standardized extract. Treatment of HepG2 cells with formononetin (1-640 µM) for 24 to 72 hours resulted in a dose- and time-dependent reduction in cell viability, as measured by the MTT assay. Specifically, 20 µM reduced cell survival to approximately 10%, while 40 µM induced near-complete cytotoxicity at 72 h. Exposure to 10 µM formononetin over five days suppressed viability, as assessed by trypan blue exclusion, indicating primarily cytostatic effects. Clonogenic assays demonstrated a significant decrease in colony formation, from approximately 75 to 55 colonies (p < 0.01). DAPI staining revealed chromatin condensation, nuclear shrinkage, and apoptotic bodies consistent with intrinsic apoptosis after 48 h. These findings demonstrate potent anticancer activity through proliferative arrest and induction of apoptosis, warranting further in vivo studies in HCC.