Optimized Mixture of Essential Oils From <i>Thymus capitatus</i> , <i>Rosmarinus officinalis</i> , and <i>Aloysia citrodora</i> : Synergistic Antidiabetic and Antioxidant Activities With Reduced Cytotoxicity

This study employed a simplex-centroid mixture design to formulate and optimize a blend of essential oils from Thymus capitatus, Rosmarinus officinalis, and Aloysia citrodora for enhanced bioactivity and reduced cytotoxicity. Essential oils were hydrodistilled from Tunisian plant material and chemically characterized via GC-MS, revealing carvacrol, 1,8-cineole, and D-limonene as the dominant compounds, respectively. The antidiabetic (α-amylase inhibition) and antioxidant activities of individual oils and their mixtures were evaluated. Polynomial models (R² > 0.96, p < 0.001) revealed significant synergistic interactions, particularly between T. capitatus and R. officinalis. Desirability optimization yielded an optimal mixture of 52% T. capitatus, 8% R. officinalis, and 40% A. citrodora, which exhibited potent activity (67.81% α-amylase inhibition, 76.99% DPPH scavenging) comparable to or better than pure T. capitatus. Crucially, this optimized blend showed significantly reduced cytotoxicity on Vero cells (IC₅₀ = 105.36 µg/mL) compared to T. capitatus alone (IC₅₀ = 82.46 µg/mL) and was over 20-fold safer than doxorubicin (IC₅₀ = 5.11 µg/mL). The results demonstrate the efficacy of mixture design in balancing potency and safety, proposing the optimized essential oil blend as a promising low-toxicity candidate for the adjuvant management of diabetes and oxidative stress, warranting further in vivo validation.