A rapid and reliable method to assay the total amount of tyrosol and hydroxytyrosol derivatives in extra virgin olive oil has been developed. The methodology intends to establish the nutritional quality of this edible oil addressing recent international health claim legislations (the European Commission Regulation No. 432/2012) and changing the classification of extra virgin olive oil to the status of nutraceutical. The method is based on the use of high-performance liquid chromatography coupled with tandem mass spectrometry and labeled internal standards preceded by a fast hydrolysis reaction step performed through the aid of microwaves under acid conditions. The overall process is particularly time saving, much shorter than any methodology previously reported. The developed approach represents a mix of rapidity and accuracy whose values have been found near 100% on different fortified vegetable oils, while the RSD% values, calculated from repeatability and reproducibility experiments, are in all cases under 7%. Graphical abstract Schematic of the methodology applied to the determination of tyrosol and hydroxytyrosol ester conjugates.

Embryos of the American lobster, Homarus americanus, are remarkably resistant to infection by the fungus Lagenidium callinectes, a pathogen of many crustaceans. The surfaces of healthy lobster embryos are covered almost exclusively by a single, Gram-negative bacterium, which grows in a dense mosaic pattern. In culture, this bacterium produces a compound that completely inhibits the growth of the pathogenic fungus in vitro at 10 mcg/ml. Large-scale fermentation, extraction, and subsequent chromatographic purification led to the identification of the antifungal substance as 4-hydroxyphenethyl alcohol (tyrosol), an antibiotic substance known to be produced by terrestrial fungi.

Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with jasmonic acid (JA) and pectin as elicitors to evaluate their effect on metabolism from two cell lines using NMR spectroscopy and multivariate data analysis. According to principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA), the chloroform extract of the pectin-treated cultures were more different than control and JA-treated cultures; but in the methanol/water extract the metabolome of the JA-treated cells showed clear differences with control and pectin-treated cultures. Tyrosol, an antioxidant metabolite, was detected in cannabis cell cultures. The tyrosol content increased after eliciting with JA.

Lipid peroxidation is a major factor in the pathogenesis of many disease states. To detect the initial stages of lipid peroxidation or evaluate antioxidant efficacy, cis-parinaric acid (cis-PnA) has been successfully used and thoroughly validated. However, cis-PnA is not very well suited for medium throughput screening of antioxidants in living cells. We recently introduced and validated a lipid peroxidation reporter molecule, C11-BODIPY(581/591). To further explore this probe, we evaluated the protective effect of 12 natural antioxidants in rat-1 fibroblasts subjected to 50 microM cumene-hydroperoxide using both probes. The same pecking order for the individual antioxidant efficacies was obtained: alpha-tocopherol approximately gamma-tocopherol>> quercetin approximately lycopene>> kaempferol>> palm oil>> hydroxy-tyrosol>>>> alpha-carotene = beta-carotene = lutein = tyrosol = chlorogenic acid. This validates the accuracy of the C11-BODIPY(581/591) method and shows that this assay is an accurate and highly flexible method for indexing lipid peroxidation or determining antioxidant efficacy in living cells in a medium throughput scenario. The antioxidant efficacy was compared with their one-electron reduction potential, hydrophobicity and Trolox C equivalent antioxidant capacity. Our results show that although these parameters are valuable for determining structure-function relationships, they have limited predictive value for antioxidant efficacy in vivo.

Rhodiola has been widely used in traditional Asian medicine. In this study, we tested the hypopigmentation effects of R. sachalinensis and its active compounds including catechin, chlorogenic acid, p-coumaric acid, and p-tyrosol. Results have shown that only p-coumaric acid inhibits melanin synthesis in B16F10 cells. However, p-coumaric acid did not inhibit tyrosinase activity when L-DOPA was used as a substrate. Instead, p-coumaric acid inhibited tyrosinase activity when L-tyrosine was used as a substrate. We further analyzed the changes of cAMP responsive element binding protein (CREB) phosphorylation and tyrosinase gene expression. The results indicate that p-coumaric acid does not affect CREB phosphorylation or tyrosinase protein production. In turn, these findings demonstrate that p-coumaric acid has no effect on the upstream regulation of tyrosinase gene expression, although p-coumaric acid showed a significant inhibitory effect on melanogenesis. Because p-coumaric acid showed different effects on tyrosinase activity according to different substrates, we tested whether tyrosinase can utilize p-coumaric acid as a substrate. Our findings revealed that competitive inhibition occurs between p-coumaric acid and tyrosine. Consequently, this finding could be a primary mechanism for the hypopigmenting action of p-coumaric acid.

