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

Abstract

This study investigated chemical composition, cytotoxicity in normal and cancer cells, and antimicrobial and antioxidant activity of the essential oil (EO) isolated by hydrodistillation from the discarded leaves of lemon (Citrus limon) plants cultivated in Sardinia (Italy). The volatile chemical composition of lemon leaf EO (LLEO) was analyzed with gas chromatography-mass spectrometry combined with flame ionization detection (GC/MS and GC/FID). The most abundant component of LLEO was limonene (260.7 mg/mL), followed by geranial (102.6 mg/mL) and neral (88.3 mg/mL). The antimicrobial activity of LLEO was tested using eight bacterial strains and two types of yeasts by a microdilution broth test. Candida albicans showed the greatest susceptibility (MIC = 0.625 μL/mL) and Listeria monocytogenes and Staphylococcus aureus were inhibited at low LLEO concentration (MIC values from 2.5 to 5 μL/mL). The C. limon leaf EO displayed radical scavenging ability (IC₅₀ value of 10.24 mg/mL) in the 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) assay. Furthermore, the LLEO impact on cell viability was explored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in cancer HeLa cells, A375 melanoma cell line, normal fibroblasts (3T3 cells), and keratinocytes (HaCaT cells). LLEO, at 24 h of incubation, significantly reduced viability from 25 μM in Hela cells (33% reduction) and A375 cells (27%), greatly affecting cell morphology, whereas this effect was found from 50 μM on 3T3 fibroblasts and keratinocytes. LLEO's pro-oxidant effect was also established in HeLa cells by 2',7'-dichlorodihydrofluorescein diacetate assay.

Keywords: Citrus limon; bioactivity; cytotoxicity; discarded leaves; leaf essential oil.

Figure 1

Digital image of Citrus limon leaves (a) and essential oil (LLEO) obtained by steam distillation of the leaves (b).

Figure 2

GC-MS chromatogram, obtained on an HP-5 capillary column, of C. limon leaf essential oil (LLEO).

Figure 3

The panel shows representative images of phase contrast of human cancer HeLa cells, A375 human melanoma cells, human HaCaT keratinocytes, and 3T3 normal murine fibroblasts. Bar = 100 μm.

Figure 4

Viability, expressed as % of the control (0), induced by incubation for 24 h with different amounts (2.5–500 μg/mL) of the essential oil obtained from C. limon leaves (LLEO) in human cancer HeLa cells (a), A375 human melanoma cells (b), healthy human HaCaT keratinocytes (c), and 3T3 normal murine fibroblasts (d) (MTT assay). Three independent experiments are performed, and data are presented as mean and SD (n = 15). *** = p < 0.001, ** = p < 0.01 versus respective controls (0) (One-way ANOVA and Bonferroni post Test).

Figure 5

The panel shows representative images of phase contrast of control HeLa cells and cells treated for 24 h with C. limon leaf essential oil (LLEO) at 2.5–500 μg/mL. Bar = 100 μm.

Figure 6

The panel shows representative images of phase contrast of control HaCaT keratinocytes and cells treated for 24 h with C. limon leaf essential oil (LLEO) at 2.5–500 μg/mL. Bar = 100 μm.

Figure 7

(a) ROS-induced fluorescence (arbitrary units), by H₂-DCF-DA assay, measured at 0, 20, 40, 60, 80, 100, and 120 min in HeLa control cells (Ctrl) and cells exposed for 2 h to different amounts of C. limon leaf essential oil (LLEO) (from 2.5 to 500 μg/mL). Data were presented as mean ± SD (n = 9); *** = p < 0.001, ** = p < 0.01, * = p < 0.05 versus the Ctrl at each time point. Evaluation of the statistical significance of differences between groups was performed by one-way ANOVA followed by the Bonferroni Multiple Comparisons Test. (b) The panel shows representative images of phase contrast of control HeLa cells and cells after 2 h incubation with LLEO at 100, 250, and 500 μg/mL. Bar = 100 μm.

Figure 8

ROS-induced fluorescence (arbitrary units), by H₂-DCF-DA assay, measured at different time points (every 5 min) in HaCaT control cells (Ctrl) and cells exposed for 1 h to different amounts of H₂O₂ (0.5, 1, and 2.5 mM) in the absence and in the presence (24 h of pre-incubation) of C. limon leaf essential oil (LLEO) at the dose of 10 μg/mL (0.5 + LLEO, 1 + LLEO, 2.5 + LLEO). Data were presented as mean ± SD (n = 9). At each time point, significant differences (p < 0.001) were observed for all oxidized samples (in the absence and in the presence of LLEO) versus the respective Ctrl. Evaluation of the statistical significance of differences between groups was performed by one-way ANOVA followed by the Bonferroni Multiple Comparisons Test.