Optimization of Oleuropein Extraction from Olive Leaves and Its Protective Effect Against TBHP-Induced Oxidative Damage in HEK-293 Cells

Olive leaves, a major byproduct of olive processing, are generated in large quantities annually yet suffer from inefficient utilization and low added value. In this study, response surface methodology (RSM) was employed to optimize the extraction conditions of oleuropein from olive leaves. Additionally, the antioxidant activity of purified oleuropein and its protective effect against tret-butyl hydroperoxide (TBHP)-induced oxidative damage in the human embryonic kidney 293 (HEK-293) cell line were investigated. The optimal extraction conditions for oleuropein were determined as follows: extraction temperature of 71 °C, extraction time of 72 min, ethanol concentration of 58%, and solid–liquid ratio of 1:27 (mg/mL), yielding an oleuropein recovery of 44.5%. The extract was purified and identified as oleuropein via Fourier transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC). Oleuropein exhibited remarkable antioxidant activity and mitigated TBHP-induced oxidative damage in HEK-293 cells by inhibiting apoptosis. TBHP treatment reduced cell viability by approximately 70%, while treatment with 100 and 200 μg/mL oleuropein restored the decreased cell viability to 100%. Morphological observations and 4′,6-diamidino-2-phenylindole (DAPI) staining revealed that TBHP induced apoptotic cell death characterized by nuclear condensation and fragmentation, and this effect was reversed by oleuropein treatment. Flow cytometry analysis showed that TBHP caused approximately 90% cell death, whereas co-treatment with oleuropein reduced cell death to only about 10%. TBHP downregulated the expression of p53, and oleuropein reactivated its expression, highlighting the role of oleuropein in the recovery of the cellular antioxidant system. This study possibly indicated the protective mechanism of oleuropein against oxidative damage-related diseases and provides a theoretical basis for the development of olive leaves as a potential ingredient in functional foods.