Abstract
To provide further insight into the antioxidant potential of procyanidins (PCs) from cocoa beans, PC extract was fractionated by several methodologies, including solid phase extraction, Sephadex LH-20 gel permeation, and preparative HPLC using C18 and diol stationary phases. All the isolated fractions were analyzed by UHPLC-QTOF-MS to determine their relative composition. According to our results, classical techniques allowed good separation of alkaloids, catechins, dimers, and trimers, but were inefficient for oligomeric PCs. Preparative C18-HPLC method allowed the attainment of high relative composition of fractions enriched with alkaloids, catechins, and PCs with degree of polymerization (DP) < 4. However, the best results were obtained by preparative diol-HPLC, providing a separation according to the increasing DP. According to the mass spectrometry fragmentation pattern, the nine isolated fractions (Fractions II-X) consisted of exclusively individual PCs and their corresponding isomers (same DP). In summary, an efficient, robust, and fast method using a preparative diol column for the isolation of PCs is proposed. Regarding DPPH• and ABTS•+ scavenging activity, it increases according to the DP; therefore, the highest activity was for cocoa extract > PCs > monomers. Thereby, cocoa procyanidins might be of interest to be used as alternative antioxidants.