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.

A descriptive review on exploiting the therapeutic significance of essential oils as a potential nutraceutical and food preservative

“…Limonene is a type of monoterpene that is commonly present in citrus fruits, particularly in oranges and lemons. It is found in high concentrations within the EOs of these fruits (Petretto et al, 2023). De Sousa, Soares Hocayen, et al (2015) conducted a study, where they examined how limonene affected guinea pig tracheas and rat aortas.…”

Protective Effects of Lemon Leaf and Limonene against Indomethacin-Induced Gastric Ulcers in Rats: An In Vivo and In Silico Study

“…These properties are considered to be due to various kinds of essential oils it contains. Because these essential oils can neutralize ROS and preventing them from causing damage to cells and tissues, and play a crucial role in protecting against oxidative stressinduced damage by reducing lipid peroxidation and enhancing cellular antioxidant defenses (Petretto et al, 2023). Recently, the essential oils obtained from Citrus L. have been popular due to their antioxidant properties and their ability to modulate oxidative stress-related pathways in the body.…”

Arzanol, a natural phloroglucinol α‐pyrone, protects HaCaT keratinocytes against H2O2‐induced oxidative stress, counteracting cytotoxicity, reactive oxygen species generation, apoptosis, and mitochondrial depolarization

“…The cytotoxicity of arzanol, DMSO, and H 2 O 2 was performed by MTT assay as previously described (Kasugai et al, 1990;Petretto et al, 2023)…”

“…which reacts with intracellular reactive species and fluoresces (Pavelescu, 2015;Petretto et al, 2023). HaCaT cells were seeded in 96-well plates at 10 5 cells/mL density in 100 μL/well of complete culture medium and cultured for 48 h. Then, fresh medium without (control cells) and with 50 μM arzanol was added, and cells were incubated for a further 24 h. Next, the cells, after washing with phosphate-buffered saline (PBS) solution (pH 7.4), were incubated with 10 μM H 2 DCFDA in PBS, at 37 C for 30 min.…”

“…The cytotoxic potential of arzanol was evaluated by using the colorimetric MTT assay, a method frequently used as an indicator of cell viability, proliferation, and cytotoxicity (Petretto et al, 2023). Figure 2 shows the viability, expressed as percent of the control, induced by incubation for 24 h with different amounts of arzanol (5-100 μM)…”