Analysis of Active Components and NRF2 Activation in Olive Leaf Pressurized Water Extract

Pressurized liquid extraction is a valuable technique for obtaining bioactive compounds from diverse natural sources. This method employs liquids at temperatures and pressures exceeding their critical points, enhancing the extraction efficiency of these compounds. While components in the pressurized water extract of olive leaves have been reported, their biological activities remain largely unexplored. Here, we investigated the major components and the activation of nuclear factor erythroid 2-related factor 2 (NRF2), a key regulator of oxidative stress response, using a dual-color real-time bioluminescence monitoring system in pressurized water, ethanol, and hot water extracts of olive leaves. Oleuropein was identified as a primary component in ethanol and hot water extracts but was absent in the pressurized water extract. Remarkably, hydroxymethylfurfural, furfural, hydroxytyrosol, and hydroxytyrosol acetate were uniquely detected in the pressurized water extract. We further evaluated the activation of the NRF2 signaling pathway in human hepatoma HepG2 cells. The pressurized water extract demonstrated typical, dose-dependent NRF2 activation without cytotoxicity. Conversely, the ethanol extract exhibited cytotoxicity, while the hot water extract showed neither NRF2 activation nor cytotoxicity. Quantitative analysis and a reporter gene assay with commercial standards revealed that hydroxytyrosol and hydroxytyrosol acetate contribute to NRF2 activation by the pressurized water extract.