Determination of steviol glycosides in commercial extracts of Stevia rebaudiana and sweeteners by ultra-high performance liquid chromatography Orbitrap mass spectrometry.

Stevia rebaudiana extracts are used as sweeteners in several countries worldwide. Several extracts of diverse composition are available on the market, and their taste depends on the contents of the various steviol glycosides. This study presents an accurate method for the qualitative and quantitative determination of steviol glycosides in 40 Stevia extracts, 7 sweeteners and 3 Stevia-sweetened beverages by a UHPLC coupled to an Orbitrap mass spectrometer. The sub-2 µm amide column provided the separation of all the target analytes in a run time of 30 min with high resolution. The effect of different eluent compositions on the ionisation efficiency of the steviol glycosides was studied. The optimal ionisation conditions were achieved in negative mode using 0.05% formic acid. Under this condition, adducts were not found, [M-H]- were the main ions and the spontaneous loss of a glucose residue at C19 was reduced. The %RSD for intra- and inter-day precision for all eleven analytes varied from 2.1 to 4.2% and 3.0-5.1%, respectively. The recoveries from spiked Stevia extract samples were greater than 95% for all analytes. Rebaudioside A was the most abundant, ranging from 23 to 102%. Nine Stevia extracts and one drink were not compliant with the European Regulation. Isosteviol was under the LOD in all samples and steviol was found in four samples in quantities in the range 0.01-0.03%.

Toxicity and antioxidant activity in vitro and in vivo of two Fucus vesiculosus extracts.

The consumption of seaweeds has increased in recent years. However, their adverse and beneficial effects have scarcely been studied. Two extracts from the brown seaweed Fucus vesiculosus containing 28.8% polyphenols or 18% polyphenols plus 0.0012% fucoxanthin have been obtained and studied to determine their toxicity in mice and rats and also their antioxidant activity. Both extracts were shown to lack any relevant toxic effects in an acute toxicity test following a 4 week daily treatment in rats. The extracts exhibited antioxidant activity in noncellular systems and in activated RAW 264.7 macrophages, as well as in ex vivo assays in plasma and erythrocytes, after the 4 week treatment in rats. Our ex vivo results indicated that compounds from extract 2 may be more easily absorbed and that the antioxidants in their parent or metabolized form are more active. These findings support the view that the daily consumption of F. vesiculosus extract 2 (Healsea) would have potential benefits to humans.

Dealcoholized red and white wines decrease oxidative stress associated with inflammation in rats.

In vitro experiments have demonstrated that polyphenols exhibit antioxidant and anti-inflammatory activities. The present study was designed to test whether dealcoholized red (DRW) and white (DWW) wines can decrease the oxidative stress associated with inflammation in vivo. Rats were fed for 15 d either a control diet or one supplemented with DRW or DWW. Finally, a granuloma was induced by subcutaneous administration of carrageenan. Although DRW showed higher antioxidant activity in vitro than DWW, both wines decreased the number of cells recruited into the granuloma pouch. Malondialdehyde decreased in plasma and inflammatory exudate from rats fed with DRW- and DWW-rich diets. Moreover, the concentration of NO increased in exudate, which correlates with the increase in the citrulline:arginine ratio. Polymorphonuclear leucocytes from the inflammatory exudate of rats fed dealcoholized wines showed decreased superoxide anion (O*2) production and increased NO production ex vivo. This change in NO production resulted from increased expression and activity of inducible NO synthase (EC 1.14.13.39). Moreover, the up regulation of cyclo-oxygenase-2 (EC 1.14.99.1) protein expression observed in rats fed the DRW-rich diet was not related to a direct effect of NO. The present results indicate that the non-alcoholic compounds of wines not only improve antioxidant status in an inflammatory situation, but also limit cell infiltration, possibly through a decrease in O*2 and an increase in NO production.

LC-APCI-MS/MS analysis of urinary 8-hydroxy-2'-deoxyguanosine.

8-Hydroxy-2'-deoxyguanosine (8OHdG) is regarded as an important biomarker of oxidative DNA damage and it may be estimated by using different techniques in various biological matrices, most notably DNA and urine. In the case of DNA, artifactual oxidation may take place during the isolation of DNA, its hydrolysis and possible derivatization (as for GC-MS), invalidating the measurement of 8OHdG. Therefore, the direct analysis of 8OHdG excreted into urine was preferred. Interferences from the urine matrix were excluded by applying LC-APCI-MS/MS in the multiple reaction monitoring (MRM) mode. The abundant fragment ion at m/z 168 arising from 8OHdG was monitored in the urine sample of volunteers supplemented with tomato concentrate for different times. The procedure allowed the detection of levels of 8OHdG as low as 1 ng/ml in urine sample.

Polyphenol pattern and antioxidant activity of different tomato lines and cultivars.

