Formononetin exerts synergistic action with artesunate against multi-drug-resistant P. falciparum arresting ring-to-schizont transition by inducing reactive oxygen species

Malaria, caused by Plasmodium falciparum, presents significant challenges for treatment due to the parasite’s complex life cycle and increasing multi-drug resistance. Artemisinin-based combination therapies (ACTs) are the current standard treatment, resistance development necessitates the exploration of new therapeutic targets. Recent evidence suggests that targeting oxidative stress to arrest blood stage ring to schizont growth progression in Plasmodium could offer a novel approach to combat drug-resistant malaria. Phytomolecules have been recognized for their potential to modulate oxidative stress with artemisinin derivatives. In the present study, we aimed to evaluate the effectiveness of formononetin (FMT), a natural isoflavonoid, alone and in combination with artesunate (ART) against multidrug-resistant P. falciparum (K1) strain and to decipher the underlying mechanism of action. The study presents compelling evidence demonstrating the anti-plasmodial action of FMT alone (IC₅₀ value 212µM) and synergistic interaction (FICI 0.13) with ART at a 1:1 ratio against the K1 strain of P. falciparum. The combination treatment affected the progression of P. falciparum from the ring stage to the schizont and showed the effect at asexual erythrocytic stages. Moreover, the combination resulted in a notable increase in reactive oxygen species (ROS) levels, both independently and in combination with ART. In combination with ART, FMT effectively modulated the total glutathione (GSH) level. Moreover, FMT and ART demonstrated the ability to induce apoptosis-like death of parasites, as evidenced by the Lipid peroxidation (malondialdehyde-MDA) and DNA fragmentation (TUNEL) levels. These results indicate that FMT could potentially ameliorate the growth of multidrug-resistant malaria parasites, enhance the effects of ART, and be suitable for developing anti-plasmodial agents from a cheap and sustainable source.