Supercritical hybrid carbon dioxide-hydrogen extraction for phenolics and antioxidants from mint using response surface methodology, modelling, and optimization

This study aims to recover phytochemicals from mint by a novel supercritical hybrid carbon dioxide-hydrogen (Sp-CO 2 -H 2 ) extraction method, along with mathematical modelling, statistical methods, and various optimization techniques. In the optimization process, time, pressure, and temperature were the independent variables, while antioxidant activity (DPPH, ABTS), total phenolic content (TPC), total flavonoid content (TFC), and phenolic compounds were the dependent variables (responses). The TPC (52.59 mg GAE/100 mL), TFC (41.93 mg RE/100 mL), t-ferulic acid (2.05 mg/L), rosmaric acid (6.10 mg/L), antioxidant activity (39.99 % for ABTS and 33.20 % for DPPH), gallic acid (19.27 mg/L), catechin (19.27 mg/L), and TPC-by-HPLC (45.01 mg/L) were obtained at optimum Sp-CO 2 -H 2 conditions of 80 bar pressure, 5 min time, and 60 °C temperature. These findings, further corroborated by comprehensive multivariate analyses (PCA, hierarchical clustering, and heatmaps) that showed a high correlation between predicted and experimental data, provided a deeper understanding of the interactions between extraction parameters and the resulting phytochemical profile, decisively confirming the optimization's success. The experimental findings were quite similar to the RSM-predicted results. The multivariate analyses revealed strong positive correlations between total phenolics, flavonoids, and antioxidant activities (r > 0.9), and clearly distinguished two main clusters of bioactives: one dominated by general phenolic–antioxidant associations (TPC, TFC, DPPH, ABTS), and another comprising hydroxycinnamic acid derivatives (t-Ferulic, p-Coumaric, and Caffeic acids). The results revealed both the natural groupings among mint bioactives (phenolics, flavonoids, and antioxidants) and the effects of extraction conditions on the mint's bioactive profile. These results suggest that the Sp-CO 2 -H 2 extraction method, when combined with response surface methodology, mathematical modeling, and optimization techniques, can be used to efficiently extract phytochemicals from mint. • Mint leaves were extracted through the Sp-CO 2 -H 2 method for the first time. • Sp-CO 2 -H 2 was optimized with RSM, mathematical modeling, and optimization techniques. • Optimum extraction conditions of phytochemicals were 80 bar, 60 °C, and 5 min. • Strong positive correlations between TPC, TFC, and antioxidant activities were found. • Sp-CO₂-H₂ showed a high degree of process selectivity and pre-fractionation potential.