Olive Oil Phenols Research Articles

Effect of a Purified Extract of Olive Mill Waste water on Endothelial Cell Proliferation, Apoptosis, Migration and Capillary-Like Structure in vitro and in vivo

Olive oil, a major feature of the Mediterranean diet, is an important source of phenolic compounds. Poliphenols are associated with inhibition of several pathological processes, including cancer. Soluble phenols are contained in the aqueous part of olive and are discarded during oil production in the ‘olive mill wastewaters’. Here we investigated the properties of a purified extract of olive mill wastewaters, named A009, as potential anti-angiogenic compound. While the strong anti-oxidant activity of olive derived phenolic compounds has been well characterized, little is known about their anti-angiogenic properties. We investigated effects of A009 on endothelial cell morphogenesis, proliferation, migration and apoptosis, comparing the results obtained with the activity of a well-characterized olive oil phenol, Hydroxytyrosol (HT). Further, we tested the effects of A009 and HT in an in vivo angiogenesis assay. We found that A009 exerted strong anti-angiogenic effects both in vitro and in vivo, and that the complex natural purified extract has stronger anti-angiogenic potential when compared to the same concentrations of HT in most of the assays performed. These data demonstrate that a novel purified, phenols enriched, extract with anti-angiogenic, and angiopreventive potential can be obtained from olive oil mill waste material, recovering useful products from an agricultural waste.

Effects of the olive oil phenol metabolite 3,4-DHPEA-EDAH2 on human erythrocyte oxidative damage.

Red blood cells (RBCs), as anucleated cells, have poor repair and biosynthetic mechanisms, suffering and accumulating oxidative lesions whenever oxidative stress develops. RBCs are particularly exposed to endogenous oxidative damage because of their specific role as oxygen carriers. However, as the most abundant blood cells, RBCs also play an important role in the oxidative status of the whole blood constituents. In previous studies by our group, the most important polyphenolic compounds found in virgin olive oil, 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-DHPEA-EDA), were shown to significantly protect RBCs from oxidative damage initiated by AAPH and H2O2, with the most active compound being 3,4-DHPEA-EDA. However, the in vivo protective effects of these phenols are dependent on their bioavailability. It has been demonstrated that 3,4-DHPEA-EDA is absorbed by intestinal cells and is then metabolized, yielding a reduced metabolite, 3,4-DHPEA-EDAH2. In order to assess the importance of VOO phenolic compound metabolites for the overall in vivo protective activity, the capacity of this phase I metabolite to protect RBCs in the presence of the radical initiators AAPH or H2O2 was evaluated in the presence and absence of the naturally occurring antioxidant, ascorbic acid. The metabolite was shown to protect RBCs from haemolysis induced by both initiators, in a dose dependent way, after 2 h and 4 h of incubation. The protective effect was however lower than that of the parental compound. The analysis of the membrane proteins of erythrocytes showed that the metabolite can interact with these biological structures.

Synthesis and antioxidant evaluation of isochroman-derivatives of hydroxytyrosol: Structure–activity relationship

Isochroman-derivatives of the natural olive oil phenol hydroxytyrosol (HT) have been synthesised via Oxa-Pictet–Spengler reaction in high yields. Lipophilicity and antioxidant activity were determined to establish the structure–activity relationship of isochromans compared to HT, BHT and α-tocopherol. Antioxidant capacity was tested in two different media: bulk oils, using the Rancimat test, and brain homogenates, by measuring malondialdehyde (MDA) levels as a lipoperoxidation biomarker. In addition, other antioxidant assays (FRAP, ABTS and ORAC) were carried out. Rancimat and MDA results show that antioxidant activity was related with lipophilicity, directly in brain homogenates and inversely in the oils, in agreement with the polar paradox. Free o-diphenolic groups positively determined the activity in the oils, whereas reducing and radical-scavenging activities were related to the number of free hydroxyl moieties. BHT and α-tocopherol showed lower antioxidant activity than isochromans and HT. We conclude that HT-isochromans present significant potential as bioactive compounds.

