Sustainable Extraction of Rosmarinic Acid and Eupatorin from Orthosiphon aristatus Using Nanoparticle-Incorporated Ethyl Lactate-Based Aqueous Two-Phase Systems

Conventional recovery of low-molecular-weight bioactive compounds such as rosmarinic acid (RA) and eupatorin (EUP) typically gives lower yields and relies on toxic organic solvents. Aqueous two-phase systems (ATPS) often suffer from low partition coefficients and yields. Incorporating nanoparticles as additives offers a promising route to enhance separation efficiency. However, their effect on the partitioning of bioactive compounds and the simultaneous extraction of multiple bioactive compounds remains underexplored. This study aims to evaluate the application of nanoparticle-incorporated ethyl lactate (EL)-based ATPS for the extraction of RA and EUP from Orthosiphon aristatus. Various ATPS parameters, including the types and concentrations of salt, the compositions of the EL-salt system, sample loading, pH, and the types and concentrations of added nanoparticles, including zinc oxide, silver, and graphene nanoplatelets, were evaluated. The highest partition coefficient and yield of RA were recorded at 85.54 ± 2.17 and 30.45 ± 0.25 mg/g, respectively, representing an increase of 54.72% in the partition coefficient of RA as compared with the EL–citrate salt system without nanoparticle addition (55.28 ± 0.84). These values were achieved using an ATPS composed of 33.7% (w/w) EL and 16.0% (w/w) citrate salt, a sample loading of 0.1% (w/w), a pH of 7, and the addition of zinc oxide nanoparticles (ZnO-NPs) at a concentration of 0.04% (w/w). FE-SEM and FT-IR analyses indicated that extract-derived material was retained on the hydroxylated ZnO-NP surfaces, while KRA varied with nanoparticle loading and passed through a maximum at 0.04% (w/w), enhancing the preferential enrichment of RA in the EL-rich phase. This study highlights the effectiveness of nanoparticle-incorporated EL-based ATPS for the extraction of RA and EUP from O. aristatus, with potential applications in biopharmaceuticals and enzyme purification.