Isolation, in vitro cytotoxicity and in silico analysis of methoxylated flavone derivatives from Kaempferia parviflora on breast cancer cell lines

Abstract Kaempferia parviflora ( K. parviflora ) is a plant native to Southeast Asia known to contain bioactive methoxylated flavone derivatives, including polymethoxyflavones (PMFs) and hydroxy-substituted methoxyflavones. This study aims to isolate and characterize methoxylated flavone derivatives from K. parviflora , evaluate their cytotoxic activity against breast cancer cell lines, and perform in silico analyses to predict their possible molecular interactions with selected cancer-related targets. PMFs were isolated from the optimized ethanolic extract of K. parviflora using gravitational column chromatography, followed by structural characterization via 1 H-NMR and 13 C-NMR spectroscopy. The cytotoxic activity of the isolated compounds was evaluated in vitro against hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cell lines, as well as NIH/3T3 mouse fibroblast cells using an MTT cytotoxicity assay . In silico analyses included molecular docking, density functional theory (DFT) calculations, drug-likeness evaluation, ADMET screening, and Pearson correlation analysis. Four PMFs and five hydroxy substituted methoxyflavones were successfully isolated and identified: 5-hydroxy-3,7-dimethoxyflavone (1), 5-hydroxy-7-methoxyflavone (2), 5-hydroxy-3,7,4′-trimethoxyflavone (3), 5-hydroxy-7,4′-dimethoxyflavone (4), 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (5), 3,5,7-trimethoxyflavone (6), 5,7-dimethoxyflavone (7), 3,5,7,3′,4′-pentamethoxyflavone (8), and 5,7,4′-trimethoxyflavone (9). The isolated compounds exhibited selective cytotoxicity, with stronger IC₅₀ effects on MCF-7 cells than MDA-MB-231 cells after 72-h treatment. PMF 4 showed the strongest cytotoxic effect against MCF-7 cells, with an IC₅₀ value of 24.12 ± 0.45 µM. Toxicity screening of selected compounds on NIH-3T3 mouse fibroblast cells at their respective MCF-7 IC 50 concentrations showed 77.86–88.41% cell viability, suggesting comparatively lower toxicity under the tested in vitro conditions. PMF 4, 7, and 9 showed predicted binding interactions with selected protein targets, particularly Bcl-xL, Bcl-2, and mTOR. DFT analysis revealed stabilized PMFs and hydroxy-substituted methoxyflavones with favorable frontier molecular orbitals, indicating strong electron-accepting capabilities. Drug-likeness and ADMET screening provided preliminary predictions of pharmacokinetic and toxicity-related properties of both PMFs and its hydroxy-substituted derivatives. Pearson correlation analysis showed exploratory associations between cytotoxic activity (IC₅₀) and predicted binding scores for selected targets, including Bcl-xL (r = 0.831) and mTOR (r = 0.860). However, these findings should be interpreted as preliminary because metabolism, achievable systemic concentrations, tissue distribution, and protein-level target validation were not investigated.