Synthesis and antioxidant activity of nitrohydroxytyrosol and its acyl derivatives.

A series of nitroderivatives has been synthesized from hydroxytyrosol, the natural olive oil phenol, to increase the assortment of compounds with putative effects against Parkinson's disease. Nitrohydroxytyrosyl esters were obtained from nitrohydroxytyrosol using a chemoselective one-step, high-yield, transesterification procedure. The antioxidant activity of these new series of nitrocatechols was evaluated using FRAP, ABTS, and ORAC assays and compared to that of free hydroxytyrosol. The nitro functional group induced a significant increase in the antioxidant activity of nitrohydroxytyrosol compared to hydroxytyrosol. Regarding nitroester derivatives, variable antioxidant activity was observed depending on the acyl side-chain length; shorter chains maintained or even enhanced the antioxidant activity compared to nitrohydroxytyrosol, decreasing the activity with longer side chains in keeping with their lipophilic nature. Therefore, it may be concluded that nitroester derivatives of hydroxytyrosol, which may be obtained by a simple, high-yield reaction, have elevated antioxidant activity and thus present potential bioactivity.

Preventive Activity of Olive Oil Phenolic Compounds on Alkene Epoxides Induced Oxidative DNA Damage on Human Peripheral Blood Mononuclear Cells

The aim of this study was to investigate the ability of epoxides of styrene (styrene-7,8-oxide; SO) and 1,3-butadiene (3,4-epoxy-1-butene; 1,2:3,4:-diepoxybutane) to cause oxidative stress and oxidative DNA damage on human peripheral blood mononuclear cells (PBMCs) and whether a complex mixture of olive oil phenols (OOPE) could prevent these effects. The DNA damage was measured by the single-cell gel electrophoresis (SCGE; comet assay). We found that the DNA damage induced by alkene epoxides could be prevented by N-acetyl-cysteine (10 mM) and catalase (100 U/ml). Alkene epoxides caused a significant (P < 0.05) increase of both peroxide concentration in extra- and intracellular environment and formamidopyrimidine DNA glycosylase (FPG)- and Endonuclease III (ENDO III)-sensitive sites in PBMCs, demonstrating the presence of oxidized bases. OOPE (1 μg of total phenols/ml) was able to prevent the alkene epoxide induced DNA damage both after 2 and 24 h of incubation. In addition, OOPE completely inhibited the SO-induced intracellular peroxide accumulation in PBMCs and prevented the oxidative DNA damage induced by SO, as evidenced by the disappearance of both FPG- and ENDO III-sensitive sites. This is the first study demonstrating the ability of OOPE to prevent the DNA damage induced by alkene epoxides providing additional information about the chemopreventive properties of olive oil.

Rapid down-regulation of hepatic lipid metabolism by phenolic fraction from extra virgin olive oil.

Regular consumption of extra virgin olive oil (EVOO) is associated with a low incidence of atherosclerotic diseases. The phenolic component contributes to the hypolipidemic action of EVOO, although the biochemical mechanisms leading this beneficial outcome are not fully understood. Since liver plays a pivotal role in the whole body lipid homeostasis, we investigated the short-term effects of EVOO extract, with a high phenol content (HPE), on lipid synthesis in primary rat hepatocytes. Refined olive oil extract, with a low phenol content, was used throughout this study as a control. Olive oil phenols isolated with methanolic extractions were subsequently analyzed by high performance liquid chromatography, electrospray ionization tandem mass spectrometry, and gas chromatography mass spectrometry. Rat hepatocytes were obtained from collagenase perfusion of liver. A colorimetric assay was performed to exclude cytotoxicity of the extracts. Radioenzymatic methods were used in order to investigate hepatic lipid metabolism. HPE, dose- (0.1-50 μg/mL) and time-dependently (0.5-4 h) inhibited both lipogenesis and cholesterogenesis (n = 6, P < 0.05), as well as triglycerides synthesis (n = 5, P < 0.05). We showed that these effects are attributable to a short-term modulation by HPE of the key enzymes implicated in the abovementioned pathways (n = 5, P < 0.05). The decrease in hepatic lipid synthesis may represent a potential mechanism underlying the hypolipidemic effect of EVOO phenols.

