In vitro and in silico guided identification of antimalarial phytoconstituent(s) in the root of Citrus maxima (Burm.) Merr.

As a part of our continuous effort to find new therapeutic agents from natural sources, the hydroalcoholic (1:1) extract of Citrus maxima (Burm.) Merr. root was selected for the identification of possible antimalarial phytoconstituents. From the extract, three flavonoids including luteolin were isolated and evaluated for in vitro antimalarial activity against the chloroquine-sensitive ( Pf 3D7) and resistant ( Pf RKL-9) strains of Plasmodium falciparum . Among these, luteolin (CM3) showed the highest antimalarial activity with IC 50 values of 2.315 ± 0.489 and 2.691 ± 0.454 µg/ml against the Pf 3D7 and Pf RKL-9 strains respectively. To assess the safety of luteolin (CM3), a cytotoxicity study against a normal human embryonic kidney cell line (HEK-293) was performed and the compound was found to be safe with a CC 50 value of 222.3 ± 1.443 µg/ml. The docking study against 26 target proteins of P. falciparum revealed that luteolin (CM3) has a better binding affinity with two proteins, viz. P. falciparum lactate dehydrogenase ( Pf LDG) and P. falciparum enoyl-ACP reductase ( Pf EAR) in comparison to the co-crystallized ligands. Furthermore, the molecular dynamics simulation study of the protein-ligand complexes also supported the binding affinity and interactions of luteolin (CM3) at the active sites. Finally, the binding free energy calculation revealed that the luteolin formed a thermodynamically more stable complex with Pf LDG (-50.955 ± 17.184 kJ/mol) than Pf EAR (-24.856 ± 13.739 kJ/mol). Overall, in this study, we identified an antimalarial marker in the hydroalcoholic extract of C. maxima root which may act by inhibiting Pf LDG.Communicated by Ramaswamy H. Sarma.