Implication of glutathione in the in vitro antiplasmodial mechanism of action of ellagic acid

Abstract

The search for new antimalarial chemotherapy has become increasingly urgent due to parasite resistance to current drugs. Ellagic acid (EA) is a polyphenol, recently found in various plant products, that has effective antimalarial activity in vitro and in vivo without toxicity. To further understand the antimalarial mechanism of action of EA in vitro, we evaluated the effects of EA, ascorbic acid and N-acetyl-L-cysteine (NAC), alone and/or in combination on the production of reactive oxygen species (ROS) during the trophozoite and schizonte stages of the erythrocytic cycle of P. falciparum. The parasitized erythrocytes were pre-labelled with DCFDA (dichlorofluorescein diacetate). We showed that NAC had no effect on ROS production, contrary to ascorbic acid and EA, which considerably reduced ROS production. Surprisingly, EA reduced the production of the ROS with concentrations (6.6×10(-9) - 6.6×10(-6) M) ten-fold lower than ascorbic acid (113×10(-6) M). Additionally, the in vitro drug sensitivity of EA with antioxidants showed that antiplasmodial activity is independent of the ROS production inside parasites, which was confirmed by the additive activity of EA and desferrioxamine. Finally, EA could act by reducing the glutathione content inside the Plasmodium parasite. This was consolidated by the decrease in the antiplasmodial efficacy of EA in the murine model Plasmodium yoelii- high GSH strain, known for its high glutathione content. Given its low toxicity and now known mechanism of action, EA appears as a promising antiplasmodial compound.

Figure 1. ROS generation in Plasmodium falciparum was detected by DCFDA probe.

All parasitized erythrocytes were labeled with DCFDA for 30 minutes. After washing, they were incubated for 3 hours with or without ellagic acid, ascorbic acid and NAC, alone and in combination. The level of ROS in the parasites was detected by DCF-dependent measurements. The data represent the mean ±SEM of four different experiments with triplicate sets in each assay. In the first column (A), the fluorescence intensity in parasites without ellagic acid is represented. The black band (control value) corresponded to the fluorescence intensity in untreated parasites incubated 3 hours. The grey and white bands corresponded to the fluorescence intensity in parasites only treated with NAC (857.10⁻⁶ M) and ascorbic acid (113.10⁻⁶ M) respectively. In the next four columns, the black bands are the fluorescence of parasites after treatment with various concentrations (between 6.61×10⁻⁹ and 6610×10⁻⁹ M) ellagic acid alone. The grey and white bands correspond to the ellagic acid treatment with NAC and ascorbic acid respectively with the same concentrations as in the first column. **p<0.01 indicates a significant difference compared with the untreated parasites. The quantity amount of ROS was compared according to the increased of EA concentration, ## p<0.05 indicates the significant difference.