Dihydrocaffeic acid,a major microbial metabolite of chlorogenic acids,shows similar protective effect than a yerba mate phenolic extract against oxidative stress in HepG2 cells

The hepatoprotective effect of a yerba mate phenolic extract (YMPE), rich in chlorogenic acids, and its main circulating metabolites dihydrocaffeic (DHCA) and dihydroferulic (DHFA) acids were assessed in human hepatoma HepG2 cells subjected to oxidative damage induced by tert-butylhydroperoxide (t-BOOH). Direct treatment of HepG2 cells with realistic concentrations of YMPE (1, 10 and 50 μg/mL), DHCA or DHFA (0.2, 1, 10 μM) for 20 h was not cytotoxic and significantly decreased ROS generation. Pre-treatment with YMPE and DHCA prevented the cytotoxicity and macromolecular damage induced by t-BOOH. Moreover, decreased levels of reduced glutathione (GSH), and increased ROS levels and antioxidant enzyme activity induced by t-BOOH were dose-dependently recovered. DHFA only showed a slight protection against cell cytotoxicity, lipid oxidation and GSH depletion. In conclusion, YMPE and one of its major microbial metabolites, DHCA, confer significant protection against oxidative damage, adding evidences to the beneficial health effects associated with mate intake.     

Assessment of the ability of seaweed extracts to protect against hydrogen peroxide and tert-butyl hydroperoxide induced cellular damage in Caco-2 cells

The ability of brown seaweed extracts, Ascophyllum nodosum, Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus and Fucus serratus to protect against tert-butyl hydroperoxide (tert-BOOH) induced stress in Caco-2 cells was investigated. Oxidative stress was determined by measuring alteration in the enzymatic activity of catalase (CAT) and superoxide dismutases (SOD) and cellular levels of glutathione (GSH). L. hyperborea, P. canaliculata and F. serratus significantly protected against tert-BOOH induced SOD reduction but did not protect against the reduction in CAT activity or the increased cellular levels of GSH. The ability of F. serratus and F. vesiculosus to protect against H₂O₂ and tert-BOOH induced DNA damage was also assessed. The DNA protective effects of the two seaweed extracts was compared to those of three metal chelators; deferoxamine mesylate (DFO), 1,10-phenanthroline (o-phen) and 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (BAPTA-AM). F. serratus and F. vesiculosus significantly protected (P < 0.05) against H₂O₂ (50 μM) induced DNA damage but not tert-BOOH induced damage.     

Direct determination of phenolic compounds and phospholipids in virgin olive oil by micellar liquid chromatography

A simple HPLC method is reported for fast separation and determination of phenolic compounds (tyrosol, caffeic acid, p-coumaric acid and oleuropeina) and phospholipids (phosphatidylethanolamine and phosphatidylcholine) in virgin olive oil samples. The samples were diluted with 2-propanol and injected into the column directly without previous extraction. Samples with an olive oil content of up to 65% were injected without any problems. The analytes were separated on a C-18 column by a micellar mobile phase containing 0.07 M SDS and 2.5% 2-propanol at pH 3, and were detected at 210 nm. Linear calibration curves [r² > 0.997] were obtained with detection limits ranging from 0.052 to 0.16 μg/g and 1 to 8.6% repeatability for the phenolic compounds. Several virgin olive oil samples were analysed and the recovery values were around 110%.     

Antifungal activity of the honey flavonoid extract against Candida albicans

Candida albicans is the most prevalent dimorphic pathogen in humans. We investigated the potential protective effect of honey flavonoid extract (HFE) on Candida dimorphism induced by RPMI 1640 medium. Yeast to hyphal transition was dose dependently prevented when Candida cultures were treated with HFE for 6 h (germ-tube formation) and 18 h (hypha elongation). Since during hyphal growth remarkable amounts of reactive oxygen species (ROS) are generated, we investigated whether HFE affects the generation of ROS, the glutathione level and the activity of glutathione-dependent enzymes. Treatment of Candida cells with 48 μg/ml (MIC₅₀) of HFE for 6 or 18 h inhibited the dimorphic conversion by supporting of the intracellular glutathione level. HFE exerts a dual protective effect inhibiting both ROS generation during germ-tube formation and γ-glutamyltranspeptidase activity, responsible for GSH degradation, during hypha elongation. These results show that HFE confers a significant protection against yeast to hyphal transition of C. albicans.     

