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Rising consumer concerns with synthetic drugs to treat non-communicable diseases (NCDs) have promoted a shift towards using natural biological active constituents that offer similar health benefits. Hairless canary seed (Phalaris canariensis L) is an emerging crop traditionally used in Mexico to treat NCDs. Peptides liberated during simulated digestion of canary seed protein are believed to be responsible for their biological activity; however, no studies have shown the effect of controlled protein hydrolysis using commercial proteases on canary seed protein’s biological activity. Therefore, this study aimed to explore the in vitro antihypertensive, antidiabetic, and anti-obesity activity of canary seed peptides derived from proteolysis with Alcalase®. Protein fractions were primarily composed of prolamins (54.07 ± 1.8%), glutelins (32.19 ± 3.18%), globulins (5.97 ± 0.52%) and albumins (5.97 ± 0.52%). The < 3 kDa and 3–10 kDa peptide fractions showed the highest inhibition capacity (p < 0.05) towards angiotensin-converting enzyme (IC50= 0.028–0.032 mg/mL) lipase (IC50= 2.15–2.27 mg/mL), α-glucosidase (IC50= 0.82–1.15 mg/mL), and dipeptidyl-peptidase-IV (IC50= 1.27–1.60 mg/mL). Additionally, these peptide fractions showed high antioxidant activity against DPPH (134.22–150.66 μmol TE/mg) and ABTS (520.92–813.33 μmol TE/mg). These results provide an insight into the potential development of functional foods using commercial enzymatic hydrolysis of canary seed proteins for treating hypertension, type-2 diabetes, and obesity.

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