Phenol hydroxylase gene cloning from the thermophilic bacteria Geobacillus thermoglucosidasius was used to develop an effective method to convert tyrosol into the high-added-value compound hydroxytyrosol by hydroxylation. Phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2 genes and strictly dependent on NADH and FAD. These two genes were subcloned together as a 2 kb fragment into Escherichia coli Rosetta cells, and the transformants were able to grow and effectively transform up to 5 mM of phenol and tyrosol using IPTG (isopropyl-β-D-thiogalactopyranoside) as inducer. In addition, when a new fragment with a 340 pb upstream pheA1 gene was subcloned, a similar biotransformation rate was attained without IPTG, confirming that this fragment encodes for a phenol hydroxylase promoter that can be recognised by E. coli. Both transformants brought about the total bioconversion of monophenols at a high concentration (5 mM), which represents an increase, both in concentration and in yield, compared with that previously described in the bibliography. The use of the transformant with its constitutive promoter was more interesting from a biotechnological point of view, since it is not necessary to use IPTG. It also gave rise to greater operational stability.

Hydroxytyrosol and other olive biophenols (OBPs) such as tyrosol, verbascoside, apigenin-7-glucoside, and alpha-taxifolin have been extracted from alperujo by using static-dynamic superheated liquids. Multivariate methodology has been used to carry out a detailed optimization of the extraction. Under the optimal working conditions no further extraction of the target analytes was achieved after 27 min (up to 2800 and 1500 mg/kg of hydroxytyrosol and tyrosol, respectively), so complete removal of them within this interval was assumed. The extract was injected into a chromatograph-photodiode array detector assembly for individual separation-quantification. The efficacy of ethanol/water mixtures to extract OBPs from alperujo has been demonstrated and compared with that of a conventional stirring-based method. These less toxic extractant mixtures are of interest with a view to future human uses of OBPs.

OBJECTIVE

We have previously reported that tyrosol (TYR) promotes lifespan extension in the nematode Caenorhabditis elegans, also inducing a stronger resistance to thermal and oxidative stress in vivo. In this study, we performed a whole-genome DNA microarray in order to narrow down the search for candidate genes or signaling pathways potentially involved in TYR effects on C. elegans longevity.

METHODS

Nematodes were treated with 0 or 250 μM TYR, total RNA was isolated at the adult stage, and derived cDNA probes were hybridized to Affymetrix C. elegans expression arrays. Microarray data analysis was performed, and relative mRNA expression of selected genes was validated using qPCR.

RESULTS

Microarray analysis identified 208 differentially expressed genes (206 over-expressed and two under-expressed) when comparing TYR-treated nematodes with vehicle-treated controls. Many of these genes are linked to processes such as regulation of growth, transcription, reproduction, lipid metabolism and body morphogenesis. Moreover, we detected an interesting overlap between the expression pattern elicited by TYR and those induced by other dietary polyphenols known to extend lifespan in C. elegans, such as quercetin and tannic acid.

CONCLUSIONS

Our results suggest that important cellular mechanisms directly related to longevity are influenced by TYR treatment in C. elegans, supporting our previous notion that this phenol might act on conserved genetic pathways to increase lifespan in a whole organism.

Experiments on rats showed that n-tyrosol limited the increase in blood viscosity during thermal exposure at a shear rate of 5-300 sec(-1) and inhibited ADP-induced platelet aggregation. The effects of n-tyrosol are comparable to that of pentoxyphylline.

OBJECTIVE

To investigate the chemical constituents from the tuber of the planted Cremastra appendiculata.

METHODS

The compounds were isolated by column chromatography over silica gel, Sephadex LH-20 and RP-HPLC, and their structures were elucidated on the basis of spectroscopic analysis.

RESULTS

Eight compounds were isolated, and identified as cirrhopetalanthrin (I), 7-hydroxy-4-methoxyphenanthrene-2-O-beta-D-glucoside (II), 4-(2-hydroxyethyl)-2-methoxyphenyl-1-O-beta-D-glucopyranoside (III), tyrosol 8-O-beta-D-gluco-pyranoside (IV), vanilloloside (V), p-hydroxybenzaldehyde (VI), sucrose (VII), adenosine (VIII).