The effect of citric acid on the phenolic contents of olive oil

Response surface methodology was used to optimise the combined effects of malaxation time (t) and aqueous citric acid solution (C_A) added at the beginning of the malaxation step on total polyphenols (TP) and o-diphenols (OD) content and the antiradical power (ARP) of the olive oil extracted from the Italian olive fruits of Coratina cultivar. Different tests were performed according to a 3² full factorial design, varying t from 30 to 90 min and the C_A from 5 to 15 ml/kg_paste. Overall optimal conditions identified by a numerical optimisation for the three responses were found to be at t = 30 min and C_A = 13.79 ml/kg_paste under which the model predicted TP = 604.52 μgCAE/g_oil, OD = 80.44 μgCAE/g_oil and ARP = 28.73 μgDPPH/μlextract. There were also linear correlations between TP (R² = 0.8176) and OD (R² = 0.8633) values of olive oil and waste water. The results of this study demonstrate that considerably short malaxation time and relatively small amounts of citric acid were required to enhance the quality of olive oil. The outcome of our study will therefore be of great value for the commercial production of olive oil with high level of polyphenols and o-diphenols.     

Physicochemical characterisation and oxidative stability of fish oil and fish oil–extra virgin olive oil microencapsulated by sugar beet pectin

Spray-dried microcapsules were prepared at 25% and 50% w/w oil load from sugar beet pectin-stabilised emulsions (pH 3) containing fish oil, and a blend of fish oil and with extra virgin olive oil (1:1 w/w). Microencapsulation efficiencies were high (≥90%). However, deterioration in microcapsule wall integrity and an increase in oil droplet size were observed during storage (25 °C, 0–3 months). Lipid oxidation increased with both increased oil load (P < 0.05) and storage duration (P < 0.05), but was independent of oil composition (P > 0.05). These results suggest that sugar beet pectin functions poorly as a wall material and its residual metal ions exacerbate omega-3 oxidation, despite the presence of endogenous antioxidants found in extra virgin olive oil. Interestingly, under accelerated storage conditions (OxiPres® at 80 °C, 0.5 bar oxygen pressure), microcapsules containing the oil blend showed the best oxidative stability (P < 0.05), irrespective of oil load. A possible explanation for the superior oxidative stability of the microencapsulated oil blend at high storage temperature is discussed.     

Cytoprotective effects of pu-erh tea on hepatotoxicity in vitro and in vivo induced by tert-butyl-hydroperoxide

The in vitro and in vivo protective effects of water extract of pu-erh tea (WEPT) on tert-butyl-hydroperoxide (t-BHP)-induced oxidative damage in hepatocytes of HepG2 cells and in rat livers were investigated. After treatment with 200 μg/ml of samples, the survival rate of HepG2 cells induced by t-BHP increased. WEPT concentration-dependently inhibited reactive oxygen species (ROS) generation in HepG2 cells in response to the oxidative challenge induced by t-BHP. Administration of WEPT (0.2, 0.5 and 1.0 g/kg of body weigh) to rats for 56 consecutive days before a single dose of t-BHP (0.5 mmol/kg, i.p.) exhibited a significant (p < 0.01) protective effect by lowering serum levels of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), as well as reducing the formation of malondialdehyde. Taken together, these results demonstrate that WEPT is able to protect against hepatic damage in vitro and in vivo, suggesting that the drinking of pu-erh tea may protect liver tissue from oxidative damage.     

