In-vitro Evaluation of Antileishmanial Activity and Toxicity of Artemether with Focus on its Apoptotic Effect

Abstract

Artemisinin and its derivatives are very important new class of antimalarial drugs. One of the most important artemisinin derivatives is artemether. The antiparasitic activity of artemether as a derivative of artemisinin is related to endoperoxide bridge in its structure. The aim of this study was the evaluation of antileishmanial effect of artemether, with more focus on its apoptotic effect. In this study we used artemether in concentration of 5, 10, 25, 50 and 100 μg/mL for promastigote assay, promastigote proliferation measurements by MTT assay, detection of apoptotic cells by Flow cytometry analysis and DNA ladder assay. The application of artemether, promastigote IC50 was measured as 25 μg/mL. The percentage of apoptotic promastigotes by using 25 μg/mL of artemether was 42.28. The results of present study showed that artemether has inhibition effect on intracellular and extracellular growth of Leishmania major. Promastigotes of Leishmania major undergo apoptosis after exposure to artemether.

Keywords: Apoptosis; Artemether; In-vitro; Leishmania major; Promastigotes.

Figure 1

Chemical structure of artemether

Figure2

The OD in 540 nm obtained from MTT assay of Leishmania major promastigotes in the presence of various concentrations of artemether in comparison with control group in 0, 24, 48 and 72 h

Figure 3

Flow cytometry analysis of promastigotes following treatment with artemether and after labeling with annexin-V and PI. Control 1, without treatinh. Control 2, with ethanol as solvent. Samples 10 μg/mL, 25 μg/mL and 50 μg/mL are samples that treated with 10 μg/mL, 25 μg/mL and 50 μg/mL of artemether respectively. Lower right region (LR) belongs to apoptotic cells (annexin positive) and upper left region (UL) belongs to necrotic cells (PI positive).

Figure 4

DNA fragmentation analysis by agarose gel electrophoresis. The DNA of promastigotes treated with 10, 25 and 50 μg of artemether after 24 h of incubation (lines 1-3). Integrated DNA (line4). Standard apoptotic ladder (line 5) and DNA marker (line 6).