CONCLUSIONS

These compounds are isolated from this plant for the first time. All compounds were evaluated against human colon cancer (HCT-8), human hepatoma (Bel7402), human stomach cancer(BGC-823), human lung adenocarcinoma (A549), human breast cancer (MCF-7), and human ovarian cancer (A2780) cell lines, and cirrhopetalanthrin (I) showed non-selective moderate cytotoxicity with IC50 values of 8.4-13.3 micromol x L(-1), and other compounds were inactive.

Biocatalysis is today a standard technology for the industrial production of several chemicals, and the number of biotransformation processes running on a commercial scale is constantly increasing. Among biocatalysts, bacterial multicomponent monooxygenases (BMMs), a diverse group of nonheme diiron enzymes that activate dioxygen, are of primary interest due to their ability to catalyze a variety of complex oxidations, including reactions of mono- and dihydroxylation of phenolic compounds. In recent years, both directed evolution and rational design have been successfully used to identify the molecular determinants responsible for BMM regioselectivity and to improve their activity toward natural and nonnatural substrates. Toluene o-xylene monooxygenase (ToMO) is a BMM isolated from Pseudomonas sp. strain OX1 which hydroxylates a wide spectrum of aromatic compounds. In this work we investigate the use of recombinant ToMO for the biosynthesis in recombinant cells of Escherichia coli strain JM109 of 4-hydroxyphenylethanol (tyrosol), an antioxidant present in olive oil, from 2-phenylethanol, a cheap and commercially available substrate. We initially found that wild-type ToMO is unable to convert 2-phenylethanol to tyrosol. This was explained by using a computational model which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h.

Olive mill wastes represent a significant environmental problem in Mediterranean areas where they are generated in huge quantities in a short period of time. Their high phenol, lipid and organic acid concentrations turn them into phytotoxic materials, but these wastes also contain valuable resources such as a large proportion of organic matter and a wide range of nutrients that could be recycled. Composting is one of the technologies used for the valorization of this effluent, producing a fertilizer useful for poor soils.The present work deals with the changes that occur in the content of phenolic compounds and the biotoxicity of the oxidized substrate which result from the composting of olive mill wastewater (OMW) sludge with sesame bark. The total organic matter decreased 52.72% while water-soluble phenol degradation decreased 72% after 7 months of processing. Gas chromatography coupled with mass spectroscopy was used to confirm the elimination of polyphenols during composting. Initially, the analysis showed three abundant polyphenolic compounds, one of which was identified as the 4-hydroxyphenyl-ethanol (tyrosol), a well-known antioxidant in OMW. After 7 months of composting, all of the phenolic compounds disappeared. The phytotoxic effects of OMW sludge, assessed by the plant index germination, increased during the composting to reach 80% after 210 days. This trend was confirmed by the correlation between physico-chemical and toxicity parameters. The results obtained confirmed the stability of the compost prepared from OMW sludge with sesame bark and indicated a gradual detoxification as the compost matured.

Calixdrugs are calix[4]arene-based clusters (chaliced shapes) of established therapeutic drugs (e.g., penicillin, cephalosporin, tyrosol, and carboplatin) that are innovatively named calixpenam, calixcephem, calixtyrosol, and calixplatin, respectively. Going over the structures of cluster compounds, the calixarene scaffold lies at the heart of the structure (chaliced shape of drug), and it is an integral part of the cluster. In fact, the monomer drugs contribute as 1/4 of the corresponding cluster. In addition, probably because of the multivalency, spatial preorganization, and synergistic effect of four impacted drug units in the structures of calixdrugs, they are more effective in interactions with the target sites over their corresponding monomers.

BACKGROUND

The recovery of phenolic compounds from olive milling is recognized as strategic for producers. The aim of this work was to evaluate the quality and stability of retentates obtained from olive mill wastewaters treated with a membrane filtration system constituted by a micro-, ultra- and nanofiltration, followed by a final reverse osmosis, over three crop seasons. Efficiency was evaluated in terms of phenolic amount in the retentates and of organic load in the final discarded waters. Phenolic compounds were quantified using tyrosol as external standard.