Comparison of protective effects of three varieties of sugarcane leaves on oxidative stress in Clone 9 cells

The protective effects of water extracts of sugarcane (Saccharum officinarum L.) leaves among three varieties, including 28NG256, wild type, and ROC10, on t-BHP-induced oxidative stress in Clone 9 cells were systematically compared. Among these three varieties, 28NG256 showed the highest protective effect against 0.2 mM t-BHP-induced oxidative stress in Clone 9 cells. In addition, 28NG256 displayed higher inhibitory effects on ROS generation than wild type and ROC10. Moreover, 28NG256 showed higher positive regulated GSH levels and antioxidant enzymes as well as higher protective potential against cell death by inhibiting caspase-3 activity and mitochondrial membrane depolarization. Chlorogenic and caffeic acids present in 28NG256 decreased significantly the generation of ROS, which may partly be responsible for the effect of Clone 9 cell growth. Thus, 28NG256, among the three varieties studied, showed the most protective effects against t-BHP-induced oxidative stress in Clone 9 cells.     

A new hydroxytyrosol metabolite identified in human plasma: Hydroxytyrosol acetate sulphate

We report progress in the study of olive oil phenolic metabolites in humans and identify a new hydroxytyrosol metabolite called hydroxytyrosol acetate sulphate, which was determined using tandem MS, after ingestion of 30 ml of olive oil with a high phenolic content (500 mg/kg oil), reaching a maximum concentration of 1.63 μM. In order to understand and explain the generation of this metabolite, two different pathways are proposed.     

High performance size-exclusion chromatography analysis of polar compounds applied to refined, mild deodorized, extra virgin olive oils and their blends: An approach to their differentiation

An investigation was carried out to evaluate the use of High Performance Size-Exclusion Chromatography (HPSEC) of polar compounds of refined, mild deodorized, extra virgin olive oils as well as of their blends, in attempting to reveal significant differences in the amounts of the substance classes constituting polar compounds among these oils. Two sets of blends were prepared by mixing an extra virgin olive oil with both refined and mild deodorized olive oils in increasing amounts. The obtained data highlighted that the triacylglycerol oligopolymers were absent or present in traces in the extra virgin olive oil, while their mean amount was equal to 0.04 g/100 g and 0.72 g/100 g in mild deodorized and refined olive oils, respectively. Oxidized triacylglycerols and diacylglycerols were more abundant in mild deodorized oil and refined oil than in extra virgin olive oil. The Factorial Discriminant Analysis of the data showed that the HPSEC analysis could reveal the presence of refined/mild deodorized oils in extra virgin olive oils. In particular, the classification functions obtained allowed designation of mixtures containing at least 30 g/100 g of mild deodorized oil and all those containing refined olive oil as deodorized oil, therefore as oils subjected to at least a mild refining treatment.     

Chemical characterization and chemo-protective activity of cranberry phenolic powders in a model cell culture. Response of the antioxidant defenses and regulation of signaling pathways

Oxidative stress and reactive oxygen species (ROS)-mediated cell damage are implicated in various chronic pathologies. Emerging studies show that polyphenols may act by increasing endogenous antioxidant defense potential. Cranberry has one of the highest polyphenol content among commonly consumed fruits. In this study, the hepato-protective activity of a cranberry juice (CJ) and cranberry extract (CE) powders against oxidative stress was screened using HepG2 cells, looking at ROS production, intracellular non-enzymatic and enzymatic antioxidant defenses by reduced glutathione concentration (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) activity and lipid peroxidation biomarker malondialdehyde (MDA). Involvement of major protein kinase signaling pathways was also evaluated. Both powders in basal conditions did not affect cell viability but decreased ROS production and increased GPx activity, conditions that may place the cells in favorable conditions against oxidative stress. Powder pre-treatment of HepG2 cells for 20 h significantly reduced cell damage induced by 400 μM tert-butylhydroperoxide (t-BOOH) for 2 h. Both powders (5–50 μg/ml) reduced t-BOOH-induced increase of MDA by 20% (CJ) and 25% (CE), and significantly reduced over-activated GPx and GR. CE, with a significantly higher amount of polyphenols than CJ, prevented a reduction in GSH and significantly reduced ROS production. CJ reversed the t-BOOH-induced increase in phospho-c-Jun N-terminal kinase. This study demonstrates that cranberry polyphenols may help protect liver cells against oxidative insult by modulating GSH concentration, ROS and MDA generation, antioxidant enzyme activity and cell signaling pathways.     

