ABSTRACT

Background and Aim: Antimalarial drug resistance highlights the need to identify alternative bioactive compounds. Azadirachta indica (neem) contains phytochemicals with potential antiplasmodial properties, although their molecular interactions remain incompletely characterized. This study aimed to characterize the metabolites of A. indica leaf extract, evaluate its antimalarial activity against Plasmodium berghei, and investigate predicted interactions between selected metabolites and parasite-associated proteins.

Materials and Methods: Ethanolic leaf extract was characterized using liquid chromatography–high-resolution mass spectrometry (LC-HRMS). Twenty-five male BALB/c mice infected with chloroquine-sensitive P. berghei ANKA were randomly assigned to five groups (n = 5). Three groups received oral extract doses of 250, 500, or 750 mg/kg body weight for four consecutive days. Artesunate (2.4 mg/kg) and distilled water served as positive and negative controls, respectively. Parasitemia suppression was evaluated using a modified Peters' 4-day suppressive test. Selected metabolites underwent molecular docking against Plasmodium falciparum dihydrofolate reductase–thymidylate synthase, chloroquine resistance transporter, and plasmepsin II, alongside exploratory host-response targets caspase-3 and Bcl-2. Molecular dynamics simulations (100 ns) and binding free-energy calculations assessed predicted interaction stability.

Results: LC-HRMS tentatively identified 33 metabolites, predominantly flavonols and flavonol glycosides. Scopoletin exhibited the greatest relative abundance, followed by rutin, trifolin, isoquercetin, quercetin 3-β-D-glucoside, and morin. Parasitemia suppression reached 78.11%, 82.08%, and 76.83% at 250, 500, and 750 mg/kg, respectively, compared with 95.75% for artesunate. The highest numerical suppression occurred at 500 mg/kg. Differences among extract-treated and artesunate groups were not statistically significant (p > 0.05), although therapeutic equivalence was not established. Computational analyses identified favorable predicted interactions involving catechin, luteolin, and 2-coumaroylquinic acid, particularly luteolin with the chloroquine resistance transporter.

Conclusion: A. indica leaf extract demonstrated preliminary antimalarial activity against chloroquine-sensitive P. berghei. Integrated metabolite profiling and computational analyses identified candidate compounds warranting further investigation. Experimental target validation, individual-compound testing, and evaluation against drug-resistant parasites are necessary to establish mechanisms and therapeutic relevance.

Keywords: Azadirachta indica, antimalarial activity, liquid chromatography–high-resolution mass spectrometry, molecular docking, molecular dynamics, neem leaf extract, Plasmodium berghei, phytochemical profiling.