This work aimed to extract essential oil from Citrus limon fruits (HEC) and to assess its biological activities, focusing on its in vitro antioxidant properties and its in vivo protective potential in rats subjected to carbon tetrachloride (CCl₄) toxicity.

The chemical analysis of the extracted oil revealed nine major compounds, representing 97.0% of the total composition. The dominant constituents were limonene (34.11%), Z-citral (32.10%), and γ-terpinene (29.90%). Fourier-transform infrared spectroscopy (FTIR) confirmed the presence of phenolic and aromatic structures, highlighting the abundance of limonene, Z-citral, and γ-terpinene. The oil contained approximately 3.6 mg gallic acid equivalents (GAE)/mL of total polyphenols and 1.9 mg catechin equivalents (CE)/mL of flavonoids. Antioxidant assays demonstrated a dose-dependent free radical scavenging effect against DPPH and inhibitory activity against superoxide anions (O₂⁻), though the potency was lower compared to vitamin C. The total antioxidant capacity was estimated at 23.20 mg vitamin C equivalents/mL.

To complement these in vitro findings, an in vivo study was conducted using a rat model of oxidative stress induced by CCl₄. The results showed that CCl₄ administration caused significant oxidative damage and hepatic injury. However, preventive treatment with Citrus limon essential oil (150 mg/kg body weight) markedly reduced these toxic effects, demonstrating its hepatoprotective and antioxidant potential.