Protective effects of sweet orange (Citrus sinensis) peel and their bioactive compounds on oxidative stress

Protective effects of sweet orange (Citrus sinensis) peel and their bioactive compounds on oxidative stress were investigated. According to HPLC-DAD and HPLC-MS/MS analysis, hesperidin (HD), hesperetin (HT), nobiletin (NT), and tangeretin (TT) were present in water extracts of sweet orange peel (WESP). The cytotoxic effect in 0.2 mM t-BHP-induced HepG2 cells was inhibited by WESP and their bioactive compounds. The protective effect of WESP and their bioactive compounds in 0.2 mM t-BHP-induced HepG2 cells may be associated with positive regulation of GSH levels and antioxidant enzymes, decrease in ROS formation and TBARS generation, increase in the mitochondria membrane potential and Bcl-2/Bax ratio, as well as decrease in caspase-3 activation. Overall, WESP displayed a significant cytoprotective effect against oxidative stress, which may be most likely because of the phenolics-related bioactive compounds in WESP, leading to maintenance of the normal redox status of cells.     

Stability of avocado oil during heating: Comparative study to olive oil

The stability of the saponifiable and unsaponifiable fractions of avocado oil, under a drastic heating treatment, was studied and compared to that of olive oil. Avocado and olive oil were characterised and compared at time 0 h and after different times of heating process (180 °C). PUFA/SFA (0.61 at t = 0) and ω-6/ω-3 (14.05 at t = 0) were higher in avocado oil than in olive oil during the whole experiment. Avocado oil was richer than olive oil in total phytosterols at time 0 h (339.64; 228.27 mg/100 g) and at 9 h (270.44; 210.30 mg/100 g) of heating. TBARs was higher in olive oil after 3 h, reaching the maximum values in both oils at 6 h of heating treatment. Vitamin E was higher in olive oil (35.52 vs. 24.5 mg/100 g) and it disappeared earlier in avocado oil (at 4 vs. 5 h). The stability of avocado oil was similar to that of olive oil.     

Impact of olive oil phenolic concentration on human plasmatic phenolic metabolites

Three different functional phenol-enriched virgin olive oils (FVOO) were prepared with a phenolic content of 250 (L-FVOO), 500 (M-FVOO), and 750 mg (H-FVOO) total phenols/kg. In a randomised, cross-over study with 12 healthy volunteers, the pharmacokinetics of phenolic biological metabolites was assessed. An increasing linear trend was observed for hydroxytyrosol sulfate, the main phenolic metabolite quantified in plasma, with C_max values of 1.35, 3.32, and 4.09 μmol/l, and AUC mean values of 263.7, 581.4, and 724.4 μmol/min for L-FVOO, M-FVOO, and H-FVOO, respectively. From our data an acute intake of phenol-enriched olive oils promotes a dose-dependent response of phenol conjugate metabolites in human plasma. Also, we point out for the first time hydroxytyrosol acetate sulfate as a main biological metabolite of hydroxytyrosol from olive oil ingestion.     

