In vivo anti-hyperuricemic and xanthine oxidase inhibitory properties of tuna protein hydrolysates and its isolated fractions

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 (IC50 = 25.7 mM). Molecular docking studies revealed the importance of two hydrogen bonds and one pi-pi stacking interaction with Phe-914 in XO for XO-peptide inhibitor binding. Phe-containing di-/tripeptides could be potent XO inhibitors against hyperuricemia.