Extraction of protein with protease inhibitor activity from Brazilwood (Caesalpinia echinata LAM.) seeds using choline-based ionic liquids

Abstract

Proteolytic inhibitors are low molecular weight proteins that prevent protein hydrolysis and are used against moderate to high severity diseases. Some plants can produce these protease inhibitors, such as Brazilwood seeds (Caesalpinia echinata LAM.). This work aimed to optimise the extraction of proteins from Brazilwood seeds with proteolytic inhibitory activity, using choline-based ionic liquid. The seeds were characterised in terms of their dimensions (15 mm in length, 11 mm in width and 4.8 mm in thickness), centesimal composition (moisture - 9.21%, ash - 3.37%, lipids - 32.6%, total fibre - 0.80%, crude proteins - 17.2%, carbohydrates - 36.8%), energy (510 Kcal.100 g-1) and major fatty acid (linoleic acid - 43%). Protein extraction was performed by maceration of the Brazilwood seeds using ionic liquids (choline bitartrate, choline chloride and choline dihydrogen citrate) and optimised using a factorial design of experiments, whose variables were the temperature and solvent concentration, with the total protein content as the response variable. The best extraction conditions were 25.42 °C, 5.42% (m/v) choline bitartrate, solid-liquid ratio 1:5, 500 rpm and pH 7.0 (1.74 mg mL−1 theoretical and 1.88 mg mL−1 experimental). The kinetic model best fitted to the experimental data was Peleg's, and the best extraction time was 15 min. Under optimised conditions, the protein extract of Brazilwood seeds provided a 60.8% inhibition of trypsin. According to the molecular docking results, the inhibition of bovine trypsin is driven by Caesalpinia echinata kallikrein inhibitor (CeKI) and the selected ionic liquid (choline bitartrate), promoting a synergistic inhibition effect.