The coupling of HPLC-ESI-QTOF-MS led to the in-depth characterisation of the olive-leaf extracts on the basis of mass accuracy, true isotopic pattern and tandem mass spectrometry (MS/MS) spectra. We may conclude therefore that this analytical tool is very valuable in the study of phenolic compounds in plant matrices.

Aging can be viewed as a quasi-programmed phenomenon driven by the overactivation of the nutrient-sensing mTOR gerogene. mTOR-driven aging can be triggered or accelerated by a decline or loss of responsiveness to activation of the energy-sensing protein AMPK, a critical gerosuppressor of mTOR. The occurrence of age-related diseases, therefore, reflects the synergistic interaction between our evolutionary path to sedentarism, which chronically increases a number of mTOR activating gero-promoters (e.g., food, growth factors, cytokines and insulin) and the "defective design" of central metabolic integrators such as mTOR and AMPK. Our laboratories at the Bioactive Food Component Platform in Spain have initiated a systematic approach to molecularly elucidate and clinically explore whether the "xenohormesis hypothesis," which states that stress-induced synthesis of plant polyphenols and many other phytochemicals provides an environmental chemical signature that upregulates stress-resistance pathways in plant consumers, can be explained in terms of the reactivity of the AMPK/mTOR-axis to so-called xenohormetins. Here, we explore the AMPK/mTOR-xenohormetic nature of complex polyphenols naturally present in extra virgin olive oil (EVOO), a pivotal component of the Mediterranean style diet that has been repeatedly associated with a reduction in age-related morbid conditions and longer life expectancy. Using crude EVOO phenolic extracts highly enriched in the secoiridoids oleuropein aglycon and decarboxymethyl oleuropein aglycon, we show for the first time that (1) the anticancer activity of EVOO secoiridoids is related to the activation of anti-aging/cellular stress-like gene signatures, including endoplasmic reticulum (ER) stress and the unfolded protein response, spermidine and polyamine metabolism, sirtuin-1 (SIRT1) and NRF2 signaling; (2) EVOO secoiridoids activate AMPK and suppress crucial genes involved in the Warburg effect and the self-renewal capacity of "immortal" cancer stem cells; (3) EVOO secoiridoids prevent age-related changes in the cell size, morphological heterogeneity, arrayed cell arrangement and senescence-associated β-galactosidase staining of normal diploid human fibroblasts at the end of their proliferative lifespans. EVOO secoiridoids, which provide an effective defense against plant attack by herbivores and pathogens, are bona fide xenohormetins that are able to activate the gerosuppressor AMPK and trigger numerous resveratrol-like anti-aging transcriptomic signatures. As such, EVOO secoiridoids constitute a new family of plant-produced gerosuppressant agents that molecularly "repair" the aimless (and harmful) AMPK/mTOR-driven quasi-program that leads to aging and aging-related diseases, including cancer.

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The aim of this work was to develop a rapid resolution liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (RRLC-ESI-TOF-MS) method followed by tetrazolium salt (MTT)-based cell viability assays for qualitative and quantitative classification of extra virgin olive oil (EVOO) varieties by phenolic and other polar compound contents as well as for rapid characterization of putative cytotoxic activities against human cancer cells. Five different Spanish EVOO varieties were analyzed, and RRLC-ESI-TOF-MS method was applied for qualitative and quantitative identification of most important phenolic compounds. We finally employed MTT-based cell viability protocol to assess the effects of crude EVOO phenolic extracts (PEs) on the metabolic status of cultured SKBR3 human breast cancer cells. MTT-based cell viability assays revealed a wide range of breast cancer cytotoxic potencies among individual crude PE obtained from EVOO monovarietals. Remarkably, breast cancer cell sensitivity to crude EVOO-PEs was up to 12 times higher in secoiridoids enriched-PE than in secoiridoids-low/null EVOO-PE.

This review describes the olive oil production process to obtain extra virgin olive oil (EVOO) enriched in polyphenol and byproducts generated as sources of antioxidants. EVOO is obtained exclusively by mechanical and physical processes including collecting, washing, and crushing of olives, malaxation of olive paste, centrifugation, storage, and filtration. The effect of each step is discussed to minimize losses of polyphenols from large quantities of wastes. Phenolic compounds including phenolic acids, alcohols, secoiridoids, lignans, and flavonoids are characterized in olive oil mill wastewater, olive pomace, storage byproducts, and filter cake. Different industrial pilot plant processes are developed to recover phenolic compounds from olive oil byproducts with antioxidant and bioactive properties. The technological information compiled in this review will help olive oil producers to improve EVOO quality and establish new processes to obtain valuable extracts enriched in polyphenols from byproducts with food ingredient applications.

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