Isolation, in-vitro Cytotoxicity and in Silico Analysis of Polymethoxyflavones From Kaempferia Parviflora on Breast Cancer Cell Lines

Kaempferia parviflora ( K. parviflora ) is a plant native to Southeast Asia known for its numerous health benefits, primarily attributed to its major constituents, polymethoxyflavones (PMFs). This study aims to isolate and characterize PMFs from K. parviflora , evaluate their cytotoxic activity against breast cancer cell lines, and perform in silico analyses to explore their potential mechanisms of action. PMFs were isolated from the optimized ethanolic extract of K. parviflora using gravitational column chromatography, followed by structural characterization via ¹H-NMR and ¹³C-NMR spectroscopy. The cytotoxic activity of the isolated PMFs 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 cytotoxicity assay. In silico analyses included molecular docking, density functional theory (DFT) calculations, drug-likeness evaluation, ADMET screening, and Pearson correlation analysis. Nine PMFs 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 ). All PMFs exhibited selective cytotoxicity, with stronger IC₅₀ effects on MCF-7 than on MDA-MB-231 cells after 72-hour treatment. PMF 4 showed the strongest cytotoxic effect, with an IC₅₀ value of 24.12 ± 0.45 µM respectively. Toxicity screening of these PMFs on NIH-3T3 mouse fibroblast cells at their respective IC ₅₀ concentrations showed > 80% cell viability ( PMF 9 : 88.41%) validating their selective cytotoxicity against cancer cells. PMF 4 , 7 , and 9 demonstrated strong binding affinities across six protein targets, particularly Bcl-XL, Bcl-2, and mTOR. DFT analysis revealed stabilized PMFs with favorable frontier molecular orbitals, indicating strong electron-accepting capabilities. Drug-likeness and ADMET screening supported the bioavailability and safety profiles of the PMFs. Pearson correlation analysis showed a significant positive correlation between cytotoxic activity (IC₅₀) and binding affinity to Bcl-XL (r = 0.831) and mTOR (r = 0.860).