Jesús Lozano-Sánchez

Xenohormetic and anti-aging activity of secoiridoid polyphenols present in extra virgin olive oil: a new family of gerosuppressant agents.

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.

Prediction of Extra Virgin Olive Oil Varieties through Their Phenolic Profile. Potential Cytotoxic Activity against Human Breast Cancer Cells

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.

HPLC-ESI-QTOF-MS as a powerful analytical tool for characterising phenolic compounds in olive-leaf extracts.

INTRODUCTION Olea europaea L. leaves may be considered a cheap, easily available natural source of phenolic compounds. In a previous study we evaluated the possibility of obtaining bioactive phenolic compounds from olive leaves by pressurised liquid extraction (PLE) for their use as natural anti-oxidants. The alimentary use of these kinds of extract makes comprehensive knowledge of their composition essential. OBJECTIVE To undertake a comprehensive characterisation of two olive-leaf extracts obtained by PLE using high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS). METHOD Olive leaves were extracted by PLE using ethanol and water as extraction solvents at 150°C and 200°C respectively. Separation was carried out in a HPLC system equipped with a C₁₈-column working in a gradient elution programme coupled to ESI-QTOF-MS operating in negative ion mode. RESULTS This analytical platform was able to detect 48 compounds and tentatively identify 31 different phenolic compounds in these extracts, including secoiridoids, simple phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. Lucidumoside C was also identified for the first time in olive leaves. CONCLUSION 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.

Literature Review on Production Process To Obtain Extra Virgin Olive Oil Enriched in Bioactive Compounds. Potential Use of Byproducts as Alternative Sources of Polyphenols

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.

Wastes generated during the storage of extra virgin olive oil as a natural source of phenolic compounds.

Phenolic compounds in extra virgin olive oil (EVOO) have been associated with beneficial effects for health. Indeed, these compounds exert strong antiproliferative effects on many pathological processes, which has stimulated chemical characterization of the large quantities of wastes generated during olive oil production. In this investigation, the potential of byproducts generated during storage of EVOO as a natural source of antioxidant compounds has been evaluated using solid-liquid and liquid-liquid extraction processes followed by rapid resolution liquid chromatography (RRLC) coupled to electrospray time-of-flight and ion trap mass spectrometry (TOF/IT-MS). These wastes contain polyphenols belonging to different classes such as phenolic acids and alcohols, secoiridoids, lignans, and flavones. The relationship between phenolic and derived compounds has been tentatively established on the basis of proposed degradation pathways. Finally, qualitative and quantitative characterizations of solid and aqueous wastes suggest that these byproducts can be considered an important natural source of phenolic compounds, mainly hydroxytyrosol, tyrosol, decarboxymethyl oleuropein aglycone, and luteolin, which, after suitable purification, could be used as food antioxidants or as ingredients in nutraceutical products due to their interesting technological and pharmaceutical properties.

Crude phenolic extracts from extra virgin olive oil circumvent de novo breast cancer resistance to HER1/ HER2-targeting drugs by inducing GADD45-sensed cellular stress, G2/M arrest and hyperacetylation of Histone H3

