Cytotoxicity and anti-Leishmania amazonensis activity of Citrus sinensis leaf extracts

• Published in: Pharmaceutical biology 2017, Vol.55 (1), p.1780-1786
• Main Authors: Garcia, Andreza R., Amaral, Ana Claudia F., Azevedo, Mariana M. B., Corte-Real, Suzana, Lopes, Rosana C., Alviano, Celuta S., Pinheiro, Anderson S., Vermelho, Alane B., Rodrigues, Igor A.
• Format: Article
• Language: English
• Subjects: Acetic acid; Amastigotes; Amphotericin B; Animals; Antileishmanial activity; Antileishmanial agents; Antimicrobial activity; Antiprotozoal Agents - isolation & purification; Antiprotozoal Agents - pharmacology; Antiprotozoal Agents - toxicity; Cell Survival - drug effects; citrus genus; Citrus sinensis; Citrus sinensis - chemistry; Citrus sinensis - toxicity; Cutaneous leishmaniasis; Cytotoxicity; Dichloromethane; Dose-Response Relationship, Drug; Ethanol; Ethyl acetate; Inhibitory Concentration 50; Leishmania - drug effects; Leishmania - growth & development; Leishmania - ultrastructure; Leishmania amazonensis; macrophage infection; Macrophages; Macrophages - parasitology; Mice; Mitochondria; Original; Parasites; Parasitic Sensitivity Tests; Phytotherapy; Plant extracts; Plant Extracts - isolation & purification; Plant Extracts - pharmacology; Plant Extracts - toxicity; Plant Leaves - chemistry; Plant Leaves - toxicity; Plants, Medicinal; Promastigotes; RAW 264.7 Cells; Solvents - chemistry; Transmission electron microscopy; Ultrastructure
• ISSN: 1388-0209; 1744-5116
• DOI: 10.1080/13880209.2017.1325380

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 μg/mL) for 120 h at 27 °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 °C using the tetrazolium assay. In addition, Leishmania-infected macrophages were treated with CH and CD/Et (10-80 μg/mL). Results: CH, CD/Et and CEA displayed antileishmanial activity with 50% inhibitory activity (IC 50 ) of 25.91 ± 4.87, 54.23 ± 3.78 and 62.74 ± 5.04 μg/mL, respectively. Parasites treated with CD/Et (131.2 μg/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 μg/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.