Roberto Fabiani

Phenolic compounds in olive oil: antioxidant, health and organoleptic activities according to their chemical structure

Abstract.Hydrophilic phenols are the most abundant natural antioxidants of virgin olive oil (VOO), in which, however, tocopherols and carotenes are also present. The prevalent classes of hydrophilic phenols found in VOO are phenolic alcohols and acids, flavonoids, lignans and secoiridoids. Among these substances the last two classes include the most concentrate phenols of VOO. Secoiridoids, like aglycone derivatives of oleuropein, demethyloleuropein and ligstroside, are present in olive fruit as most abundant VOO phenolic antioxidants. Several important biological properties (antioxidant, anti-inflammatory, chemopreventive and anti-cancer) and the characteristic pungent and bitter tasty properties have been attributed to VOO phenols. Relationships between polyphenols activities and their chemical structures are discussed in this paper.

Cancer chemoprevention by hydroxytyrosol isolated from virgin olive oil through G1 cell cycle arrest and apoptosis.

Recent epidemiological evidence and animal studies suggest a relationship between the intake of olive oil and a reduced risk of several malignancies. The present study assesses the effect of hydroxytyrosol, a major antioxidant compound of virgin olive oil, on proliferation, apoptosis and cell cycle of tumour cells. Hydroxytyrosol inhibited proliferation of both human promyelocytic leukaemia cells HL60 and colon adenocarcinoma cells HT29 and HT29 clone 19A. The con-centrations of hydroxytyrosol which inhibited 50% of cell proliferation were ∼50 and ∼750 μmol/l for HL60 and both HT29 and HT29 clone 19A cells, respectively. At concentrations ranging from 50 to 100 μmol/l, hydroxytyrosol induced an appreciable apoptosis in HL60 cells after 24 h of incubation as evidenced by flow cytometry, fluorescence microscopy and internucleosomal DNA fragmentation. Interestingly, no effect on apoptosis was observed after similar treatment of freshly isolated human lymphocytes and polymorphonuclear cells. The DNA cell cycle analysis, quantified by flow cytometry, showed that the treatment of HL60 cells with hydroxytyrosol 50–100 μmol/l arrested the cells in the G0/G1 phase with a concomitant decrease in the cell percentage in the S and G2/M phases. These results support the hypothesis that hydroxytyrosol may exert a protective activity against cancer by arresting the cell cycle and inducing apoptosis in tumour cells, and suggest that hydroxytyrosol, an important component of virgin olive oil, may be responsible for its anticancer activity.

Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes

Recent in vitro and in vivo studies suggest that the anti-inflammatory properties of extra virgin olive oil may be involved in the prevention of chronic degenerative diseases. In this study, the ability of olive oil phenols to influence the release of superoxide anions (O2-), prostaglandin E2 (PGE2) and tumor necrosis factor α (TNFα) and the expression of cyclooxygenase2 (COX2) in human monocytes, freshly isolated from healthy donors, was investigated. O2- were measured by superoxide dismutase-inhibitable cytochrome c reduction and PGE2 and TNFα production were determined in culture medium with appropriate enzyme immunoassay kits. COX2 mRNA and protein were evaluated by quantitative reverse transcription-polymerase chain reaction and Western immunoblotting, respectively. Treatment of monocytes for 24 h with 100 μM of hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and their secoiridoid derivatives (3,4-DHPEA and p-HPEA linked to the dialdehydic form of elenolic acid: 3,4-DHPEA-EDA and p-HPEA-EDA, respectively) significantly (P<.05) inhibited the production of O2(-) as follows: 3,4-DHPEA (40%,), p-HPEA (9%), 3,4-DHPEA-EDA (25%) and p-HPEA-EDA (36%). Hydroxytyrosol also considerably reduced the expression of COX2 at both the mRNA and protein level (P<.05) and caused a clear dose-dependent reduction of PGE2 released into the culture medium (45% and 71% at 50 and 100 μM, respectively, P<.05). The COX2 mRNA was also efficiently inhibited by the secoiridoids. Moreover, it was shown that hydroxytyrosol increased the monocytes TNFα production. In addition to other chemopreventive properties, these results suggest that the health effects of olive oil phenols may be related to their ability to modulate the production of pro-inflammatory molecules, a property common to non-steroidal anti-inflammatory drugs.

