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.

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.