Antioxidant activity of semen cassiae protein hydrolysate: thermal and gastrointestinal stability, peptide identification, and in silico analysis

Abstract

Semen cassiae (SC), seeds of Cassia obtusifolia, is used as treatments for various diseases in Traditional Chinese Medicine. This study evaluated the antioxidant activity and stability of SC hydrolysate fractions and identified peptides in the most active fraction. SC proteins were hydrolyzed by alcalase for 2-6 hours; < 3000 u peptide fractions were produced via membrane ultrafiltration. UF 2 h (fraction produced from 2-hour hydrolysate) exhibited the strongest ABTS•+ scavenging (EC50=229 µg/mL) and iron chelating (EC50=89 µg/mL) activities. Its ABTS•+ scavenging activity was retained after thermal treatment and simulated gastrointestinal (GI) digestion, but iron chelating activity was fully retained only after GI digestion. Peptides were purified from UF 2 h by using solid phase extraction, gel filtration chromatography and high performance liquid chromatography analysis, guided by ABTS•+ scavenging assay. Four peptides were identified by mass spectrometric analysis: PMPVR (599.29 u), FETLPF (752.34 u), KMRDNL (775.37 u), and LDESKRF (893.50 u). In silico analysis predicted that before simulated GI digestion, all four peptides were non-toxic and non-allergenic. After GI digestion, peptide fragments released from the four peptides were predicted to be still non-toxic, whereas fragments of only PMPVR and KMRDNL were predicted to be non-allergenic. Taken together, UF 2 h and the peptides contained in it are promising antioxidants for the development of functional food and nutraceuticals.