Genotoxic effect of bile acids on human normal and tumour colon cells and protection by dietary antioxidants and butyrate

BackgroundColorectal cancer is the second cause of death for tumour worldwide. Among the risk factors for this disease the dietary habits seem to have a pivotal role. An elevated intake of fats causes a high release in the gut lumen of bile acids that are positively correlated with colorectal cancer, since they act as detergents and proliferation promoters. Recently, it was evidenced that bile acids can also be able to induce DNA damage.Aim of the studyIn this study the genotoxicity of deoxycholic acid (DCA) and chenodeoxycholic acid CDCA) has been evaluated in human normal colonocytes derived from 60 colon biopsies and in tumour cells. The involvement of reactive oxygen species (ROS) and the oxidative DNA damage was assessed. In addition, the protective effect exerted by both two well-known antioxidants commonly present in the diet, β-carotene and α-tocopherol, and butyrate which is known to be involved in the regulation of several cellular functions, has also been tested.MethodsThe DNA damage was evaluated by the “comet assay” or single cell gel electrophoresis (SCGE) both in its conventional use and by the Endonuclease III modified method, which allow to detect the presence of oxidized pyrimidines.ResultsBile acids (CDA and CDCA) resulted genotoxic on both normal and tumour human colon cells. The inclusion of the endonuclease III digestion step in the comet assay demonstrated that bile acids induced an oxidative DNA damage. In addition, treatment of colonocytes with bile acids in the presence of the antioxidants (β-carotene, α-tocopherol) and Na-butyrate caused a reduction of DNA damage.ConclusionOur results suggest that bile acids may be involved in the tumour initiation by inducing a DNA oxidative damage, and so add further evidences to the preventive properties of antioxidants present in the Mediterranean diet.

Fusion of prostasomes to human spermatozoa stimulates the acrosome reaction

OBJECTIVE To determine the effect of the fusion of prostasomes to spermatozoa on the acrosome reaction. DESIGN In vitro study of human spermatozoa. SETTING Healthy volunteers in an academic research environment. PATIENT(S) Healthy volunteer men, 25 to 35 years old. INTERVENTION(S) Human semen was fractionated into spermatozoa and prostasomes. Fusion of prostasome to spermatozoa was performed at pH 5.5. Progesterone (1 microM) was added when required. MAIN OUTCOME MEASURE(S) Evaluation of the acrosome reaction by fluorescence microscopy. RESULTS(S) The percentage of spontaneously acrosome-reacted cells was very low unless the Ca(2+)-ionophore A 23187 was added. The treatment of spermatozoa with 1 microM of progesterone scarcely affected the acrosome reaction; a pretreatment in conditions permitting fusion increased it. The addition of progesterone to prostasome-fused spermatozoa further increased the extent of the acrosome reaction. CONCLUSION(S) The H(+)-dependent fusion with prostasomes makes spermatozoa more sensitive to the effect of progesterone on acrosome-reaction induction.

Production of hydrogen peroxide is responsible for the induction of apoptosis by hydroxytyrosol on HL60 cells

Hydroxytyrosol [3,4-dihydroxyphenylethanol (3,4-DHPEA)], a phenolic compound found exclusively in olive oil, exerts growth-suppressive and pro-apoptotic effects on different cancer cells. Although some molecular mechanisms involved in the pro-apoptotic activity of 3,4-DHPEA have been proposed, the initial stress signals responsible of this phenomenon are not known. Our aim was to assess the involvement of reactive oxygen species as mediators of apoptosis induced by 3,4-DHPEA on HL60 cells. Apoptosis was determined by analyzing the nuclear fragmentation by both fluorescence microscopy and flow cytometry. The externalization of phosphatidylserine was evidenced using an Annexin V-FITC kit. The concentration of H(2)O(2) in the culture medium was measured by the ferrous ion oxidation-xylenol orange method. The pro-apoptotic effect of 3,4-DHPEA (100 muM) was prevented by N-acetyl-cysteine, ascorbate, and alpha-tocopherol. Catalase suppressed the 3,4-DHPEA-induced apoptosis, while the Fe(II)-chelating reagent o-phenantroline showed no effect, suggesting the involvement of H(2)O(2 )but not of OH(*). Indeed, 3,4-DHPEA caused accumulation of H(2)O(2) in the culture medium. Tyrosol (p-hydroxyphenylethanol) and caffeic acid, compounds structurally similar to 3,4-DHPEA but not able to generate H(2)O(2), did not induce an appreciable apoptotic effect. This is the first study demonstrating that apoptosis induction by 3,4-DHPEA is mediated by the extracellular production of H(2)O(2).

Anti-proliferative and pro-apoptotic activities of hydroxytyrosol on different tumour cells: the role of extracellular production of hydrogen peroxide

