Comparative analysis of <i>Citrus sinensis</i> (L) leaf and fruit peel essential oils using solvent-free microwave and hydro-distillation extraction methods, multi-enzymatic inhibitory and antioxidant capacity profiles

Citrus species are known for their therapeutic properties in managing inflammatory conditions. This study presents the first systematic evaluation of the bioactivity of essential oils (EOs) extracted from the Sri Lankan variety of Citrus sinensis L. using two extraction methods: solvent-free microwave extraction (SFME) and hydro-distillation (HD). The leaf and fruit peel EOs were assessed for their anti-inflammatory properties through in vitro arachidonate 5-lipoxygenase (A5-LOX), arachidonate 15-lipoxygenase (A15-LOX), hyaluronidase, and xanthine oxidase (XO) enzyme inhibition assays. The inhibition of nitric oxide (NO) production in RAW 264.7 macrophages, and antioxidant capacity were determined through DPPH, FRAP, ORAC, TPC and TFC. The chemical composition of EOs was analyzed using GC-MS and the major compounds, from leaf EO were β-linalool, trans-β-ocimene, sabinene, and D-limonene from SFME, β-linalool, D-limonene, sabinene, and β-elemene from HD. D-limonene was the dominant compound in C. sinensis fruit peels, with 81.46 ± 0.34% and 60.26 ± 0.48% in SFME and HD techniques. Both leaf and fruit peel EOs showed promising A5-LOX inhibitory activity with IC50 values ranging from 5.84 ± 0.06 μg/mL to 21.65 ± 1.15 μg/mL and good anti-hyaluronidase activity with percentage inhibition between 33.05 ± 3.64% and 76.85 ± 6.72% at concentration of 500 μg/mL. The fruit peel EOs exhibited the lowest IC50 values for NO inhibition in RAW 264.7 macrophages, where standard D-limonene recorded IC50 values of 148.80 ± 3.74 μg/mL. The EOs from the leaves and fruit peels of C. sinensis exhibited significant anti-inflammatory activity, positioning them as selective potential natural candidates for the development of novel anti-inflammatory agents.