Obesity is a worldwide problem, and lowering pancreatic lipase (PL) activity is an effective strategy to counteract it. In this study, pancreatic lipase-inhibitory (PL-I) peptides were isolated and purified from Chlorella pyrenoidosa protein hydrolysates (CPPHs) using ultrafiltration and Sephadex gel chromatography (Sephadex G-25). A total of 858 peptides were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Four novel PL-I peptides (FLSQPF, VWTPI, IVGPF, and IPYPL) were virtually screened using molecular docking and subsequently synthesized, with VWTPI exhibiting the highest PL inhibition. Moreover, the inhibition of the enzyme by VWTPI was a mixture of competitive and non-competitive inhibition, with an inhibition constant (Ki) of 7.27 mg/mL. Molecular docking showed that VWTPI interacts with the PL active center by hydrogen bonding, hydrophobic contacts, van der Waals forces, and π-π stacking. This study suggests that peptides from Chlorella pyrenoidosa could be used as lipid-lowering agents to prevent and cure obesity.

Plant-derived bioactive peptides have garnered widespread interest for their functions in managing obesity and associated metabolic disorders. This study investigated the lipid-lowering activity and underlying mechanisms of VPIIMH, a hexapeptide derived from Chlorella pyrenoids, using in vitro enzymatic assays, Caenorhabditis elegans models, and network pharmacology. In vitro, VPIIMH acted as a reversible non-competitive inhibitor of pancreatic lipase, achieving an inhibition rate of 43.17 ± 1.47% at 8.0 mg/mL. Molecular docking revealed that this inhibition likely occurs through ionic bonds between VPIIMH and PL (1LPB) at Arg256. In a high-fat C. elegans model, treatment with 0.5 mg/mL VPIIMH significantly reduced fat accumulation by 37.2% and triglyceride levels by 26.9%. Furthermore, VPIIMH extended the lifespan of C. elegans under oxidant stress by 40.3% and under heat stress by 17.5%. Network pharmacology predicted that VPIIMH targets nine core proteins, which were classified into three synergistic modules: the SIRT1-PPAR for core regulation, the RAS for systemic coordination, and the inflammatory target (CCR5, MMP9, EGFR) for microenvironment support. This study elucidates the multi-target and multi-pathway mechanism of VPIIMH, suggesting its potential application in combating obesity and related lipid metabolism disorders. These findings provide a scientific basis for the development of VPIIMH as a functional food ingredient targeting metabolic health.