Faecal microbial metabolism of olive oil phenolic compounds: in vitro and in vivo approaches.

In the present study, the individual colonic metabolism of the main components of the virgin olive oil phenolic fraction was evaluated by an in vitro model using human faecal microbiota. To assess differences in metabolism related to the molecular structure, four phenolic standards were selected, tyrosol, hydroxytyrosol, hydroxytyrosol acetate and oleuropein. After studying the in vitro colonic metabolism pathways of the individual phenols, the presence of their colonic metabolites was investigated in human faecal samples obtained before and after the sustained intake (3 weeks) of a daily dose of 25 mL of a phenol-enriched olive oil. The in vitro colon fermentation of the four individual phenolic compounds revealed (i) an increase in phenolic acids, (ii) the stability of hydroxytyrosol and tyrosol and (iii) the high degradation of hydroxytyrosol acetate and oleuropein. Additionally, a moderate intake of a phenol-rich olive oil raised the concentration in human faeces of free hydroxytyrosol and phenylacetic and phenylpropionic acids. The products of colonic catabolism of olive oil phenolic compounds could be good candidates for novel preventive strategies and open a promising line of research into the preventive action of olive oil phenols in colon and other bowel diseases.

The effect of olive oil on osteoporosis prevention

The incidence of osteoporosis and associated fractures is found to be lower in countries where the Mediterranean diet is predominant. These observations might be mediated by the active constituents of olive oil and especially phenolic compounds. Objective: To review current knowledge by searching for all relevant publications since 2001 in the MEDLINE, EMBASE and Cochrane Library databases, using the descriptors: Mediterranean diet, virgin olive oil, phenols, bone, osteoblast and osteoporosis. Results and conclusions: Published evidence suggests that olive oil phenols can be beneficial by preventing the loss of bone mass. It has been demonstrated that they can modulate the proliferative capacity and cell maturation of osteoblasts by increasing alkaline phosphatase activity and depositing calcium ions in the extracellular matrix. Further research on this issue is warranted, given the prevalence of osteoporosis and the few data available on the action of olive oil on bone.

Metabolite profiling of olive oil and thyme phenols after a sustained intake of two phenol-enriched olive oils by humans: Identification of compliance markers

An understanding of causal relations between phenol intake and its beneficial effects on health is hindered by the lack of robust biological markers of its exposure. This is particularly relevant in mid/long-term nutritional intervention studies. An analytical methodology based on UPLC–MS/MS has been developed to determine the metabolites of the phenolic compounds from olive oil and thyme in biological fluids after a sustained intake of two phenol-enriched olive oils for their further use as compliance biomarkers. In a randomized, double-blind, controlled, cross-over trial, 33 hypercholesterolemic volunteers received during 3weeks 25mL/day of (1) raw Virgin Olive Oil with a low phenolic content as a control (80mg total phenols / kg oil; VOO), (2) Functional Virgin Olive Oil enriched with its own phenolics (500mg total phenols / kg oil; FVOO), and (3) Functional Virgin Olive Oil enriched with its own phenolics plus complementary phenolics from Thyme (500mg total phenols / kg oil, 50% from olive oil and 50% from thyme respectively; FVOOT). Plasma and 24h-urine samples were collected. The results showed that some hydroxytyrosol (HT) metabolites presented low specificity as biomarkers of intake. However, hydroxytyrosol sulfate and hydroxytyrosol acetate sulfate appeared to be suitable biomarkers for monitoring compliance with olive oil intake as their values in plasma or/and 24-h urine were significantly higher after FVOO compared to baseline pre-intervention concentrations. They were also significantly correlated with the monitored level of compliance. On the other hand, metabolites derived from thyme were more specific, thymol sulfate and hydroxyphenylpropionic acid sulfate being the metabolites with the largest increase in both plasma and 24-h urine, whereas urinary p-cymene-diol glucuronide presented the greatest increase post-treatment. Their urinary excretion values also displayed significant correlations with the level of compliance and they were defined as FVOOT compliance biomarkers. This study enabled robust quantitative and qualitative compliance biomarkers after the ingestion of two phenol-enriched olive oils to be determined and provided a thorough analysis of the true phenolic exposure after a sustained consumption that could be further related to expected biological effects.

