Synergistic inhibition of pancreatic lipase by olive phenolics isolated from olive oil by-products: hydroxytyrosol, 3,4-dihydroxyphenylglycol, and comselogoside

Obesity remains a major global health challenge, and pancreatic lipase (PL) inhibition represents a key strategy to reduce dietary fat absorption. This study evaluated the individual and combined inhibitory effects of three important olive phenolic compounds—hydroxytyrosol (HT), 3,4-dihydroxyphenylglycol (DHPG), and comselogoside (COM)—on PL activity in vitro. All three compounds showed concentration-dependent inhibition, with HT being the most potent (IC₅₀ = 0.294 mM), followed by DHPG (IC₅₀ = 0.464 mM) and COM (IC₅₀ = 0.858 mM). Kinetic analyses revealed competitive inhibition for HT and non-competitive inhibition for DHPG.Binary mixtures of HT:DHPG and HT:COM were tested at seven molar ratios (10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 1:10). Both combinations showed clear dose-dependent inhibition and marked synergistic effects across most molar ratios and inhibition levels (50–97%), as demonstrated by combination index (CI < 1) and isobologram analysis. The strongest synergism occurred at the 1:1 ratio, with CI values between 0.09 and 0.30 and high dose-reduction index (DRI) values (5–83-fold), indicating enhanced inhibitory potency at lower concentrations. Kinetic analyses confirmed that the HT:DHPG mixture followed a non-competitive pattern, whereas HT:COM maintained a competitive mechanism.These findings demonstrate that olive phenolics can act cooperatively to enhance PL inhibition, highlighting their potential as natural and sustainable agents for the development of functional foods and nutraceuticals aimed at lipid digestion and obesity management.