Isolation, Characterization, and Anti-Inflammatory Effects of Carthamus tinctorius L. Leaf-Derived Exosome-like Nanoparticles in ETEC-Challenged IPEC-J2 Cells.

Safflower (Carthamus tinctorius L.) is a cash crop grown worldwide. Its seeds and flowers are primarily processed for edible oil and medicinal raw materials, while stems and leaves are discarded as agricultural waste. Recently, plant-derived exosome-like nanoparticles (PELNs) have gained growing research interest in food nutrition owing to their exceptional biocompatibility and relatively low cost for large-scale production. This study aimed to explore the potential functional value of safflower leaf agricultural waste by isolating safflower-derived PELNs. Firstly, a protocol was established for the isolation and purification of safflower (Carthamus tinctorius L.)-derived exosome-like nanoparticles (Ct-ELNs) from safflower leaves using sucrose density gradient ultracentrifugation. NTA analysis revealed that the 30-45% sucrose fraction enriched with Ct-ELNs exhibited the most uniform particle size, highest particle concentration, and optimal purity. Transmission electron microscopy confirmed typical PELNs ultrastructure: disc- or cup-shaped vesicles surrounded by bilayer lipid membranes. FM4-64 fluorescence suggests time-dependent association and likely cellular uptake of labeled Ct-ELNs by IPEC-J2 cells. Cellular assays demonstrated that Ct-ELNs elevated IPEC-J2 cell metabolic activity under matched protein-normalized dosing conditions. The 30-45% sucrose fraction showed the most favorable physicochemical profile and preliminary in vitro protective effects, including improved IPEC-J2 cell metabolic activity and modulation of Enterotoxigenic Escherichia coli (ETEC)-induced inflammation-related gene expression. Overall, this study established an optimized isolation protocol for Ct-ELNs derived from safflower leaves. These data indicate that safflower-derived Ct-ELNs confer preliminary cytoprotective transcriptional regulatory effects on intestinal epithelial cells under in vitro culture conditions.