Olive oil phenolic compounds decrease the postprandial inflammatory response by reducing postprandial plasma lipopolysaccharide levels

We investigated the molecular mechanisms by which phenolic compounds (phenols) in virgin olive oil reduce the postprandial inflammatory response with the aim of identifying the transcription factor involved and the downstream effects. Olive oil-based breakfasts prepared with virgin olive oil (VOO) with high (398ppm), intermediate (149ppm) and low (70ppm) phenol content were administered to 49 metabolic syndrome patients following a randomized crossover design. The consumption of a high-phenol VOO-based breakfast limited the increase of lipopolysaccharide plasma levels, TLR4, and SOCS3 proteins (p<0.001, p=0.041 and p=0.008, respectively), the activation of NF-κB (p=0.016) and the IL6 (p=0.007 and p=0.048, low and intermediate oil, respectively), IL1B (p=0.002, intermediate oil), and CXCL1 (p=0.001) postprandial gene expression, in peripheral blood mononuclear cells, as compared with the consumption of a breakfast prepared with the same oil but with low or intermediate phenol content. Virgin olive oil phenolic compounds reduce the postprandial inflammatory response in association with postprandial plasma lipopolysaccharide levels.

Graphene quantum dots as sensor for phenols in olive oil

A new method for the determination of the phenol fraction of olive oil is reported. An optical nanosensor based on graphene quantum dots, obtained by pyrolysis of citric acid, was specifically developed for this purpose. The ensuing fluorescence sensing method, which is simple, and highly sensitive and reproducible, was used here to determine gallic acid and oleuropein as model analytes commonly found in olive oils, as well as the phenolic concentration of olive oil real samples. The detection limits were lower than 0.12mgL−1 and the precision, expressed as relative standard deviation, lower than 1.7%.

Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes

Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects.Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (−25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (−49%) = oleuropein (−48%) > tyrosol (−30%). A phenol-induced reduction of triglyceride synthesis was also detected.To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, −41%, = oleuropein, −38%, > tyrosol, −17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil.

Virgin olive oil phenolics extract inhibit invasion of HT115 human colon cancer cells in vitro and in vivo

The decreased cancer risk associated with consumption of olive oil may be due to the presence of phenolics which can modulate pathways including apoptosis and invasion that are relevant to carcinogenesis. We have previously shown that a virgin olive oil phenolics extract (OVP) inhibited invasion of HT115 colon cancer cells in vitro. In the current study we assessed the in vitro effects of OVP (25 μg mL(-1)) on HT115 cell migration, spreading and integrin expression. Furthermore, the anti-metastatic activity of OVP - at a dose equivalent to 25 mg per kg per day for 2, 8 or 10 weeks - was assessed in a Severe Combined ImmunoDeficiency (SCID) Balb-c mouse model. After 24 h OVP did not inhibit cell migration but significantly reduced cell spreading on fibronectin (65% of control; p < 0.05) and expression of a range of α and β integrins was modulated. In vivo, OVP by gavage significantly (p < 0.05) decreased not only tumour volume but also the number of metastases in SCID Balb-c mice. Collectively, the data suggest that - possibly through modulation of integrin expression - OVP decreases invasion in vitro and also inhibits metastasis in vivo.

Biological Activities of Phenolic Compounds of Extra Virgin Olive Oil.

