Effects of Ellagic Acid on Hemoglobin Ingestion, Transport, and Digestion in the Digestive Vacuole of Plasmodium falciparum.

Context: Malaria, caused by Plasmodium parasites, has been a persistent threat to human health for centuries, causing significant illnesses and fatalities. The rise of drug-resistant strains has diminished the efficacy of conventional antimalarial medications, necessitating the exploration of alternative treatments. Ellagic acid has emerged as a promising candidate, demonstrating effectiveness in laboratory experiments by impeding beta-hematin (hemozoin) formation in mature Plasmodium falciparum stages. This research endeavors to elucidate the ellagic acid's mechanism of action against malaria parasites by examining its impact on hemoglobin ingestion, transport, and digestion in both chloroquine-sensitive (3D7) and chloroquine-resistant (W2) P. falciparum strains in vitro. Aim: This study aimed to elucidate the mechanism of ellagic acid antimalarial action against 3D7 and W2 strains of P. falciparum in vitro by using a transmission electron microscope. Materials and Methods: 3D7 and W2 strains of P. falciparum were cultured in flasks with a complete culture medium and washed red blood cells (type O+) at a hematocrit of 2%. Synchronized ring-stage parasite-infected erythrocytes, approximately 2 h post-synchronization, were utilized for the antimalarial activity assay. Based on the half-maximal inhibitory concentration (IC50) value of ellagic acid, three different sub-lethal concentrations of the compound against the 3D7 and W2 strains. Samples were prepared for real-time microscopic analysis. Samples were immediately fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4) for 2 h. Then, the samples were washed, postfixed with 2% osmium tetroxide in 0.1 M phosphate buffer for 1 h, washed with distilled water, dehydrated, infiltrated, and embedded. The sample blocks were cut by using an ultramicrotome. These thin sections were stained with 1% lead citrate and 1% uranyl acetate. Statistical Analysis Used: The IC50 of ellagic acid against the 3D7 and W2 parasites was analyzed using computerized GraphPad Prism 8.0 software. Results: Findings show significant inhibition by ellagic acid (P < 0.001) against 3D7 and W2 parasites. The IC50 values were 1.0 ± 0.7 and 1.3 ± 0.1 nM, respectively, surpassing the artemisinin's effectiveness (IC50-3D7 parasite = 2.2 ± 0.2 nM; IC50-W2 parasite = 4.3 ± 0.3 nM). Transmission electron microscopy elucidated the ellagic acid's impact on the early trophozoite stage of 3D7 and W2 parasites, highlighting the disruption of hemoglobin metabolism within the parasites. Conclusion: These findings underscore the ellagic acid's potential as a promising antimalarial agent, targeting hemoglobin processes in both 3D7 and W2 P. falciparum strains.