BACKGROUND/AIMS: Besides antioxidant vitamins and minerals, fruits and vegetables contain flavonoids and related phenolics. The biological activities of these polyphenols have become well known in recent years evidencing their beneficial effects on human health. In this context, the characterization of the flavonoids present in tomatoes is of great interest. Thus the polyphenol pattern (including flavonols, flavanones and cinnamate derivatives), lycopene and beta-carotene concentrations and the total antioxidant activity (TAA) of the phenolic fraction from different tomato lines and cultivars have been determined. METHODS: The characterization was obtained by means of spectrophotometry and HPLC analyses. RESULTS: Mean values for single flavonoids were 0.68 +/- 0.16 for naringenin, 0.74 +/- 0.12 for rutin and 0.32 +/- 0.06 for a rutin-pentoside. Mean total polyphenol content was 13.15 +/- 1.15 mg/100 g and mean TAA value was 1.3 +/- 0.10 mmol/g. The obtained TAA values resulted in good accordance with the total polyphenol content (R(2) = 0.7928). The main phenolic acids were chlorogenic (mean +/- SE 0.20 +/- 0.03) and caffeic acid (mean +/- SE 0.03 +/- 0.01). Mean levels of lycopene and beta-carotene were 5.38 +/- 0.90 and 1.18 +/- 0.40 mg/100 g, respectively. CONCLUSIONS: Almost all the lines characterised by low carotenoid content produce high levels of polyphenols, and consequently have the most powerful antioxidant potential.

Plasma levels of caffeic acid and antioxidant status after red wine intake.

The aim of this study was to evaluate the bioavailability of caffeic acid and the modification of plasma antioxidant status following red wine intake. Five healthy male volunteers consumed 100, 200, and 300 mL of red wine corresponding to approximately 0.9, 1.8, and 2.7 mg of caffeic acid, respectively. Plasma samples collected at different times (0-300 min) were evaluated for their content of caffeic acid and their total antioxidant status. Both these parameters, i.e., plasma concentration of caffeic acid and antioxidant potential, were dose-dependent and the C(max) was reached at about 60 min after red wine intake. The results indicate that caffeic acid is bioavailable and it may be correlated with the antioxidant potential of plasma.

Liquid chromatography/atmospheric pressure chemical ionization mass spectrometry of terpene lactones in plasma of volunteers dosed with Ginkgo biloba L. extracts.

Liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-ITMS) was applied to evaluate the levels of ginkgolides A and B and bilobalide in plasma of volunteers after administration of Ginkgo biloba extracts in free (Ginkgoselect) or phospholipid complex (Ginkgoselect Phytosome) forms, providing 9.6 mg of total terpene lactones. The maximum plasma concentrations, C(max), of total ginkgolides A, B and bilobalide were 85.0 and 181.8 microg/mL for Ginkgoselect and Ginkgoselect Phytosome, respectively. The C(max) values were reached at 120 min for the free form and at 180--240 min for the phospholipid complex form. In both cases, the mean elimination half-life of each terpene lactone was in the range 120--180 min. Due to its sensitivity (about 1 ng/mL) and specificity, LC/APCI-ITMS proved to be a very powerful tool for pharmacokinetic studies of these phytochemicals.

Polyphenol content and total antioxidant activity of vini novelli (young red wines).

Eight commercial Italian vini novelli (red wines prepared by carbonic maceration and supposed to be consumed within three months from their wine-making) were evaluated for their total antioxidant activity. The wines had an average total phenol content (1605.4 +/- 337.4 mg/L gallic acid equivalents) lower than that of wines prepared by traditional maceration and consumable after aging (2057. 3 +/- 524.0 mg/L gallic acid equivalents). The average flavanol content (424.7 +/- 121.3 mg/L catechin equivalents) and the total antioxidant activity (16.8 +/- 3.8 mmol/L Trolox equivalents) of vini novelli were higher than the corresponding values (382.7 +/- 174.5 mg/L catechin equivalents and 12.3 +/- 3.3 mmol/L Trolox equivalents) found for aged wines. Three couples of experimental wines were prepared from the same grapes by traditional or carbonic maceration. These wines showed a different phenolic pattern, anthocyanins being more abundant in vini novelli. However, the average total antioxidant activities of the wines were similar, suggesting that aging (and not the wine-making technique) is the main factor influencing the antioxidant activity of red wines.

Liquid chromatography/electrospray ionization mass spectrometric characterization of flavonol glycosides in tomato extracts and human plasma.

