Valorization of flaxseed Cyclolinopeptides from byproduct: Optimization of extraction and evaluation of cellular protective effects

Cyclolinopeptides (CLs), a class of cyclic peptides derived from flaxseed (<i>Linum usitatissimum L</i>.), exhibit significant bioactivities. However, over 98% of CLs are discarded during flaxseed oil refining. This study hypothesized that an optimized methanol-K₂HPO₄ aqueous two-phase extraction (ATPE) system could efficiently recover CLs from flaxseed meal, and that these peptides might exhibit significant cellular protective effects against oxidative stress, underscoring their valorization potential. The ATPE conditions were optimized (14% K₂HPO₄, 44% methanol, feed-to-liquid ratio 1,30 <i>w</i>/w, pH 8.7), achieving a recovery rate of approximately 79%. Subsequently, the anti-senescence potential of CLs was systematically evaluated using an H₂O₂-induced senescence model in human dermal fibroblasts (HSFs). Results demonstrated that both the CL mixture (CLMIX, 120 μg/mL) and its predominant monomer CLA (20 μg/mL) significantly suppressed senescence-associated β-galactosidase (SA-β-gal) activity, inhibited excessive reactive oxygen species (ROS) generation, and attenuated apoptosis by upregulating BCL2 (B-cell lymphoma 2) and downregulating BAX (BCL2-associated X protein) expression. Furthermore, CLMIX and CLA mitigated collagen degradation via downregulation of MMP-1/MMP-3. Mechanistically, CLA counteracted oxidative stress-driven apoptosis by modulating the BCL2/BAX axis. These findings suggest that CLs, particularly CLA, could serve as functional food-grade ingredients with antioxidant and anti-senescence properties, enabling valorization of flaxseed processing waste.