PHENOLIC PROFILE, BIOACCESSIBILITY, AND ANTICANCER POTENTIAL OF BRAZILIAN OLIVE LEAF INFUSIONS (OLEA EUROPAEA L.) AFTER SIMULATED GASTROINTESTINAL DIGESTION

Olive leaves are an abundant agro-industrial by-product and an important source of phenolic compounds with recognized antioxidant and health-promoting properties. Because dietary bioactive compounds play a central role in colorectal cancer prevention and may also contribute to the prevention of other diet-related cancers, such as hepatocellular carcinoma, these compounds have attracted increasing interest as potential chemopreventive agents. However, their bioactivity depends not only on their original composition but also on the structural modifications they undergo during gastrointestinal digestion, which determines their bioaccessibility. Despite the growing interest in olive leaf phenolics, studies integrating metabolite profiling before and after digestion with functional biological evaluation remain scarce, particularly for Brazilian olive cultivars. Therefore, this study investigated the effects of in vitro simulated gastrointestinal digestion on the phenolic profile, bioaccessibility, antioxidant activity, and anticancer potential of Brazilian olive leaf infusions. Olive leaves from the Coratina and Picual cultivars were extracted by aqueous infusion and subjected to in vitro gastrointestinal digestion according to the standardized INFOGEST 2.0 protocol. Phenolic compounds were characterized by UHPLC-ESI-MS/MS and total phenolic content by the Folin-Ciocalteu assay. Antioxidant activity was determined using DPPH and ABTS assays. Anticancer activity was evaluated in colorectal (HCT116 and HT29) and hepatocellular (HepG2) carcinoma cells by Trypan Blue, intracellular ROS production, Annexin V/PI apoptosis, and clonogenic assays. Spearman correlation analysis was performed to investigate the relationship between intracellular ROS production and apoptosis. UHPLC-ESI-MS/MS identified extensive remodeling of the phenolic profile throughout digestion. Native secoiridoids, particularly oleoside, together with oleuropein derivatives and elenolic acid glucoside, predominated in the original infusions, whereas digestion generated a distinct bioaccessible profile enriched in ligstroside aglycone derivative, dialdehydic form of decarboxymethyl elenolic acid, and oleoside 11-methyl ester. Only a few compounds were detected after the gastric phase (~13%), possibly due to reversible interactions between phenolic compounds and digestive enzymes that may have limited their detection under the UHPLC analytical conditions employed. Phenolic recovery increased during the duodenal phase, reaching ~44% bioaccessibility. Despite these compositional changes, antioxidant activity increased after digestion. Treatment with the digested fraction (2%, v/v) preserved biological activity, reducing cell viability, decreasing clonogenic capacity by approximately 50%, increasing intracellular ROS (1.2–2.7-fold), and inducing apoptosis (20–50%) in colorectal and hepatocellular carcinoma cells. Spearman correlation analysis demonstrated a significant positive correlation between intracellular ROS production and apoptosis, supporting oxidative stress as a mechanism associated with the biological effects of the digested infusions. These findings demonstrate that in vitro gastrointestinal digestion acts as a biotransformation process, generating new bioaccessible secoiridoid derivatives while preserving antioxidant and anticancer activities. Overall, these results reinforce the use of olive leaf infusion as a simple, sustainable, green, and home-based strategy for the intake of bioaccessible bioactive compounds.