RESULTS

Our study highlighted a reproducibility of the process over years and a low organic load in permeates from reverse osmosis. Hydroxytyrosol was very stable in the liquid products at 18-28 °C over 24 months of storage. The retentates from reverse osmosis showed the highest phenolic content (78.6 mg g-1 dry matter in 2015), associated with a potassium content of 22 g kg-1 .

CONCLUSIONS

The liquid concentrated retentates showed an unexpected stability over time of their bioactive phenolic compounds, particularly of hydroxytyrosol. These samples recovered from olive mill wastewaters can be good sources of natural antioxidants and potassium to guarantee the correct intake and to formulate new food ingredients or food products. © 2017 Society of Chemical Industry.

The effect of olive stone removal before processing on the degradation level, secoiridoid and tocopherol contents, and antioxidant activity of monovarietal extra virgin olive oils (EVOOs) was studied. EVOOs were extracted from olives of the Leccino, Moraiolo, Frantoio, Pendolino, Taggiasca, and Colombaia varieties both in the presence and in the absence of the stones. The degradation level of EVOOs was evaluated by acidity, peroxide number, and spectroscopic indices K(232) and K(270), according to EU regulation. The secoiridoid compounds typical of EVOO, namely, the oleuropein and ligstroside derivatives, hydroxytyrosol, tyrosol, and tocopherols were analyzed by HPLC. The antioxidant activity was evaluated by the xanthine oxidase/xanthine system, generating superoxide radical and hydrogen peroxide, and by the 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl test. Results showed that EVOOs obtained from both stoned and destoned olives had a very low degradation level, which was not affected by destoning. Destoning lowered slightly the alpha-tocopherol content in EVOOs but increased the total secoiridoid content and the antioxidant activity of EVOOs (up to 3.5-fold). However, these effects were variety-dependent and negligible in some conditions. It was concluded that a better knowledge of the reactions occurring during olive processing, and particularly on the involvement of endogenous pulp and stone enzymes, is essential to predict the effect of destoning on EVOO quality.

Surface erosion has been recognized as a valuable design tool for resorbable biomaterials within the context of drug delivery devices, surface coatings, and when precise control of strength retention is critical. Here we report on high tensile strength, aromatic-aliphatic polycarbonates based on natural phenols, tyrosol (Ty) and homovanillyl alcohol (Hva), that exhibit enzymatic surface erosion by lipase. The Young's moduli of the polymers for dry and fully hydrated samples are 1.0 to 1.2 GPa and 0.8 to 1.2 GPa, respectively. Typical characteristics of enzymatic surface erosion were confirmed for poly(tyrosol carbonate) films with concomitant mass-loss and thickness-loss at linear rates of 0.14 ± 0.01 mg cm(-2) d(-1) and 3.0 ± 0.8 μm d(-1), respectively. The molecular weight and the mechanical properties of the residual films remained constant. Changing the ratio of Ty and Hva provided control over the glass transition temperature (T(g)) and the enzymatic surface erosion: increasing the Hva content in the polymers resulted in higher T(g) and lower enzymatic erosion rate. Polymers with more than 50 mol % Hva were stable at 37 °C in enzyme solution. Analysis on thin films using quartz crystal microbalance with dissipation (QCM-D) demonstrated that the onset temperature of the enzymatic erosion was approximately 20 °C lower than the wet T(g) for all tested polymers. This new finding demonstrates that relatively high tensile strength polycarbonates can undergo enzymatic surface erosion. Moreover, it also sheds light on the connection between T(g) and enzymatic degradation and explains why few of the high strength polymers follow an enzyme-meditated degradation pathway.

The transformation by an oxidoreductase (a laccase from Rhus vernificera) of a mixture of four phenols (catechol, methylcatechol, m-tyrosol and hydroxytyrosol) that simulates a typical wastewater derived from an olive oil factory was investigated. Results achieved in this study confirm that laccase-mediated transformation of phenols depends on the nature and the initial concentration of the involved phenol, the time course of the reaction, and mainly, on the complexity of the phenolic incubation mixture. Actually, the four phenols each have a completely different response to enzyme action both in terms of quantitative and kinetic transformation. For example, after 24-h incubation, methylcatechol was completely removed, whereas 30% of untransformed hydroxytyrosol and catechol and more than 65% of m-tyrosol were still present in the reaction mixture. A reduction of enzyme activity occurred for all phenols after enzymatic oxidation. No correspondence between phenol transformation and disappearance of enzymatic activity was observed, thus suggesting that different mechanisms are probably involved in the laccase-mediated transformation of the four phenols. The behavior of phenols became more complex when an increasing number of phenols was present in the reaction mixture, and even more so when different concentrations of phenols were used. Competitive effects may arise when more than one phenol is present in the reaction solution and interacts with the enzyme.

