Delivery of polyphenol-rich plant extracts using lactose-free whey permeate: cytotoxic and mitochondrial effects in Caco-2 cells

The development of functional food matrices for the delivery of bioactive compounds, together with the valorization of agro-industrial by-products, is a growing research area. Whey permeate (WP), an abundant dairy by-product suitable for human consumption, has attracted interest as a potential food-compatible matrix, although its use for incorporating bioactive compounds remains limited. This study investigated the biological activity of polyphenol-rich plant extracts incorporated into a lactose-free WP matrix on human colorectal adenocarcinoma Caco-2 cells. Extracts from peanut skin (Arachis hypogaea L.), Marcela del campo (Achyrocline satureioides (Lam.) D.C.), and yerba mate (Ilex paraguariensis A. St. Hil) were evaluated. Cytotoxicity (CC₅₀) was determined in Caco-2 and non-tumor Vero cells by Neutral Red uptake and Resazurin reduction assays. WP-based formulations exhibited cytotoxic activity against tumor cells, with CC₅₀ values ranging from 15.9 to 28.5 mg/mL of dairy carbohydrates and from 124 to 223 μg/mL of plant extracts, while preserving viability in non-tumor cells. Mechanistic analyses showed increased superoxide anion generation (121.88-139.82% of cell control), loss of mitochondrial membrane potential (53.9-74.7% of cell control), and ultrastructural mitochondrial damage, with the strongest effects observed with WP and peanut skin extract. These findings suggest that lactose-free WP model can serve as a potential food-compatible matrix for polyphenol-rich extracts, enabling cytotoxic effects on tumor cells, possibly with mitochondrial involvement. This approach provides a basis for further investigation of WP systems and support the valorization of dairy and peanut by-products for future food applications.