Over the last few decades, multiple biological properties, providing antioxidant, anti-inflammatory, chemopreventive and anti-cancer benefits, as well as the characteristic pungent and bitter taste, have been attributed to Extra Virgin Olive Oil (EVOO) phenols. In particular, growing efforts have been devoted to the study of the antioxidants of EVOO, due to their importance from health, biological and sensory points of view. Hydrophilic and lipophilic phenols represent the main antioxidants of EVOO, and they include a large variety of compounds. Among them, the most concentrated phenols are lignans and secoiridoids, with the latter found exclusively in the Oleaceae family, of which the drupe is the only edible fruit. In recent years, therefore, we have tackled the study of the main properties of phenols, including the relationships between their biological activity and the related chemical structure. This review, in fact, focuses on the phenolic compounds of EVOO, and, in particular, on their biological properties, sensory aspects and antioxidant capacity, with a particular emphasis on the extension of the product shelf-life.

Hydroxytyrosol attenuates tunicamycin‐induced endoplasmic reticulum stress in human hepatocarcinoma cells

Hydroxytyrosol (HT) is a phenolic compound peculiarly abundant in olives and it is being recognized as a protector of LDL from oxidation. In addition to lipid oxidation, one emerging risk factor for cardiovascular disease is ER stress. We tested the effect of HT on the modulation of ER stress in HepG2 cells. HepG2 cells were treated with 1 μM and 5 μM of HT and 100 μM lipoic acid (LA) and glutathione-ethyl ester (GSH), for 24 h. Induction of the unfolded protein response (UPR) was initiated by treatment with 2 μg/mL tunicamycin for 4 h. Real time RT-PCR analyses followed by Western blot and ELISA of different ER stress markers revealed that the protective activities of HT were superior to those of two known thiolic antioxidants, i.e., LA and GSH. Mounting evidence indicates the ER as an important target of dietary or pharmacological intervention. In this paper, we report the modulatory activities of physiological concentrations of HT toward ER stress and we shed some light on pathways alternative to the well-known antioxidant mechanisms, through which olive oil phenolics modulate cell signaling and could impact cardiovascular health and degenerative diseases.

An Up-date of Olive Oil Phenols in Inflammation and Cancer: Molecular Mechanisms and Clinical Implications

Olive oil (OO), the main fatty component of the Mediterranean diet, exhibits numerous biological functions which are beneficial for the state of health. In addition to monounsaturated fatty acid (MUFA) evidences have accumulated on the favorable properties of its minor though highly bioactive components, particularly the phenolic compounds, which have shown a broad spectrum of bioactive properties, including antioxidant and anti-inflammatory effects both associated with the origin of the main chronic diseases. Additional studies have demonstrated that the health effects of olive oil polyphenols have been also associated with their, neuroprotective, antiaging and antiatherogenic effects. On the other hand, because of their ability to modulate cell death, olive polyphenols have been proposed as chemopreventive and therapeutic agents. Thus, the purpose of this article is to review the chemistry, bioavailability and pharmacokinetic characteristics of OO polyphenols, in addition to provide the reader an up-date of their putative antioxidant, anti-inflammatory and anti-cancer activities as well as the plausible action mechanisms involved.

Quantitation of hydroxytyrosol in food products using a sequential injection analysis fluorescence optosensor

Hydroxytyrosol (HXT), one of the most important phenols in olive oil, has been reported to present beneficial effects for human health, such as antioxidant and antimicrobial properties and beneficial effects on the cardio-vascular system. As a result, exhaustive research is being carried out in order to use this compound in enriched foods, such as tomato juice or milk. In this paper, we propose a sequential injection analysis (SIA) optosensor for the fluorimetric (λexc/λem=281/318nm) determination of hydroxytyrosol in several foods. The use of a solid support in the flow-cell increases the sensitivity of the system (detection limit of 0.6μgmL−1) and, especially, the selectivity. In this way, using a QuEChERS extraction procedure, the proposed method was applied to the determination of the analyte in hydroxytyrosol-enriched foods (tomato juice/soup, milk and infant formula). The recoveries (100±10%) and relative standard deviations obtained (3–5%) show the potentiality of the developed system as an alternative to chromatographic methods.

