Flaxseed protein hydrolysate as a potential anti-obesity agent: Combined animal study and in silico investigation

This study investigated the metabolic benefits of flaxseed protein hydrolysate (FPIH) in comparison with its parent protein, flaxseed protein isolate (FPI), and explored the underlying mechanisms using an integrated in vivo animal model and in silico approach. FPIH was produced via alcalase hydrolysis of FPI (final DH ∼ 21%), and both treatments were administered at 300 mg/kg body weight (BW) to Wistar rats fed a high-fat diet (HFD) for 10 weeks. FPIH showed an antioxidant potential higher than that of FPI and an inhibitory effect against pancreatic lipase in vitro (IC₅₀ = 1.2 mg/mL). These findings aligned with the in vivo efficacy, marked by the effect of FPIH on decreasing BW gain, blood glucose level and restoring liver, lipid and oxidative stress markers to normal levels. To better understand these effects, the active fraction of FPIH from ultrafiltration (<3 kDa) was fractionated by RP-HPLC and analyzed via LC-MS/MS, identifying 15 novel peptides with high spectral probabilities. Molecular docking studies revealed that these peptides exhibited high-affinity interactions with key metabolic enzymes. Specifically, LVNLGGSKSIS and GGGGFGGGGIGGGGF showed strong binding energies with the active sites of dipeptidyl peptidase-IV (DPP-IV) and α-amylase, respectively, with binding energies of -9.874, -9.430 kcal/mol. In addition, RDNGQLVR effectively interacted with pancreatic lipase with -8.213 kcal/mol. These findings provided a robust molecular explanation for the observed anti-obesity effects, supporting the potential application of flaxseed-derived peptides as promising candidate for the management of metabolic syndrome and offering a natural alternative complementing synthetic drugs in the market.