Comprehensive Analysis of Bioactive Peptides From Spleen Amaranth <i>(Amaranthus dubius)</i> and Multi‐Functional Bioactivity Evaluation

Amaranthus dubius, commonly known as spleen amaranth, is a valuable nutritional source rich in protein, vitamins, and minerals, especially in regions such as India. This study investigated the protein content of leaves, stems, and seeds, with the seeds showing the highest protein concentration. Protein isolates from seeds were enzymatically hydrolyzed using proteolytic enzymes. The resulting hydrolysates were characterized by FTIR, DSC, and SEM techniques to analyze their structural properties. The peptides were further purified using molecular weight cut-off (MWCO) filters and assessed for various bioactivities. Seed protein isolates demonstrated significant antioxidant properties, including DPPH (56.65 ± 0.027 µg AAE/mg), ABTS (31.37 ± 0.955 µg AAE/mg), FRAP (23.986 ± 0.031 µM/mg), and Fe²⁺ chelation (32.03 ± 0.020%). Additionally, membrane stabilization activity was highest in seed isolates (25.56 ± 0.045%). Among the hydrolysates, chymotrypsin-derived fractions larger than 50 kDa showed the strongest DPPH activity, whereas alcalase hydrolysates in the 10-50 kDa range exhibited the highest ABTS scavenging activity. The highest ferric reducing power was observed in alcalase >50 kDa fractions, and Fe²⁺ chelating activity was greatest in <3 kDa chymotrypsin hydrolysates. The most potent anti-inflammatory effect was observed in <3 kDa alcalase hydrolysates, whereas antithrombotic activity peaked in 10-50 kDa chymotrypsin fractions. Antibacterial activity was notable against E. coli in <3 kDa alcalase fractions and S. aureus in 10-50 kDa chymotrypsin fractions. These multi-functional bioactivities highlight the potential of Amaranthus dubius seed proteins and peptides for applications in food, nutraceutical, and pharmaceutical industries. This study underscores the promising health benefits of A. dubius proteins and encourages further research into their therapeutic uses.