Development of a reversed-phase high-performance liquid chromatography analytical methodology for the determination of antihypertensive peptides in maize crops.

The aim of this work was to estimate the content of three highly antihypertensive peptides (LQP, LSP, and LRP) in different maize crops. For that purpose, a method consisting of the extraction of the protein containing these peptides (α-zeins), releasing of peptides by thermolysin digestion, and separation and detection of peptides was designed. The rapid and efficient ultrasound assisted extraction of α-zeins proteins from whole maize kernels was achieved using 70% of ethanol followed by precipitation with acetone. A 10 mM Tris-HCl (pH 8.0) buffer containing 8M urea enabled to dissolve the precipitated α-zeins. This buffer was diluted to reach a 6 M urea concentration before digestion to keep active the enzyme. Other digestion parameters that were optimized were: enzyme to substrate ratio (5:100 was selected), digestion temperature (50°C) and digestion time (6 h). The RP-HPLC separation in a fused-core column was also optimized allowing the separation of the three peptides extracted from maize kernels in 6 min. The presence of the three antihypertensive peptides in the digested extract was confirmed using HPLC-Q-TOF-MS analysis and by comparison with peptide standards. Clear differences were observed in the content of the three antihypertensive peptides and, thus, in the antihypertensive activity of the analyzed crops. The content of LRP peptide was very low regardless of the maize variety while the content of LQP and LSP significantly varied among studied maize lines.