Waxy fraction containing long-chain aliphatic aldehydes in virgin olive oils

Long-chain aliphatic aldehydes are natural minor components occurring in the cuticle of numerous plant species and also evidenced in virgin olive oils. The fraction containing these compounds can be isolated from the oil samples by using a solid-phase extraction silica-gel cartridge and then directly analysed by GC on a 5% diphenyl-95% dimethylsiloxane capillary column, using an on column-injection system. The proposed methodology showed that extra virgin olive oils contain long-chain aliphatic aldehydes, with even carbon-atom numbers from C₂₂ to C₃₀. Quantitative results, using the synthesised aldehyde C₂₁ as internal standard, give concentrations of total long-chain aliphatic aldehydes in a variable range below 116 mg kg⁻¹, being hexacosanal (C₂₆-al) the most abundant aldehyde. The different experimental conditions utilised during olive oil extraction processes influence the total aldehydes concentration. Besides contribution to the knowledge of the minor-component composition present in olive oil, their interest and relationship with wax esters, aliphatic alcohols and n-alkanes are discussed.     

Determination of acrylamide in potato chips by a reversed-phase LC–MS method based on a stable isotope dilution assay

Potato-based products represent an important part of the daily intake of food-derived acrylamide, mainly on adolescent population from western countries. A reversed-phase liquid chromatography-mass spectrometry based on a stable isotope dilution assay was used for acrylamide analysis. Aqueous sample extraction, cleaning with Carrez solution and solid phase extraction with methanol was applied. The ratio potato/NaCl solution is critical during extraction where the optimum ratio is 0.125 g/ml NaCl 2 M solution. The use of virgin olive oil, as retaining matrix, during methanol desiccation was critical to achieve high recoveries. The method performance was validated for limit of detection (23.2 μg/kg) and quantitation (91.8 μg/kg), linearity (r > 0.999, 25–1000 μg/kg), recovery (98.8%). The method was applied on commercial potato chips; the intra-day repeatability was set at 3.9% and values were corrected with a labeled internal standard (¹³C₃-acrylamide). No significant differences on the acrylamide content were observed between industrial-scale and local-scale processed potato chips.     

Determination of herbicide residues in olive oil by gas chromatography–tandem mass spectrometry

This paper reports a method for the analysis in olive oil of multiresidues of the herbicides of low-medium polarity most widely used by Andalusian olive growers. The method, which uses gas chromatography/tandem mass spectrometry (GC–MS/MS), was developed within the framework of Project CAO00-005, which spanned the period from 2000 to 2004. The results obtained for more than 3000 samples of virgin olive oil and organic olive oil analyzed over such a period are reported. Samples were extracted with an acetonitrile/n-hexane mixture and cleaned up by passage through Florisil columns prior to analysis. A linear determination range for the herbicides from 1 to 500 μg kg⁻¹ and a correlation coefficient better than 0.996 were achieved. The reproducibility, as relative standard deviation, was quite acceptable (8–11%), and so were herbicide recoveries (90–102%). The proposed method has been transferred to both public and private laboratories in the Andalusian region.     

Separation and determination of sterols in olive oil by HPLC-MS

This study presents the first liquid chromatographic method for the identification and quantification of seven phytosterols in olive oil. Sterols were identified and quantified by liquid chromatography with mass spectrometry detection in positive APCI (atmospheric pressure chemical ionisation) mode. The samples were saponified by refluxing with 2 N ethanolic KOH, and the non-saponificable fraction was extracted with diethyl ether. This fraction was subjected to thin layer chromatography (TLC) on silica gel plates and then the band of sterols was isolated and extracted with methanol. Sterols were quantified by LC-MS, on a Waters Atlantis 5 μm dC₁₈2,1 × 150 mm column with a gradient of acetonitrile/water (0.01% acetic acid) at a flow of 0.5 mL min⁻¹; column temperature 30 °C. The method presents values between 123 and 677 ng mL⁻¹ for detection limits, with relative standard deviations between 4.0% and 5.4% at a concentration of 5 mg L⁻¹ for each sterol. Sterol contents were determined in a virgin olive oil, a refined olive oil, an olive-pomace oil and a crude olive-pomace oil.