Characterization of the molecular function of complex phenols naturally present in extra virgin olive oil (EVOO) against the HER2-gene amplified JIMT-1 cell line, a unique breast cancer model that inherently exhibits cross-resistance to multiple HER1/2-targeted drugs including trastuzumab, gefitinib, erlotinib and lapatinib, may underscore innovative cancer molecules with novel therapeutic targets because they should efficiently circumvent de novo resistance to HER1/2 inhibitors in order to elicit tumoricidal effects. We identified pivotal signaling pathways associated with the efficacy of crude phenolic extracts (PEs) obtained from 14 monovarietals of Spanish EVOOs. i) MTT-based cell viability and HPLC coupled to time-of-flight (TOF) mass spectrometry assays revealed that anti-cancer activity of EVOO PEs positively correlated with the phenolic index (i.e., total content of phenolics) and with a higher presence of the complex polyphenols secoiridoids instead of lignans. ii) Genome-wide analyses using 44 K Agilents whole human arrays followed by Gene Set Enrichment Analysis (GSEA)-based screening of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database revealed a differential modulation of the JIMT-1 transcriptome at the level of the cell cycle and p53 pathways. EVOO PEs differentially impacted the expression status of stress-sensing, G2-M check-point-related GADD45 genes and of p53-related CDKN1A, CDKN1C and PMAIP-1 genes. iii) Cell cycle and fluorescence microscopy analyses confirmed that secoiridoid-rich EVOO PE inhibited mitosis to promote G2-M cell cycle arrest. This was accompanied with the appearance of diffuse, even DNA staining with γH2AX and pan-nuclear hyperacetylation of Histone H3 at Lysine 18. iv) Semi-quantitative Signaling Node Multi-Target ELISAs determined that secoiridoid-rich EVOO PE drastically activated the mitogen-activated protein kinases MEK1 and p38 MAPK, a GADD45-related kinase involved in Histone H3 acetylation. Secoiridoids, a family of complex polyphenols characteristic of Oleaceae plants, appear to permit histones to remain in hyperacetylated states and through the resulting alterations in gene regulation to reduce mitotic viability and metabolic competence of breast cancer cells inherently refractory to HER-targeting therapies ab initio. Oleaceae secoiridoids could provide a valuable phytochemical platform for the design of more pharmacologically active second-generation phytopharmaceutical anti-breast cancer molecules with a unique mode of action.

Phytochemical Characterisation of Green Beans (Phaseolus vulgaris L.) by Using High-performance Liquid Chromatography Coupled with Time-of-flight Mass Spectrometry

INTRODUCTION Although green beans (Phaseolus vulgaris L.) constitute a major agro-industrial crop worldwide and involve many bean varieties, the phytochemical composition and how this fluctuates among varieties is not well known. OBJECTIVE The purpose of this work was to conduct a comprehensive characterisation of the phytochemical compounds found in three varieties of green bean. METHODOLOGY Hydromethanol extracts from green beans were analysed by high-performance liquid chromatography (HPLC) coupled with electrospray time-of-flight mass spectrometry (ESI-TOF-MS) in order to characterise phytochemical compounds from different varieties of P. vulgaris L. The compounds were characterised based on interpreting their mass spectrum provided by the TOF-MS as well as by comparison with information from the literature (some compounds have been described previously in Fabaceae). RESULTS In this work, 72 phytochemical compounds were tentatively characterised by HPLC-ESI-TOF-MS. These compounds were classified as, 10 phenolic acids, 59 flavonoids, two lignans and an iridoid. Notably, out of the 72 compounds, 54 are new and their isomers have been characterised for the first time in green beans. CONCLUSION The phytochemical composition of three different varieties of P. vulgaris L have been characterised using HPLC-ESI-TOF-MS. A total of 72 phytochemical compounds were characterised, 54 being reported in green beans for the first time. Among these were the main flavonoids detected. These results highlight the influence that variety can exert on the quality of phytochemicals. Given that new phytochemical compounds have been characterised, this study offers a useful approach for improving and updating the food-composition tables.

Phenolic Secoiridoids in Extra Virgin Olive Oil Impede Fibrogenic and Oncogenic Epithelial-to-Mesenchymal Transition: Extra Virgin Olive Oil As a Source of Novel Antiaging Phytochemicals

