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Bioactive peptides have received significant attention due to their natural origin, low toxicity, and targeting specificity in the past decade. This study identified highly active ACE/XO inhibitors using molecular simulation and online databases and validated their in vitro antioxidant activity and the mechanisms of molecular interactions. According to computer predictions, Asp-Gly-Gly (DGG) and Asp-Gly-Met (DGGM) were identified as potential hydrolysates of common gastrointestinal peptidases with well water-soluble, non-toxic, and non-allergenic. Fourier transform infrared spectroscopy showed that the two peptides altered the enzyme's secondary structure, decreasing α-helix content by about 13 %, along with increasing β-sheet, randam coli, and β-turns content. Molecular docking and molecular dynamics simulations showed that hydrogen bonding and electrostatic interactions caused DGG and DGGM to form stable and dense complexes with the two enzymes. This study provides a new way for economical and efficient screening of new ACE and XO inhibitory peptides from natural proteins. • New ACE and XO inhibitory peptides in milk were screened and identified. • DGG showed stronger inhibitory activity against ACE than captopril in vitro. • The inhibitory activity of DGGM against XO was stronger than allopurinol in vitro. • Hydrogen bonding and electrostatic interactions enable DGG and DGGM to form stable and dense complexes with the two enzymes. [ABSTRACT FROM AUTHOR]

Highlights • Tuna protein hydrolysate (TPH) obtained by Alcalase was anti-hyperuricemic in rats. • Ethanol-soluble fraction (ESF) of TPH had peptides <1000 Da (13 di-/tri-peptides). • Xanthine oxidase (XO) inhibition: ESF > TPH > ethanol-insoluble fraction. • Phenylalanine (Phe)-di/tri-peptides are XO inhibitors (Phe-histidine is strongest). • 2 H-bonds and 1 π-π stacking with Phe-914 in XO affect XO-peptide inhibitor binding. Abstract This study follows recent attempts to discover natural xanthine oxidase (XO) inhibitors from foods, focusing herein on under-researched fish proteins. The anti-hyperuricemic function of tuna flesh hydrolysate (TPH) produced using Alcalase 2.4L was confirmed in potassium oxonate-induced hyperuricemic rats. TPH was separated using 80 wt% aqueous ethanol. The ethanol-soluble fraction (ESF) abundant in small peptides (<1000 Da) afforded the highest XO inhibition. Separation of ESF by Sephadex G-15 and UPLC/MS/MS revealed 13 di-/tri-peptides (12 are newly identified XO inhibitors). Their XO inhibitory activities were assessed using corresponding synthetic peptides via an improved HPLC method. Results indicate that Phe-containing peptides were more potent XO inhibitors than Trp-containing peptides, with Phe-His having the highest XO inhibitory activity (IC 50 = 25.7 mM). Molecular docking studies revealed the importance of two hydrogen bonds and one π-π stacking interaction with Phe-914 in XO for XO-peptide inhibitor binding. Phe-containing di-/tri-peptides could be potent XO inhibitors against hyperuricemia. [ABSTRACT FROM AUTHOR]

• Eight primary polyphenols were identified and quantified in Tartary buckwheat. • Five xanthine oxidase inhibitors were verified in Tartary buckwheat. • Quercetin made a considerable contribution (77.63%) to the inhibitory effect. • Kaempferol-quercetin complex revealed a sub-additive inhibitory effect. The xanthine oxidase (XO) inhibitory activity is an important way to evaluate the anti-hyperuricemia effect of natural products. In the present work, the XO inhibitory effect of Tartary buckwheat was elucidated by polyphenols determination, omission experiment, interaction assay, inhibition types, fluorescence spectroscopy, and molecular docking. The results revealed that eight primary polyphenols were identified, including rutin (544 mg/100 g) and quercetin (261 mg/100 g). Quercetin (IC 50 = 0.03 mg/mL) was a mixed-type inhibitor and exhibited the strongest inhibitory effect, followed by kaempferol (IC 50 = 0.11 mg/mL). Moreover, a sub-additive effect was exhibited in the complex of quercetin-kaempferol, but the combination of quercetin and other polyphenols caused interference or antagonism effects. Furthermore, quercetin and kaempferol showed an obvious fluorescence quenching effect on XO, and the bindings were mainly driven by hydrophobic forces and hydrogen bonds. This study shows that Tartary buckwheat may be a potential functional food to inhibit XO activity. [ABSTRACT FROM AUTHOR]