Anti-inflammatory, antibacterial activity and chemical composition of essential oil extracted from Citrus aurantium L. leaves using solvent-free microwave extraction technique

Abstract

Citrus aurantium is known for its therapeutic properties in managing inflammation associated with disease conditions. Its essential oil (EO) is valued for antiseptic, antimicrobial, astringent properties and is used in aromatherapy, to alleviate anxiety and pain. This study presents the first systematic evaluation of the chemical composition, anti-inflammatory and antibacterial activity of EO extracted from the leaves of C. aurantium, using solvent-free microwave extraction (SFME) technique. Fresh leaves (200 g) were subjected to SFME using the NEOS GR microwave extractor to obtain the EO. Gas Chromatography-Mass Spectrometry (GC-MS) was used to analyze the chemical composition. Anti inflammatory activity was evaluated by the inhibitions of enzymes arachidonate 5-lipoxygenase (A5 LOX), arachidonate 15-lipoxygenase (A15-LOX) activities, and inhibition of nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages. Antibacterial activity of EO was evaluated by agar well diffusion method against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella typhimurium, and Candida albicans. GC-MS analysis identified D limonene (25.35%) and α-Citral (17.00%) as major compounds in C. aurantium EO. Dose-dependent inhibitions of A5-LOX and A15-LOX activities were recorded with IC50 values of 6.97±0.85 and 411.24±2.23 μg/mL respectively (r=0.995 & 0.979; p<0.05) which were significantly higher compared to the reference standard baicalein (IC50: 2.09±0.19 μg/mL; p<0.05). The C. aurantium EO dose dependently inhibited NO production (r=0.987; p<0.05), with 74.44±0.58% inhibition at a non-toxic concentration of 1000 μg/mL in LPS-stimulated RAW 264.7 cells, with IC50 value of 391.06±31.43 μg/mL. The EO exhibited moderate antimicrobial activity with an inhibition zone of 10 mm for both E. coli, and B. subtilis at 1000 μg/mL compared to the standard antibiotic, ciprofloxacin (5 mcg/disc). These findings on significant inhibition of A5-LOX, and NO production may support the therapeutic potential of C. aurantium leaf EO and emphasize the importance of sustainable and efficient extraction methods for its industrial applications in pharmaceuticals and cosmetics.