LC-MS- and Bioactivity-Guided Discovery of Novel Cyclohexenyl Chalcones from the Edible Rhizomes of Boesenbergia rotunda and Evaluation of Their Anticancer Activities against Colorectal Cancer

Abstract

Cyclohexenyl chalcones represent prenylated natural products characterized by a cyclohexene ring fused to the chalcone. In this study, eight new cyclohexenyl chalcones, rotundatins A–H (1–8), were purified from the edible rhizome of Boesenbergia rotunda. Their chemical structures were established through analyses of 1D and 2D NMR, HR-ESIMS, and DP4+ calculations. Notably, rotundatin A features an unprecedented oxocan-4-one ring system, and rotundatins B and E incorporate hydroperoxide moieties. All isolated cyclohexenyl chalcones were evaluated for cytotoxicity against HCT116 colorectal cancer cells, with 2, 9, 10, 12, and 14 showing antiproliferative activity (IC50 = 13.87–54.61 μM) compared with 5-fluorouracil as a positive control (IC50 = 10.32 μM). Morphological analysis and live/dead fluorescence staining supported membrane damage and cell death. Western blotting showed caspase-3 activation and PARP cleavage, indicating apoptosis-related cell death. Together, these findings highlighted the potential of food-derived cyclohexenyl chalcones as potential candidates for anticancer activity against colorectal malignancies.