Proteomics analysis inCaenorhabditis elegansto elucidate the response induced by tyrosol, an olive phenol that stimulates longevity and stress resistance

Tyrosol (TYR, 2-(4-hydroxyphenyl)ethanol), one of the main phenols in olive oil and olive fruit, significantly strengthens resistance to thermal and oxidative stress in the nematode Caenorhabditis elegans and extends its lifespan. To elucidate the cellular functions regulated by TYR, we have used a proteomic procedure based on 2DE coupled with MS with the aim to identify the proteins differentially expressed in nematodes grown in a medium containing 250 μM TYR. After the comparison of the protein profiles from 250 μM TYR and from control, 28 protein spots were found to be altered in abundance (≥twofold). Analysis by MALDI-TOF/TOF and PMF allowed the unambiguous identification of 17 spots, corresponding to 13 different proteins. These proteins were as follows: vitellogenin-5, vitellogenin-2, bifunctional glyoxylate cycle protein, acyl CoA dehydrogenase-3, alcohol dehydrogenase 1, adenosylhomocysteinase, elongation factor 2, GTP-binding nuclear protein ran-1, HSP-4, protein ENPL-1 isoform b, vacuolar H ATPase 12, vacuolar H ATPase 13, GST 4. Western-blot analysis of yolk protein 170, ras-related nuclear protein, elongation factor 2, and vacuolar H ATPase H subunit supported the proteome evidence.

Mass spectrometry to evaluate the effect of the ripening process on phenols of virgin olive oils

SummaryA global approach has been developed to study the influence of ripening of olive fruits on the phenolic fraction present in virgin olive oil from different genotypes of olives. For this purpose, a non‐targeted method based on the analysis of phenolic extracts by MS in high resolution mode was applied. The phenolic profiles obtained by liquid chromatography ‐ quadrupole‐quadrupole‐time‐of‐flight (LC‐QqTOF) were compared by statistical multivariate analysis tools such as principal component analysis and heat correlation maps. In overall terms, discrimination was only observed for specific crop dates and ripening index values, which should be ascribed to a strong influence of the genotype. The ripening process of each genotype was also studied to demonstrate the variation of phenolic profile in relation to the ripening index. This study was also extended to a panel of representative phenols appreciated by their health and nutritional properties. The application of omics technologies to plant breeding programs could be considered as one of the main pillars on which improvement of products quality can be supported.Practical applications: Olive oil phenols are of a great interest since they contribute to autoxidation stability, health properties, and organoleptic characteristics of virgin olive oil. These phenols are considered a key to assess virgin olive oil quality. This study is focused on the effect of the ripening process on the phenolic fraction from a metabolomics point of view. This advanced omics technology (foodomics) applied to plant breeding programs can be one of the pillars to improve products quality.

Zeytinyağı ve Sağlık: Biyoaktif Bileşenleri, Antioksidan Özellikleri ve Klinik Etkileri

Mediterranean countries have lower rates of mortality from cardiovascular disease and cancer than other European countries. Olive oil is the major source of dietary fat in the Mediterranean diet, and its regular consumption is thought to have various beneficial effects on human health. The biological features ascribed to olive oil consumption are associated in part to its phenolics constituents, and mainly linked to the direct or indirect antioxidant activity of olive oil phenolics and other components such as oleic acid and squalene. The major phenolic compounds identified and quantified in olive oil belong to three groups which all of them have potent antioxidant properties; (oleuropein) and the lignans. Among these substances the last two classes include the most concentrate phenols of virgin olive oil. As observed in traditional Mediterranean populations, it has been confirmed that extra virgin olive oil is beneficial when consumed properly. Many evidence indicates, however, that olive oil and its components contribute significantly to the health benefits of the Mediterranean diet, with more of an effect on prevention than treatment. This paper summarizes the evidence supporting the potentially beneficial effects of olive oil phenolics and other bioactive components