Flavonoids continue to attract wide attention as possible very useful agents for combating free radical pathologies, i.e. the pathological states associated with free radical overproduction. Commonly used methods for the analysis of plant flavonoids include high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). On the other hand, the soft-ionization approach based on electrospray ionization (ESI-MS) permits highly selective analysis of complex matrices. In this work, we examined firstly the ESI-MS behaviour of representative aglycones and glycosides of flavonols, flavones and isoflavones with the aim of suggesting a possible relationship between structure and mass spectra. Using HPLC coupled to a diode array detector (DAD) for on-line UV spectra acquisition, and in parallel to ESI-MS for mass spectra (LC/DAD-ESI-MS), we have developed methodology to observe flavonols directly in tomato puree extract. In this way, it has been possible to detect intact flavonol glycosides in tomato extracts and to characterize a flavonol trisaccharide. For the first time, using LC/ESI-MS, it has been possible to detect intact flavonol glycosides in plasma of healthy volunteers and to provide further evidence on the absorption of flavonoid glycosides after consumption of common vegetables like tomatoes.

Relationship between rate and extent of catechin absorption and plasma antioxidant status.

Flavonoids are described to exert a large array of biological activities, which are mostly ascribed to their radical-scavenging, metal chelating and enzyme modulation ability. Most of these evidences have been obtained by in vitro studies on individual compounds and at doses largely exceeding those dietary. Little is known about a possible relationship between rate and extent of the absorption and modifications of plasma antioxidants. To elucidate this aspect, human volunteers were supplemented with single doses of green tea catechins in free (Greenselect) or phospholipid complex form (Greenselect Phytosome) equivalent to 400 mg epigallocatechingallate (EGCg). EGCg was chosen as biomarker for green tea catechin absorption, and its time course plasma concentration was correlated to the subsequent percent variations of plasma ascorbate, total glutathione, alpha-tocopherol, beta-carotene and Total Radical Antioxidant Parameter (TRAP). Green tea catechins were absorbed more extensively when administered as phospholipid complex rather than as free catechins. Single dose intake of both forms of catechins produced a transient decrease (10-20%) of plasma ascorbate and total glutathione and an increase of plasma TRAP (16-19%). These variations were consistent with the plasmatic levels of EGCg, ascorbate and total glutathione.

Catechin metabolites after intake of green tea infusions.

Green tea contains relatively large amounts of catechins, that have been recognized to be efficient free-radical scavengers. In spite of a largely described antioxidant effect, the metabolic fate of catechins in humans has been scarcely studied. An infusion of green tea (about 400 mg of catechins) was given to healthy volunteers; plasma and urine samples were collected for 5 h and 2 days, respectively. Epigallocatechin gallate and epicatechin gallate were detected in plasma samples, reaching the maximum concentration (2 microM) at 2 h. Urine samples collected at 6-48 h contained detectable amounts of final catechin metabolites, including 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). The total content of these metabolites averaged 60 mg. The levels of free plasma catechins account only partly for the increased (approximately +20%) total radical-trapping antioxidant parameter (TRAP) detected after green tea intake. Catechin conjugates (glucuronide and sulphate) and metabolites may add further contribution and explain the measured TRAP increase.

Composition of platelet phospholipids after moderate consumption of red wine in healthy volunteers.

OBJECTIVE: To evaluate the effect of moderate consumption of red wine on composition of platelet phospholipids, discriminating the effect of alcohol from that of non-alcoholic components. DESIGN: A randomised crossover study. SETTING: The Department of Food Science and Technology, University of Milan. SUBJECTS: Eleven healthy male volunteers who were moderate drinkers. INTERVENTIONS: For three periods of 4 weeks, subjects drank three different beverages [320 ml of red wine (providing 30 g/day of alcohol), 30 g/day of alcohol diluted in 320 ml of clear fruit juice or 320 ml of dealcoholised red wine] during the two main meals. Each treatment was preceded by a period of 4 weeks of complete withdrawal from any alcoholic beverage. At the end of each period the fatty acid composition of individual phospholipids was determined on isolated platelets. RESULTS: Consumption for a period of 4 weeks of non-alcoholic components either from 320 ml of red wine or from the same amount of dealcoholised red wine resulted in similar increases in polyunsaturated fatty acids in all phospholipid fractions of platelet, with the exception of sphingomyelin. No differences were detected when we compared the composition of phospholipids at the end of red wine and alcohol treatments with findings at the end of dealcoholised treatment and abstinence. CONCLUSIONS: The increase of polyunsaturated fatty acids in platelet phospholipids due to the non-alcoholic components of red wine suggests an antioxidant effect that could be relevant in justifying the protective effect of red wine shown in epidemiological studies.

Effects of moderate consumption of red wine on platelet aggregation and haemostatic variables in healthy volunteers.

