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

Abstract

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.

Waste Citrus limon Leaves as Source of Essential Oil Rich in Limonene and Citral: Chemical Characterization, Antimicrobial and Antioxidant Properties, and Effects on Cancer Cell Viability

“…Citrus plants constitute one of the main valuable sources of essential oils (EOs) in the world [ 2 , 3 , 8 , 9 ]. Citrus EOs, primarily extracted from the peels [ 3 , 4 , 8 , 9 ] but also from leaves [ 9 , 10 , 11 ], flowers [ 1 , 9 ], young shoot [ 3 ], buds [ 3 , 9 ], seeds, and roots [ 9 ], are aromatic volatile liquids, easily extracted by steam distillation [ 3 , 9 ]. Citrus EOs are very complex mixtures of organic components with monoterpenes and sesquiterpenes as well as their oxygenated derivatives, aliphatic aldehydes, alcohols, and esters constituting the major fractions, and are amply used for their numerous biological activities [ 1 , 2 , 3 , 4 , 5 , 8 , 9 , 10 , 11 , 12 ].…”

“…Lemon EOs, obtained by cold pressing of the peel or distillation of leaves, are broadly applied as an aroma enhancer in beverages, bakery, and food products, as a flavoring agent in pharmaceutical preparations, and as a fragrance in perfumery/cosmetic industries [ 13 , 15 ]. Lemon leaves (evergreen and lanceolate) represent an important source of EO, which ranges approximately between 0.33–0.34% [ 10 , 16 ] and 0.56% [ 17 ] (by weight) and varies greatly with the variety and/or cultivars, the geographic origin, and the harvesting period [ 3 , 13 , 15 ]. The EO of the C. limon leaves differs in composition from oil obtained from pericarp in which limonene generally represents the major component [ 4 , 13 , 18 ].…”

“…The EO of the C. limon leaves differs in composition from oil obtained from pericarp in which limonene generally represents the major component [ 4 , 13 , 18 ]. Whereas the C. limon leaf EO is generally rich in limonene [ 11 , 13 , 18 ], in some cases, other compounds have been identified as the major constituents [ 10 , 15 , 16 , 17 , 19 ]. Other reported main compounds include neral, geranial, sabinene, citronellal, linalool, (E)-β-ocimene, geranyl acetate, geraniol, alpha-terpineol, linalyl acetate, and myrcene [ 10 , 11 , 13 , 15 , 16 , 17 , 18 , 19 ]; however, significant quali-quantitative differences in C. limon leaf EO chemical composition have emerged among data in the literature [ 10 , 11 , 13 , 15 , 16 , 17 , 18 , 19 ].…”

“…Several studies have been performed on analgesic [ 9 ], antimicrobial [ 11 , 15 , 16 , 17 , 19 , 20 ], anti-leishmanial [ 10 ], insecticidal [ 16 ], and antioxidant activities [ 11 , 15 , 18 ] of the EOs obtained from the leaves of various species and/or cultivars of lemon around the world. Moreover, C. limon EOs and their aroma components (limonene, linalyl acetate, geranial, and neral) have drawn the attention of researchers for their anticancer activity [ 9 , 21 , 22 , 23 , 24 , 25 , 26 ]; however, limited research has been specifically conducted on the antitumor properties and cytotoxicity of lemon leaf EO [ 27 , 28 ].…”

The effects of different extraction methods on essential oils from orange and tangor: From the peel to the essential oil

“…Citrus essential oil (CEO) is a concentrated organic soluble plant extract that has been used for various purposes, such as producing perfumes, cosmetics, and medicines. The aroma quality, biological effects (Bouabdallah et al., 2022 ), and yield of CEOs can be determined by the extraction methods. CEOs are mainly obtained using distillation (hydrodistillation, steam distillation, and dry distillation) and nonthermal extraction (cold pressing, supercritical CO 2 fluid extraction, and solvent extraction; Filly et al., 2016 ).…”