Cytotoxicity and Anti-Leishmania Amazonensis Activity of Citrus Sinensis Leaf Extracts.

Context: Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease characterized by lesional polymorphism and the commitment of skin surface. Previous reports demonstrated that the Citrus genus possess antimicrobial activity.Objective: This study evaluated the anti-L. amazonensis activity of Citrus sinensis (L.) Osbeck (Rutaceae) extracts.Materials and methods: Citrus sinensis dried leaves were subjected to maceration with hexane (CH), ethyl acetate (CEA), dichloromethane/ethanol (CD/Et - 1:1) or ethanol/water (CEt/W - 7:3). Leishmania amazonensis promastigotes were treated with C. sinensis extracts (1-525 mu g/mL) for 120h at 27 degrees C. Ultrastructure alterations of treated parasites were evaluated by transmission electron microscopy. Cytotoxicity of the extracts was assessed on RAW 264.7 and J774.G8 macrophages after 48-h treatment at 37 degrees C using the tetrazolium assay. In addition, Leishmania-infected macrophages were treated with CH and CD/Et (10-80 mu g/mL).Results: CH, CD/Et and CEA displayed antileishmanial activity with 50% inhibitory activity (IC50) of 25.91 +/- 4.87, 54.23 +/- 3.78 and 62.74 +/- 5.04 mu g/mL, respectively. Parasites treated with CD/Et (131.2 mu g/mL) presented severe alterations including mitochondrial swelling, lipid body formation and intense cytoplasmic vacuolization. CH and CD/Et demonstrated cytotoxic effects similar to that of amphotericin B in the anti-amastigote assays (SI of 2.16, 1.98 and 1.35, respectively). Triterpene amyrins were the main substances in CH and CD/Et extracts. In addition, 80 mu g/mL of CD/Et reduced the number of intracellular amastigotes and the percentage of infected macrophages in 63% and 36%, respectively.Conclusion: The results presented here highlight C. sinensis as a promising source of antileishmanial agents.