Supercritical CO2 extraction of Phoradendron leucarpum (American mistletoe): optimizing oleanolic acid and polyphenol yields, monitoring ROS-specific neutralization

Phoradendron leucarpum (Ph. leucarpum) is a hemiparasitic plant native to Mexico that has been used in folk medicine for years to treat cancer, diabetes, and cardiovascular disease in different extraction forms. In this study, the effect of different parameters, including temperature, pressure, and co-solvent, on extract yield obtained by the supercritical CO 2 technique is studied, using 15 conditions defined through a Box-Behnken design. The level of the total polyphenols was studied using the Folin-Ciocalteu assay. High-performance liquid chromatography was performed to identify and quantify oleanolic acid (OA) yielded per condition. Ultimately, a novel technique is employed to evaluate the profile of each extract against physiologically relevant ROS, including singlet oxygen ( 1 O 2 ), hydroxyl radical ( OH • ), hydrogen peroxide ( H 2 O 2 ), and superoxide anion ( O 2 • − ). Our results reveal optimal extraction conditions for enhanced OA yield and synergistic antioxidant responses of an extract with respect to a standard molecule. The response surface methodology analysis also demonstrated the significant effects of pressure, temperature, and co-solvent on polyphenol levels. Additionally, this work highlights an important disparity with traditional methods (e.g., ABTS and DPPH assays) for antioxidant capacity determination and raises important questions about the typical examination of ROS neutralization in the search for novel antioxidant compounds.