This study explores the chemical composition and anti-inflammatory potential of the essential oil extracted from the aerial parts of Satureja calamintha, a medicinal and aromatic plant belonging to the Lamiaceae family. The essential oil was analyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS), which led to the identification of twenty compounds, representing 96.7% of the total oil. The composition was dominated by oxygenated monoterpenes (74%) and, to a lesser extent, by hydrocarbon monoterpenes (7.4%) and sesquiterpenes (9.52%). Pulegone was identified as the major constituent (50%), followed by iso-menthone, β-caryophyllene, menthol, and cubebol.

To better understand the contribution of pulegone to the biological activity of the oil, a pulegone-rich fraction (F2) was obtained through column chromatography and subsequently characterized by GC-MS. This fraction contained pulegone as the predominant compound (80%), along with E-β-caryophyllene and pipertenone oxide. Anti-inflammatory activity was assessed using the heat-induced albumin denaturation method. The enriched fraction exhibited a significantly higher inhibitory effect (IC₅₀ = 0.128 mg/mL) compared to the crude oil (IC₅₀ = 0.86 mg/mL), suggesting antagonistic interactions in the unfractionated oil. Furthermore, combination studies with diclofenac sodium revealed a pronounced synergistic effect. Notably, the pulegone-rich fraction combined with diclofenac displayed enhanced activity (IC₅₀ = 0.085 mg/mL), more than twice as effective as diclofenac alone (IC₅₀ = 0.194 mg/mL). Additionally, hemolysis assays conducted on human red blood cells indicated a low cytotoxic potential for both the crude essential oil and the F2 fraction, supporting their potential as safe and effective natural anti-inflammatory agents.