The monoterpene 1,8-cineole prevents cerebral edema in a murine model of severe malaria

Abstract

1,8-Cineole is a naturally occurring compound found in essential oils of different plants and has well-known anti-inflammatory and antimicrobial activities. In the present work, we aimed to investigate its potential antimalarial effect, using the following experimental models: (1) the erythrocytic cycle of Plasmodium falciparum; (2) an adhesion assay using brain microvascular endothelial cells; and (3) an experimental cerebral malaria animal model induced by Plasmodium berghei ANKA infection in susceptible mice. Using the erythrocytic cycle of Plasmodium falciparum, we characterized the schizonticidal effect of 1,8-cineole. This compound decreased parasitemia in a dose-dependent manner with a half maximal inhibitory concentration of 1045.53 ± 63.30 μM. The inhibitory effect of 972 μM 1,8-cineole was irreversible and independent of parasitemia. Moreover, 1,8-cineole reduced the progression of intracellular development of the parasite over 2 cycles, inducing important morphological changes. Ultrastructure analysis revealed a massive loss of integrity of endomembranes and hemozoin crystals in infected erythrocytes treated with 1,8-cineole. The monoterpene reduced the adhesion index of infected erythrocytes to brain microvascular endothelial cells by 60%. Using the experimental cerebral malaria model, treatment of infected mice for 6 consecutive days with 100 mg/kg/day 1,8-cineole reduced cerebral edema with a 50% reduction in parasitemia. Our data suggest a potential antimalarial effect of 1,8-cineole with an impact on the parasite erythrocytic cycle and severe disease.

Fig 1. The monoterpene 1,8-cineole decreased parasitemia levels in vitro.

Synchronized cultures of erythrocytes infected with mature forms of P. falciparum were incubated with 1,8-cineole for 24 h to monitor parasitemia. (A and B) Dose-response effect of 1,8-cineole on parasitemia (n = 4). (A) Representative images of the different experimental conditions. Arrows indicate infected erythrocytes with ring form. (B) Infected erythrocytes (1% parasitemia, 3%–5% hematocrit) were incubated for 24 h with increasing concentrations of 1,8-cineole (65–6483 μM). After incubation, the percentage of ring forms was determined by optical microscopy as described in the Materials and methods section. (C) The inhibitory effect of 1,8-cineole was not dependent on the level of parasitemia (n = 5). The number of rings was monitored after treating erythrocytes infected with increasing amounts of schizonts (0.5%–3% parasitemia) with 972 μM 1,8-cineole for 24 h. The results are presented as the mean ± SD. *P < 0.05 versus control. n.s., not significant.

Fig 2. Assessment of the effect of 1,8-cineole on specific stages of the P. falciparum life cycle and evolution.

(A) Experimental design. Different evolutive forms of P. falciparum were treated with 972 μM 1,8-cineole, and parasitemia was determined by optical microscopy (analysis). The arrows indicate when treatment with 1,8-cineole started. S, schizonts; R, rings; T, trophozoites. (B) Determination of parasitemia right after differentiation (n = 5). S → R, evolution from schizont to ring; R → T, evolution from ring to trophozoite; T → S, evolution from trophozoite to schizont. The results are presented as the mean ± SD. *P < 0.05 versus control. n.s., not significant.

Fig 3. Treatment with 1,8-cineole impaired the intracellular development of Plasmodium falciparum.

(A) Experimental design. Non-synchronized cultures of P. falciparum (1% parasitemia) were treated daily (white arrows) or not with 972 μM 1,8-cineole for 96 h. Parasitemia was determined by optical microscopy every 24 h (analysis). (B) Assessment of parasitemia (n = 6). (C) Representative images showing that treatment with 1,8-cineole arrests intracellular development of the parasite. (D) Assessment of the distribution of evolutive forms of the parasite in the presence of 972 μM 1,8-cineole (n = 6). Troph, trophozoite; schiz, schizont. The results are presented as the mean ± SD. *P < 0.05 versus control.

Fig 4. Treatment with 1,8-cineole induced ultrastructural changes in Plasmodium falciparum.

Control cells (A–D) showed the characteristic structure of trophozoites, including an extensive endoplasmic reticulum network (A, white rectangle; B) and digestive vacuoles filled with hemozoin crystals (A, C, and D arrows). In contrast, cells incubated with 972 μM 1,8-cineole showed loss of endomembrane integrity (E, white rectangle; F) and empty vacuoles resembling residual food vacuoles (E, G, and H, asterisks).

Fig 5. The inhibitory effect of 1,8-cineole on the erythrocytic cycle of Plasmodium falciparum is not reversible.

(A) Experimental design. Synchronized cultures of erythrocytes infected with P. falciparum schizonts were incubated or not with 972 or 3241 μM 1,8-cineole (white arrows) for 24 h. Cells were harvested, and parasitemia was determined by optical microscopy (analysis). The cells were then washed, and parasitemia was adjusted to 0.5% before reculturing them in absence of the compound for an additional 3 consecutive days. Parasitemia was determined every 24 h (analysis at 24, 48, 72, and 96 h after the first washing). (B) The inhibitory effect of 1,8-cineole on parasitemia levels in the first 24 h, before washing out the compound (n = 3). (C) The effect of the time course of 1,8-cineole on parasitemia after washing out the compound from the cells (n = 3). The results are presented as the mean ± SD. *P < 0.05 versus control, #P < 0.05 versus 972 μM 1,8-cineole.

Fig 6. 1,8-cineole inhibited the adhesion of Plasmodium falciparum-infected erythrocytes to brain macrovascular epithelial cell monolayers.

(A) Experimental design. BMECs were cultured in 24-well plates (4 × 10⁵ cells/well) for 24 h. The erythrocytes were pre-treated with 1,8-cineole for 2 h and then 5 × 10⁴ erythrocytes/well were cocultured with BMECs for an additional 2 h (adhesion assay), generating 3 different experimental groups: (1) non-infected red blood cells (RBC, control); (2) infected red blood cells (iRBCs, 5% parasitemia); or (3) iRBCs in the presence of 972 μM 1,8-cineole. The adhesion assay was performed as described in the Materials and methods section. (B) Representative images of erythrocytes adhered to BMEC monolayers. Red arrows indicate adhered iRBCs. Scale bar, 50 μm. (C) Adhesion index. Experiments were performed in triplicate, using 3 independent cell suspensions. Results are expressed as the mean ± SD. *P < 0.05 versus RBCs, #P < 0.05 versus iRBCs.

Fig 7. Treatment with 1,8-cineole reduced infection and cerebral edema in a model of experimental cerebral malaria.

C57BL/6 mice were infected with P. berghei ANKA (PbA) before treatment with 100 mg/kg/day of 1,8-cineole or 10 mg/kg/day of artesunate via intraperitoneal injection for 6 consecutive days. (A) Peripheral blood parasitemia was determined in hematologic stained thin blood smears by optical microscopy (n = 5). As depicted, parasitemia was determined at days 3, 4, 5 and 6 post-infection. (B) Assessment of cerebral edema (n = 4). At day 6 post-infection, cerebral edema was assessed by Evans blue dye extravasation assay as described in the Materials and methods section. The results are expressed as the mean ± SD. (A) *P < 0.05 versus PbA; ^#P < 0.05 versus PbA + artesunate. (B) *P < 0.05 versus control mice (non-infected mice); ^#P < 0.05 versus PbA; ^&P < 0.05 versus PbA + artesunate.