The epithelial-to-mesenchymal transition (EMT) genetic program is a molecular convergence point in the life-threatening progression of organ fibrosis and cancer toward organ failure and metastasis, respectively. Here, we employed the EMT process as a functional screen for testing crude natural extracts for accelerated drug development in fibrosis and cancer. Because extra virgin olive oil (EVOO) (i.e., the juice derived from the first cold pressing of the olives without any further refining process) naturally contains high levels of phenolic compounds associated with the health benefits derived from consuming an EVOO-rich Mediterranean diet, we have tested the ability of an EVOO-derived crude phenolic extract to regulate fibrogenic and oncogenic EMT in vitro. High-performance liquid chromatography (HPLC) coupled to time-of-flight (TOF) mass spectrometry assays revealed that the EVOO phenolic extract was mainly composed (∼70%) of two members of the secoiridoid family of complex polyphenols, namely oleuropein aglycone-the bitter principle of olives-and its derivative decarboxymethyl oleuropein aglycone. EVOO secoiridoids efficiently prevented loss of proteins associated with polarized epithelial phenotype (i.e., E-cadherin) as well as de novo synthesis of proteins associated with mesenchymal migratory morphology of transitioning cells (i.e., vimentin). The ability of EVOO to impede transforming growth factor-β (TGF-β)-induced disintegration of E-cadherin-mediated cell-cell contacts apparently occurred as a consequence of the ability of EVOO phenolics to prevent the upregulation of SMAD4-a critical mediator of TGF-β signaling-and of the SMAD transcriptional cofactor SNAIL2 (Slug)-a well-recognized epithelial repressor. Indeed, EVOO phenolics efficiently prevented crucial TGF-β-induced EMT transcriptional events, including upregulation of SNAI2, TCF4, VIM (Vimentin), FN (fibronectin), and SERPINE1 genes. While awaiting a better mechanistic understanding of how EVOO phenolics molecularly shut down the EMT differentiation process, it seems reasonable to suggest that nontoxic Oleaceae secoiridoids certainly merit to be considered for aging studies and, perhaps, for ulterior design of more pharmacologically active second-generation anti-EMT molecules.

Recovering bioactive compounds from olive oil filter cake by advanced extraction techniques.

The potential of by-products generated during extra-virgin olive oil (EVOO) filtration as a natural source of phenolic compounds (with demonstrated bioactivity) has been evaluated using pressurized liquid extraction (PLE) and considering mixtures of two GRAS (generally recognized as safe) solvents (ethanol and water) at temperatures ranging from 40 to 175 °C. The extracts were characterized by high-performance liquid chromatography (HPLC) coupled to diode array detection (DAD) and electrospray time-of-flight mass spectrometry (HPLC-DAD-ESI-TOF/MS) to determine the phenolic-composition of the filter cake. The best isolation procedure to extract the phenolic fraction from the filter cake was accomplished using ethanol and water (50:50, v/v) at 120 °C. The main phenolic compounds identified in the samples were characterized as phenolic alcohols or derivatives (hydroxytyrosol and its oxidation product), secoiridoids (decarboxymethylated and hydroxylated forms of oleuropein and ligstroside aglycones), flavones (luteolin and apigenin) and elenolic acid derivatives. The PLE extraction process can be applied to produce enriched extracts with applications as bioactive food ingredients, as well as nutraceuticals.

New filtration systems for extra-virgin olive oil: effect on antioxidant compounds, oxidative stability, and physicochemical and sensory properties.

The purpose of this work was to evaluate some new filtration systems in relation to the quality of extra-virgin olive oil (EVOO). Filtration processes were undertaken using a polypropylene filter bag and two different inert gas flows as filter aids (argon and nitrogen). Qualitative and quantitative variations of the glyceride composition, antioxidant and pro-oxidant compounds, and water content were correlated with the oxidative stability to establish the effect on EVOO shelf life. The influence on physicochemical and sensorial properties was also evaluated. After filtration, the oxidative stability was reduced. The behavior of the polyphenols and water content on the filtration process could explain the lowest oxidative stability of filtered EVOO. Moreover, the results of the sensorial analysis confirmed that filtration using inert gases did not decrease the intensity of the main positive sensory attributes. The results could help olive-oil producers to improve EVOO quality and establish optimal storage conditions.