OBJECTIVE: To evaluate the effect of moderate consumption of red wine on platelet aggregation and haemostatic variables, discriminating the effect of alcohol from that of non-alcoholic components. DESIGN: A randomised crossover study. SETTING: The Department of Food Science and Technology, University of Milan. SUBJECTS: Eleven healthy male volunteers who were moderate drinkers. INTERVENTIONS: For three periods of four weeks, subjects drank three different beverages [320 ml of red wine (providing 30 g/day of alcohol), 30 g/day of alcohol diluted in 320 ml of clear fruit juice or 320 ml of dealcoholised red wine] during the two main meals. Each treatment was preceded by a period of four weeks of complete withdrawal from any alcoholic beverage. At the end of each period platelet aggregation after collagen and ADP stimulus, and levels of fibrinogen, plasminogen, tissue-type plasminogen activator (t-PA) antigen and von Willebrand factor (vWF) were determined. RESULTS: Consumption for a period of four weeks of 30 g/day of alcohol either from red wine or alcohol resulted in similar decreases of collagen-induced platelet aggregation and fibrinogen levels. ADP-induced platelet aggregation, t-PA antigen, vWF and plasminogen levels were not affected by any treatment. No differences were detected when we compared platelet function and the other haemostatic variables at the end of red wine and dealcoholised treatments with findings at the end of alcohol treatment and abstinence. CONCLUSIONS: The well known positive effect of moderate consumption of red wine on haemostasis seems due to alcohol and not to the non-alcoholic fraction present in red wine.

Red wine effects on peroxidation indexes of rat plasma and erythrocytes.

Moderate wine intake has been associated with low risk for cardiovascular disease, possibly due to its polyphenol content. We investigated the influence of these compounds on peroxidative indices of blood. Forty Sprague-Dawley rats were divided into four groups given the same AIN-76 diet but different types of isocaloric beverage: red wine, alcohol and dealcoholated wine and water with added sucrose. Rats consumed about 35 ml day(-1) of beverage and 17 g day(-1) of diet for a total of about 95 kcal day(-1). Plasma alpha-tocopherol was higher in the alcohol group and the peroxidation kinetic slope was higher in the control and dealcoholated wine groups. Ascorbic acid in whole blood was higher in the alcohol group than the others. Erythrocytic glutathione peroxidase activity was lower in the red wine group. The other variables studied, were not significantly different in the four groups. Despite the significant differences observed, the oxidative status of blood does not appear seriously altered by heavy ingestion of wine and thus of polyphenols. Probably the balanced diet and adequate intake of micronutrients were sufficient to counteract any oxidative damage.

Effect of red wine consumption on rat liver peroxidation.

To evaluate the role of wine polyphenols and that of alcohol on lipid peroxidation indexes and membrane composition in the liver, 40 Sprague-Dawley rats were fed for 28 days with a commercial AIN-76 diet to which was added one of four different beverages: red wine, alcohol solution, dealcoholated wine, or water. The beverage provided 26% of the caloric intake. Peroxidation indexes and antioxidative enzymes were determined: no significant differences were detected in catalase and glutathione peroxidase whereas superoxide dismutase was significantly lower in the wine-treated animals (220.3 +/- 15.4 vs. 342.2 +/- 43.0 U/mg protein of controls). The following significant differences in hepatic variables were observed: increased alpha-tocopherol concentration in the alcohol group (0.17 +/- 0.02 vs. 0.11 +/- 0.01 microgram/mg protein of controls); increased concentration of cytochrome P450 in the rats given wine (0.75 +/- 0.06 vs. 0.51 +/- 0.08 nmol/mg protein of the alcohol group); increased concentration of cytochrome b5 in wine and dealcoholated wine treatment groups (0.30 +/- 0.01 vs. 0.23 +/- 0.02 nmol/mg protein of controls). The liver membrane fatty acid composition of the wine and dealcoholated wine groups was similar and showed an increase in the saturated fatty acid percentage and a decrease in the polyunsaturated one. The data presented indicate that the main action of polyphenols seems to be an induction of cytochrome activity and that the modality of red wine administration adopted combined with an adequate diet does not provoke any apparent physiological effect on the animals.

Dietary selenium intake and selenoproteins in rat plasma.

Selenium (Se) is vital for animals and humans, as it is an essential component of Se-dependent glutathione peroxidase (GPx), an enzyme that reduces peroxides and protects cells against the damaging effects of oxidation. Se has, however, been found in rat plasma even when the enzymatic activity of GPx is very low, supporting the hypothesis that Se is also bound to other proteic structures. The purpose of this work was partially to purify the selenium-containing proteins in plasma, without denaturation, by isoelectrofocusing. We observed two pH intervals in the plasma where Se-containing proteins concentrated upon focusing: the first, at pH 6.0 +/- 0.2 and with GPx activity, and the second, between pH 4.6 and 5.4, with no enzymatic activity. We can infer therefore that other Se-containing proteins are present at a lower pH than 6, and in particular between 4.6 and 5.4