Erythrina crista-galli (Fabaceae) is used in Argentinean ethnopharmacology as anti-inflammatory medication, narcotic, desinfectant, and for the treatment of wounds. The common name of the tree is "ceibo" or coral tree. The dominating endophytes in E. crista-galli all belong to the genus Phomopsis as identified by microscopic features and the analysis of their ITS sequences. To investigate a possible contribution of Phomopsis spp. to the metabolites found in the plant, twelve different isolates were cultivated in different media. Besides several new metabolites a number of known compounds were detected: mellein, nectriapyrone, 4-hydroxymellein, scytalone, tyrosol, clavatol, mevinic acid, and mevalonolactone.

Although oak wood is the main material used in cooperage, other species are being considered as possible sources of wood for the production of wines and their derived products. In this work we have compared the phenolic composition of acacia (Robinia pseudoacacia), chestnut (Castanea sativa), cherry (Prunus avium) and ash (Fraxinus excelsior and F. americana) heartwoods, by using HPLC-DAD/ESI-MS/MS (some of these data have been showed in previous paper), as well as the changes that toasting intensity at cooperage produce in each polyphenolic profile. Before toasting, each wood shows a different and specific polyphenolic profile, with both qualitative and quantitative differences among them. Toasting notably changed these profiles, in general, proportionally to toasting intensity and led to a minor differentiation among species in toasted woods, although we also found phenolic markers in toasted woods. Thus, methyl syringate, benzoic acid, methyl vanillate, p-hydroxybenzoic acid, 3,4,5-trimethylphenol and p-coumaric acid, condensed tannins of the procyanidin type, and the flavonoids naringenin, aromadendrin, isosakuranetin and taxifolin will be a good tool to identify cherry wood. In acacia wood the chemical markers will be the aldehydes gallic and β-resorcylic and two not fully identified hydroxycinnamic compounds, condensed tannins of the prorobinetin type, and when using untoasted wood, dihydrorobinetin, and in toasted acacia wood, robinetin. In untoasted ash wood, the presence of secoiridoids, phenylethanoid glycosides, or di and oligolignols will be a good tool, especially oleuropein, ligstroside and olivil, together verbascoside and isoverbascoside in F. excelsior, and oleoside in F. americana. In toasted ash wood, tyrosol, syringaresinol, cyclolovil, verbascoside and olivil, could be used to identify the botanical origin. In addition, in ash wood, seasoned and toasted, neither hydrolysable nor condensed tannins were detected. Lastly, in chestnut wood, gallic and ellagic acids and hydrolysable tannins of both the gallotannin and ellagitannin type, can be used as chemical markers.

The phytochemical constituents of a biologically active, standardized, 80% ethanol extract of Rhodiola heterodonta were characterized. The extract was fractionated over a Sephadex LH-20 column to afford two main fractions representing two classes of secondary metabolites: phenylethanoids and proanthocyanidins. This fractionation facilitated the identification and quantification of individual compounds in the fractions and sub-fractions using HPLC, and LC-MS. The major compounds in the phenylethanoid fraction were heterodontoside, tyrosol methyl ether, salidroside, viridoside, mongrhoside, tyrosol, and the cyanogenic glucoside rhodiocyanoside A. These seven compounds comprised 17.4% of the EtOH extract. Proanthocyanidins ranged from oligomers to polymers based on epigallocatechin and gallate units. The main identified oligomeric compounds in the proanthocyanidin fraction were epigallocatechin gallate, epigallocatechin-epigallocatechin-3-O-gallate and 3-O-galloyl-epigallocatechin-epigallocatechin-3-O-gallate, which constituted 1.75% of the ethanol extract. Tyrosol methyl ether, mongrhoside, and the two proanthocyanidin dimers were reported for the first time from this species in this study. Intraperitoneal injection of the 80% ethanol extract increased survival time of mice under hypoxia by 192%, as an indication of adaptogenic activity.

