Isolation, structure elucidation, and in silico Topoisomerase II inhibition of medicarpins isolated from Alfalfa (Medicago sativa).

Phytochemical investigation of Medicago sativa (alfalfa) extract led to the isolation of sixteen compounds. Their structures were elucidated using NMR and comparison with reported data. Structural analysis of medicarpin, 10-methoxymedicarpin, and 4-methoxymedicarpin isolated from alfalfa revealed that these compounds share key features with reported Topoisomerase II inhibitors, including a bicyclic non-planar framework and two distinct aromatic groups, suggesting their potential as non-intercalative Topoisomerase II inhibitors. A flexible alignment study demonstrated significant structural similarity between medicarpin and podophyllotoxin, a known Topo II inhibitor. 10-Methoxymedicarpin exhibited the highest binding affinity to Topoisomerase II-DNA complex, along with a stable binding mode and favourable interaction dynamics. Molecular dynamics simulations over 100 ns confirmed the stability of 10-methoxymedicarpin-Topo II-DNA complex, with minimal fluctuations. MM-GBSA, ProLIF, PCAT, and FEL studies further validated the compound's strong binding potential and stability. These findings suggest that 10-methoxymedicarpin is a promising candidate for further development as a Topoisomerase II inhibitor and a potential anticancer candidate.