OPTIMIZATION OF SOLVENT COMPOSITION AND EXTRACTION CYCLES FOR PHENOLIC COMPOUNDS FROM PAJURO

Pajuro (Erythrina edulis) is an Andean legume with potential as a source of phenolic compounds for functional food ingredients. Efficient characterization requires an extraction procedure that accounts for the different polarities of phenolic compounds and establishes when further extraction provides little additional recovery. This study optimized the solvent composition for extracting phenolic compounds from pajuro flour and determined the number of consecutive extraction cycles needed to recover at least 95% of the total response obtained after eight cycles. A four-component simplex-centroid mixture design evaluated water, methanol, ethanol, and acetone as independent variables. The matrix included four pure solvents, six binary mixtures, four ternary mixtures, four formulations enriched in one component, and four replicates of the mixture containing 25% of each solvent, totaling 22 trials. A sample-to-solvent ratio equivalent to 100 mg of sample per 1000 µL was used. Absorbance at 750 nm (Folin-Ciocalteu method) was the response variable and served as an indicator of extraction performance, rather than a direct measure of phenolic concentration. Across the experimental mixtures, mean absorbance ranged from 0.151 ± 0.008 to 0.548 ± 0.036. The lowest response was obtained with equal proportions of water and ethanol, whereas the highest was achieved with equal proportions of water, methanol, and acetone. Pure ethanol yielded an absorbance of 0.175 ± 0.009, compared with 0.286 ± 0.025 for water, 0.330 ± 0.029 for methanol, and 0.337 ± 0.012 for acetone. The water–methanol, methanol–ethanol, and methanol–acetone binary mixtures produced higher responses than their respective pure components, indicating that solvent combinations can improve the recovery of compounds contributing to the analytical signal (P < 0.05; R² > 0.85). Optimization yielded a formulation containing 23.9% water, 29.7% methanol, 1.12% ethanol, and 45.2% acetone with a desirability of 1.0. Its experimentally observed absorbance was 0.568 ± 0.036 at 750 nm. Exhaustive extraction was then evaluated over eight consecutive cycles in triplicate. The sixth cycle increased cumulative recovery to 95.44%, providing a practical stopping point for determining phenolic compounds in pajuro, achieving satisfactory recovery while limiting solvent use and processing time. Overall, the mixture design identified a solvent composition that enhanced the analytical response for pajuro extracts, while the exhaustive extraction study established enough cycles. Together, these findings provide a defined procedure for subsequent quantification and comparison of phenolic compounds in pajuro flour.

Funding: This work was funded by CONCYTEC through the PROCIENCIA program, within the framework of the Financial Scheme E041-2025-02 titled “Proyectos de Investigación Aplicada” (Grant PE501096410-2025). Thanks to CNPq for the research scholarship provided to M.S. (312759/2025-8).