Parasitostatic effect of maslinic acid. I. Growth arrest of Plasmodium falciparum intraerythrocytic stages

Abstract

Background: Natural products have played an important role as leads for the development of new drugs against malaria. Recent studies have shown that maslinic acid (MA), a natural triterpene obtained from olive pomace, which displays multiple biological and antimicrobial activities, also exerts inhibitory effects on the development of some Apicomplexan, including Eimeria, Toxoplasma and Neospora. To ascertain if MA displays anti-malarial activity, the main objective of this study was to asses the effect of MA on Plasmodium falciparum-infected erythrocytes in vitro.

Methods: Synchronized P. falciparum-infected erythrocyte cultures were incubated under different conditions with MA, and compared to chloroquine and atovaquone treated cultures. The effects on parasite growth were determined by monitoring the parasitaemia and the accumulation of the different infective stages visualized in thin blood smears.

Results: MA inhibits the growth of P. falciparum Dd2 and 3D7 strains in infected erythrocytes in, dose-dependent manner, leading to the accumulation of immature forms at IC50 concentrations, while higher doses produced non-viable parasite cells. MA-treated infected-erythrocyte cultures were compared to those treated with chloroquine or atovaquone, showing significant differences in the pattern of accumulation of parasitic stages. Transient MA treatment at different parasite stages showed that the compound targeted intra-erythrocytic processes from early-ring to schizont stage. These results indicate that MA has a parasitostatic effect, which does not inactivate permanently P. falciparum, as the removal of the compound allowed the infection to continue

Conclusions: MA displays anti-malarial activity at multiple intraerythrocytic stages of the parasite and, depending on the dose and incubation time, behaves as a plasmodial parasitostatic compound. This novel parasitostatic effect appears to be unrelated to previous mechanisms proposed for current anti-malarial drugs, and may be relevant to uncover new prospective plasmodial targets and opens novel possibilities of therapies associated to host immune response.

Figure 1

Dose-response curve of maslinic acid on P. falciparum infected erythrocytes. A. Structure of maslinic acid. B. P. falciparum Dd2 (black circles) and 3D7 (white circles) strains were grown in erythrocyte cultures at 1% initial parasitaemia and incubated with different concentrations of maslinic acid. The DNA content was determined at 48 hours by microfluorimetry. The calculated 50% inhibitory concentration (IC₅₀) values were 32 ± 2 for Dd2 and 26 ± 3 μM for 3D7. Results are the mean of three independent cultures.

Figure 2

Assay of β-haematin polymerization. Haem was incubated in the presence of increasing amounts of maslinic acid (open circle, dashed line), chloroquine (black circle, solid line) and atovaquone (triangle, dotted line) for 48 h, and the formation of β-haematin was determined spectrophotometrically at 405 nm. Results are the mean ± SD of three independent experiments.

Figure 3

Dose-dependent effects of maslinic acid on P. falciparum cultures compared to chloroquine and atovaquone. Synchronized cultures of Dd2 at mature ring stage (22 h, 1% parasitaemia) were incubated for 48 hours with the compounds at the indicated concentrations and compared to an untreated control. Cultures were evaluated by microscopic inspection of Wright's-stained thin blood smears. A. Microscopic images of P. falciparum cultures incubated with different concentrations of maslinic acid, chloroquine and atovaquone. Delayed trophozoites (Y) and pyknotic forms (X) showing condensation of the parasite nucleus are highlighted. B. Parasitaemia of the previous cultures after 48 h MA treatment. Results are the mean ± SD of three independent experiments. C. Parasitic stages observed in cultures treated with different concentrations of MA, chloroquine and atovaquone for 48 hours. Bars show percentage of rings (white), trophozoites (grey), schizonts (black) and pyknotic (hatched) forms observed in Dd2 cultures treated at the indicated compound concentrations. The percent of each group accounts for the fraction of cells showing every parasite stage from a total of 1000 erythrocytes ± SD. Results are representative of 2 experiments.

Figure 4

Effect of transient treatment with MA at different plasmodial stages. A. Development of parasitaemia after maslinic acid treatment at different growth stages of P. falciparum 3D7. Twelve-hours ring-stage synchronized cultures at 10% parasitaemia were incubated in the presence of MA 100 μM for 12 hours at times corresponding to ring (R+MA), trophozoite (T+MA) and schizont (S+MA) stages. After treatment, MA was removed and parasitaemia was monitored up to 86 h after culture synchronization: untreated (white triangles), R+MA (white circles), T+MA (black circles), S+MA (black triangles), not-removed MA (black squares). Cycle time indicates the elapsed hours of the culture started with synchronized 12 hour-old rings. Results are presented as arithmetic mean of three independent experiments ± SD. B. Morphological changes observed in the transiently-treated cultures. Representative light-microscopy fields of cultures subjected to the treatment are shown after Wright's-staining at the times indicated.

Figure 5

Effect of maslinic acid treatment at different infection periods in the accumulation of P. falciparum erythrocytic stages. Bars show the percentage of rings (white), trophozoites (grey), schizonts (black) and pyknotic (hatched) forms observed after 12 h incubation with 100 μM MA at ring (R+MA), trophozoite (T+MA) and schizont (S+MA) stages. Data collected by microscopic inspection of Wright's-stained blood smears and compared with controls untreated (top) and cultivated in the presence of 100 μM MA throughout the whole experiment (bottom). The percent of each group accounts for the fraction of cells showing every parasite stage out of a total of 1,000 erythrocytes. Results are representative of two experiments.