Two new clerodane diterpenoids 2 beta-methoxy-cleroda-3,13-dien-18-carboxy-15,16-olide (1) and 15 xi-methoxy-cleroda-3,12-dien-18-carboxy-15,16-olide (2) and one new nitrogen-containing clerodane diterpenoid 15-oxo-echinophyllin A (3), along with six known compounds, namely, echinophyllin A, (-)-patagonic acid, tyrosol, oplopanone, 4-hydroxy-4-methyl-2-[(1R)-1-(1-methylethyl)-4-oxopentyl]-2-cyclohexen-1-one and 1 beta,6 alpha-dihydroxy-eudesman-4(15)-ene were isolated from the leaves of Casearia sylvestris. Their structures were determined by extensive NMR techniques.

For the provision of oleocanthal (OLC), a phenolic compound with very promising pharmacological properties, isolation from olive oil is a very important option. Due to the compound's sensitivity to decomposition upon exposure to oxygen and light, a very gentle isolation method has been developed under use of high performance countercurrent chromatography (HPCCC). By partition of olive oil between hexane and methanol, an extract enriched in phenolics was prepared and subjected to a two-step HPCCC separation under use of heptane-EtOAc-MeOH-H2O mixtures in normal-phase and reverse phase mode, respectively. With this method, the isolation of tyrosol, hydroxytyrosol, and the mixture of (3S,4E)- and (3S,4Z)-OLC was achieved in approx. 70 min for each step. By one- and two-dimensional NMR-experiments and LC-MS, the equilibrium of (3S,4E)- and (3S,4Z)-OLC in such olive oil extracts has unambiguously been proven for the first time.

Aromatic substrates tyrosol (p-hydroxyphenylethanol) and 2,6-dihydroxynaphthalene (2,6-DHN) were converted into chromane derivatives by means of chemoenzymatic reactions catalyzed by the aromatic prenyltransferase of bacterial origin NovQ, using dimethylallyl bromide as allylic substrate instead of the natural isoprenyl pyrophosphate substrate. Stereoselective prenylation occurred in o-position with respect to the phenol hydroxyl in both compounds. Prenylated derivatives were readily converted into chromane products via a selective 6-endo-trig cyclization involving the oxygen atom from the phenol moiety and the double bond of the prenyl substituent, a process catalyzed by FeCl(3). These findings set up the basis of a most convenient two-step, one-pot process which allows for easy recovery of the chromane products in high yields. The chromane derivatives thus obtained were tested for cytotoxicity and pro-apoptotic activity using LoVo WT cells, a line of human colon adenocarcinoma.

We report the results of in vivo studies in Caenorhabditis elegans nematodes in which addition of extra virgin olive oil (EVOO) to their diet significantly increased their life span with respect to the control group. Furthermore, when nematodes were exposed to the pesticide paraquat, they started to die after two days, but after the addition of EVOO to their diet, both survival percentage and lifespans of paraquat-exposed nematodes increased. Since paraquat is associated with superoxide radical production, a test for scavenging this radical was performed using cyclovoltammetry and the EVOO efficiently scavenged the superoxide. Thus, a linear correlation (y = -0.0838x +19.73, regression factor = 0.99348) was observed for superoxide presence (y) in the voltaic cell as a function of aliquot (x) additions of EVOO, 10 μL each. The originally generated supoeroxide was approximately halved after 10 aliquots (100 μL total). The superoxide scavenging ability was analyzed, theoretically, using Density Functional Theory for tyrosol and hydroxytyrosol, two components of EVOO and was also confirmed experimentally for the galvinoxyl radical, using Electron Paramagnetic Resonance (EPR) spectroscopy. The galvinoxyl signal disappeared after adding 1 μL of EVOO to the EPR cell in 10 minutes. In addition, EVOO significantly decreased the proliferation of human leukemic THP-1 cells, while it kept the proliferation at about normal levels in rat L6 myoblasts, a non-tumoral skeletal muscle cell line. The protection due to EVOO was also assessed in L6 cells and THP-1 exposed to the radical generator cumene hydroperoxide, in which cell viability was reduced. Also in this case the oxidative stress was ameliorated by EVOO, in line with results obtained with tetrazolium dye reduction assays, cell cycle analysis and reactive oxygen species measurements. We ascribe these beneficial effects to EVOO antioxidant properties and our results are in agreement with a clear health benefit of EVOO use in the Mediterranean diet.