**Background:** Oregano (Origanum vulgare ssp. hirtum) is an aromatic herb rich in phenolic compounds, including flavonoids such as apigenin, luteolin, and kaempferol, which are associated with antioxidant, anti-inflammatory, and anticancer properties. The study focuses on oregano cultivated on the Greek island of Lemnos, aiming to optimize ultrasound-assisted extraction (UAE) of total phenolic compounds and antioxidant activity using response surface methodology (RSM), and to identify specific phenolic compounds in the optimized extracts.
**Methods:** A Box–Behnken design (BBD) with three factors—extraction temperature (40, 60, 80 °C), time (20, 30, 40 min), and ethanol concentration (60%, 70%, 80% v/v)—was employed, resulting in 15 experimental runs including three center points. UAE was performed using a 750 W ultrasonic processor with 60% amplitude and 1:1 pulse. Total phenolic content (TPC) was measured by the Folin–Ciocalteu method, and antioxidant activity was evaluated by DPPH, ABTS, FRAP, and CUPRAC assays. Identification and quantification of apigenin, luteolin, and kaempferol were performed using HPLC-PDA and UHPLC-QTOF-MS. Model fitting and optimization were conducted using Minitab® statistical software, with ANOVA used to assess significance (p < 0.05).
**Key Results:** The experimental TPC values ranged from 25.0 to 250.0 mg GAE/g dry oregano, and DPPH values ranged from 22.2 to 95.6 mg TE/g dry oregano. After data transformation and model reduction, ANOVA showed the refined models were significant for both TPC (F-value = 80.68, p = 0.000) and DPPH (F-value = 17.30, p = 0.001). The linear terms temperature, time, and ethanol concentration were all significant (p < 0.05). Quadratic terms for time, temperature, and ethanol concentration were also significant. The interaction of time and ethanol concentration (p = 0.009) and temperature and ethanol concentration (p = 0.011) were significant for TPC. The R² values were 0.9772 for TPC and 0.9390 for DPPH, with adjusted R² of 0.9544 and 0.8780, respectively. Lack-of-fit was not significant for either model (p = 0.973 for TPC; p = 0.953 for DPPH). The optimal extraction conditions were determined as 80 °C, 40 min, and 60% ethanol (v/v). At these conditions, the predicted values were 363.0 mg GAE/g for TPC and 108.5 mg TE/g for DPPH, while experimental values were 362.1 ± 1.8 mg GAE/g and 108.6 ± 0.9 mg TE/g, with no significant difference (p > 0.05) and desirability of 1.000. Additional antioxidant assays on the optimized extract yielded: ABTS 115.2 ± 1.2 mg TE/g, FRAP 13.7 ± 0.8 mg TE/g, and CUPRAC 1.2 ± 0.2 mg TE/g. HPLC-PDA quantification of specific phenolics in the optimized extract gave: luteolin 1.30 ± 0.05 mg/g, apigenin 1.43 ± 0.06 mg/g, and kaempferol 0.40 ± 0.03 mg/g dry oregano. UHPLC-QTOF-MS confirmed the identity of these compounds via precursor ions (apigenin m/z 269, kaempferol m/z 285, luteolin m/z 285) and fragmentation patterns.
**Clinical Implications:** This study demonstrates that UAE with green solvents (ethanol/water) can efficiently extract high levels of phenolic compounds from oregano, yielding extracts with substantial antioxidant capacity. The quantified presence of bioactive flavonoids (apigenin, luteolin, kaempferol) supports the potential use of these extracts for food enrichment and development of functional foods aimed at improving consumer health. The green extraction approach enables direct application in food products without safety concerns associated with synthetic antioxidants (e.g., BHA, BHT). However, further research on bioavailability, metabolism, and stability (e.g., encapsulation) is needed before commercial implementation.