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Honey is known for its medicinal benefits and receiving renewed attention as natural medicine. Studies on health benefits of honey attributed its antioxidant activity to phenolic compounds, but the contribution of proteins and peptides to the antioxidant activity of honey is lacking. The aim of this study was to explore the contribution of proteins and peptides to the antioxidant activity of honey, which remained obscure for decades. Total honey proteins (THP) were isolated by dialysis method and hydrolyzed through simulated gastrointestinal digestion. The hydrolysates were fractionated using size-exclusion chromatography. The antioxidant activity was determined by using superoxide radical-scavenging, DPPH reduction and intracellular ROS assays. THP was shown to exhibit superoxide-scavenging activity but its pepsin-hydrolysate (HP-p) showed superior scavenging activity. The HP-p produced five peptide fractions (P1~P5) when fractionated on Sephacryl S-100 size-exclusion column. The five fractions showed superoxide-scavenging activities and DPPH reducing activities, whereas the slow-eluting peptide fractions (P3 and P4) were the most potent. MALDI-TOF/MS analysis identified a pentapeptide (TSNTF) as the dominant peptide in the active fractions P3 and P4. Human colonic epithelial cells treated with P3 and P4 peptides exhibited lower intracellular ROS, when oxidative stress was induced by H 2 O 2 or diethyl maleate (DEM), indicating strong tolerance to oxidative stress. The viabilities of human cells or yeast cells were largely decreases under oxidative stress, but treated cells with P3 and P4 showed higher viability compared with the untreated cells. The results are the first to describe a novel antioxidant peptide from honey that confer ex vivo anti-oxidative function within a complicated milieu of eukaryotic cells and pave the way for its potential as nutraceutical or therapeutic peptide for risk-reduction of oxidative-stress and related diseases. Graphical abstract

Potent antioxidant peptides derived from honey major protein enhance tolerance of eukaryotic cells toward oxidative stress