Optimization of Ultrasound-Assisted Extraction of (-)-Stepholidine from Onychopetalum amazonicum Leaves Using...

(-)-Stepholidine, a naturally occurring alkaloid found in the leaves of Onychopetalum amazonicum (Annonaceae), has shown significant pharmacological potential and serves as an important precursor in the synthesis of bioactive compounds. Currently, ultrasound-assisted extraction (UAE), combined with optimization through response surface methodology (RSM), has emerged as a promising strategy to enhance the efficiency of extraction processes. This study aimed to develop an efficient method for extracting (-)-stepholidine from the leaves of O. amazonicum. Powdered leaves were extracted using methanol-based systems assisted by ultrasound, following a central composite rotatable design (CCRD) with three factors: plant-to-solvent ratio (X₁), methanol concentration (X₂), and extraction time (X₃). The samples were quantified by NMR using the PULCON method. The model was validated by ANOVA, and the optimal conditions were determined using RSM. The factors X₂ ² (methanol percentage), X₃ (extraction time), and the interaction X₁·X₃ (plant-to-solvent ratio × extraction time) significantly influenced (-)-stepholidine concentration (p < 0.05). The model showed a good fit (R² = 0.728) and was statistically significant (p = 0.0005). The optimal conditions identified were as follows: a plant-to-solvent ratio of 1:10, 100% methanol, and 20 min of extraction. Experimental validation yielded an average (-)-stepholidine concentration of 82.8 ± 1.3 mg per g of extract, close to the predicted value of 81.2 mg/g. This study demonstrates that optimizing extraction parameters, such as the plant-to-solvent ratio, methanol percentage, and extraction time, is crucial for maximizing (-)-stepholidine recovery. These findings also support the potential of UAE combined with qNMR as a reliable and reproducible approach for the extraction and quantification of bioactive compounds from plant matrices.