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Anti-leishmanial drugs extracted from natural sources have not been sufficiently explored in the literature. Until now, leishmaniasis treatments have been limited to synthetic and expensive drugs. This study investigated, for the first time, the anti-leishmanial efficacy of essential oils (EOs) from the leaves of Citrus species ( C. sinensis , C. limon , and C. clementina ). Essential oils were extracted from three species by solvent free microwave extraction (SFME); in addition, lemon oil was also isolated by hydro-distillation (HD). These were investigated using gas chromatography coupled with mass spectrometry (GC-MS) and evaluated against Leishmania species, namely Leishmania major and Leishmania infantum , using a mitochondrial tetrazolium test (MTT) assay. The chemical compositions of Citrus limon EOs obtained by HD and SFME showed some differences. The identified peaks of C. limon (SFME) represented 93.96%, where linalool was the major peak (44.21%), followed by sabinene (14.22%) and ocimene (6.09%). While the hydro-distilled oil of C. limon contained geranial (30.08%), limonene (27.09%), and neral (22.87%) in the identified peaks (96.67%). The identified components of C. clementina leaves oil (68.54%) showed twenty-six compounds, where the predominant compound was geranial (42.40%), followed by neral (26.79%) and limonene (14.48%). However, 89.82% C. sinensis oil was identified, where the major peaks were for neral (27.52%), linalool (25.83%), and geranial (23.44%). HD oil of lemon showed the highest activity against L. major , with moderate toxicity on murine macrophage (RAW 264.7) cells, and possessed the best selectivity index on both Leishmanial species (SI: 3.68; 6.38), followed by C. clementina oil and C. limon using SFME (0.9 ± 0.29, 1.03 ± 0.27, and 1.13 ± 0.3), respectively. C. clementina oil induced the greatest activity on Leishmania infantum , followed by HD lemon and SFME lemon oils (0.32 ± 0.18, 0.52 ± 0.15, and 0.57 ± 0.09, respectively) when compared to Amphotericin B (0.80 ± 0.18 and 0.23 ± 0.13) as a positive control, on both species, respectively. Our study suggests a potent anti-leishmanial activity of lemon oil (HD) on L. major , followed by C. clementina . With the same potency on L. infantum shown by C. clementina oil, followed by HD lemon oil. This effect could be attributed to the major compounds of limonene, citral, and neral, as well as the synergistic effect of other different compounds. These observations could be a starting point for the building of new anti-leishmanial drugs from natural origins, and which combine different EOs containing Citrus cultivars.

Sustainable Extraction, Chemical Profile, Cytotoxic and Antileishmanial Activities In-Vitro of Some Citrus Species Leaves Essential Oils