PurposeSeveral recently published data suggest that the anti-proliferative and pro-apoptotic properties of hydroxytyrosol [3,4-dihydroxyphenyl ethanol (3,4-DHPEA)] on HL60 cells may be mediated by the accumulation of hydrogen peroxide (H2O2) in the culture medium. The aim of this study was to clarify the role played by H2O2 in the chemopreventive activities of 3,4-DHPEA on breast (MDA and MCF-7), prostate (LNCap and PC3) and colon (SW480 and HCT116) cancer cell lines and to investigate the effects of cell culture medium components and the possible mechanisms at the basis of the H2O2-producing properties of 3,4-DHPEA.MethodsThe proliferation was measured by the MTT assay and the apoptosis by both fluorescence microscopy and flow cytometry. The concentration of H2O2 in the culture medium was measured by the ferrous ion oxidation–xylenol orange method.ResultsIt was found that the H2O2-inducing ability of 3,4-DHPEA is completely prevented by pyruvate and that the exposure of cells to conditions not supporting the H2O2 accumulation (addition of either catalase or pyruvate to the culture medium) inhibited the anti-proliferative effect of 3,4-DHPEA. Accordingly, the sensitivity of the different cell lines to the anti-proliferative effect of 3,4-DHPEA was inversely correlated with their ability to remove H2O2 from the culture medium. With regard to the mechanism by which 3,4-DHPEA causes the H2O2 accumulation, it was found that superoxide dismutase increased the H2O2 production while tyrosinase, slightly acidic pH (6,8) and absence of oxygen (O2) completely prevented this activity. In addition, different transition metal-chelating compounds did not modify the H2O2-producing activity of 3,4-DHPEA.ConclusionsThe pro-oxidant activity of 3,4-DHPEA deeply influences its ‘in vitro’ chemopreventive activities. The main initiation step in the H2O2-producing activity is the auto-oxidation of 3,4-DHPEA by O2 with the formation of the semiquinone, superoxide ions (O2−) and 2H+.

Nutrigenomics of extra-virgin olive oil: A review

Nutrigenomics data on the functional components of olive oil are still sparse, but rapidly increasing. Olive oil is the main source of fat and health‐promoting component of the Mediterranean diet. Positive effects have been observed on genes involved in the pathobiology of most prevalent age‐ and lifestyle‐related human conditions, such as cancer, cardiovascular disease and neurodegeneration. Other effects on health‐promoting genes have been identified for bioactive components of olives and olive leafs. Omics technologies are offering unique opportunities to identify nutritional and health biomarkers associated with these gene responses, the use of which in personalized and even predictive protocols of investigation, is a main breakthrough in modern medicine and nutrition. Gene regulation properties of the functional components of olive oil, such as oleic acid, biophenols and vitamin E, point to a role for these molecules as natural homeostatic and even hormetic factors with applications as prevention agents in conditions of premature and pathologic aging. Therapeutic applications can be foreseen in conditions of chronic inflammation, and particularly in cancer, which will be discussed in detail in this review paper as major clinical target of nutritional interventions with olive oil and its functional components.

Influence of culture conditions on the DNA-damaging effect of benzene and its metabolites in human peripheral blood mononuclear cells

The DNA‐damaging ability of benzene and its metabolites on peripheral blood mononuclear cells (PBMC) has been investigated by using the alkaline comet assay. The PBMC were incubated with different compounds in two different media for 2 and 24 hr at concentrations that did not affect cell viability and the DNA damage was quantified by a computerized image analysis system. Benzene and phenol (5 mM) did not show any genotoxic activity after 2 hr of incubation in the two media tested, phosphate‐buffered saline (PBS) and RPMI containing 5% of heat‐inactivated fetal calf serum (RPMI + 5% FCS), whereas phenol was genotoxic and cytotoxic at 10 mM after 24 hr of incubation in RPMI + 5% FCS. All other benzene metabolites were genotoxic at micromolar concentrations when incubated in PBS with the following decreasing order of potency: benzenetriol, catechol, hydroquinone, and benzoquinone. When the PBMC were incubated in RPMI + 5% FCS, the effect of catechol (200–600 μM) and benzenetriol (10 μM) was reduced, whereas the genotoxicity of benzenetriol at high concentrations (50–100 μM) and hydroquinone (150–2500 μM) was not affected. In contrast, the effect of benzoquinone at 5 and 10 μM was greatly enhanced when the cells were incubated in RPMI + 5% FCS. This effect resulted mainly from the presence of serum in the medium and it was almost completely inhibited by boiling the serum (100°C, 5 min) and was partially reduced by extensive dialysis. Benzoquinone was the most damaging compound when tested under more physiological conditions, thereby supporting the general observation that it is the most myelotoxic benzene metabolite. Environ. Mol. Mutagen. 37:1–6, 2001

Deoxycholic acid and SCFA-induced apoptosis in the human tumor cell-line HT-29 and possible mechanisms

Short chain fatty acids (propionate and butyrate) and deoxycholic acid (DCA) are able to induce apoptosis in HT-29 colonic tumor cell line, but DCA induces a much higher level of apoptosis than butyrate and propionate. Mixtures of DCA with butyrate or propionate enhance the effect of the single components. Apoptosis is not affected by the PKC, PTK or de novo mRNA and protein synthesis inhibitors, so that the involvement of these enzymes and processes is ruled out. In contrast, DCA-induced apoptosis is directly related to [Ca2+]i concentration as demonstrated by the apoptosis inhibition caused by [Ca2+]i chelator BAPTA/AM.