Procyanidin oligomers and polyphenol oxidase: Methods for extraction optimization, enzymatic inhibition, and application as a natural inhibitor

The extraction of procyanidins from a food is influenced by food matrix, extraction solvent, sample particle size, sample to solvent ratio, as well as other factors. Many of these parameters can be controlled in a laboratory to improve or target the extraction of low or high molecular weight oligomers. This study first examines the influence of solvent polarity and composition, solvent-solute ratio and particle size on the extraction of procyanidin oligomers monomer through decamer. The solvents evaluated included dimethyl sulfoxide (DMSO), acetone and methanol. Reversed-phase high-performance liquid chromatography (HPLC) and a diol-stationary phase were used to qualitatively compare extraction efficiencies of the procyanidin oligomers. Results indicate that using 70% acetone, solute-solvent ratio 3g-15 mL, and particle size <0.99 mm optimized procyanidin extraction from cocoa beans, yielding 55.2mg/g procyanidins per gram defatted cocoa. These optimized extraction methods were used with cocoa hulls but resulted in roughly 5% of the amount of procyanidin oligomers extracted from the cocoa bean; when the solvent volume was doubled, 8.5% was recovered. However, the profile of oligomers extracted changed, as the hull provided a more uniform distribution of higher oligomers extracted than the cocoa bean. Oligomeric procyanidin extracts from cocoa pods and cocoa hulls were studied for their ability to inhibit polyphenol oxidase. Pentameric procyanidins from cocoa beans showed mixed, non-competitive inhibition with a KI of ∼0.1-0.3 mM. Cocoa hull extracts showed inhibition in a controlled assay, but did not show significant inhibition when applied to a food model. Upon re-extraction of the procyanidins from the food-model, it was discovered that the oligomers were no longer detectable by HPLC chromatography. While procyanidin oligomers can act as polydentate ligands and bind the enzyme at several sites on the protein surface, interactions between the oligomers and the food-matrix lead to loss of oligomeric structure, as well as loss of inhibition function.