Andreja Vanzo

The stomach as a site for anthocyanins absorption from food1

The ability of anthocyanins to permeate the gastric mucosa can be suggested as a possible explanation of the fast kinetics of plasma appearance of anthocyanins in rats and humans. This paper presents an in vivo experiment aimed to prove the involvement of the stomach in the absorption of grape anthocyanins in rats. The required analytical selectivity and sensitivity was achieved by high‐performance liquid chromatography, diode array detection and mass spectrometry. Malvidin 3‐glucoside appeared in both portal and systemic plasma after only 6 min. The average concentrations measured in portal and systemic plasma were 0.650±0.162 μM and 0.234±0.083 μM (mean±S.E.M.), respectively.

Bioavailability of Flavonoids: A Review of Their Membrane Transport and the Function of Bilitranslocase in Animal and Plant Organisms

Fruits and vegetables are rich in flavonoids, and ample epidemiological data show that diets rich in fruits and vegetables confer protection against cardiovascular, neurodegenerative and inflammatory diseases, and cancer. However, flavonoid bioavailability is reportedly very low in mammals and the molecular mechanisms of their action are still poorly known. This review focuses on membrane transport of flavonoids, a critical determinant of their bioavailability. Cellular influx and efflux transporters are reviewed for their involvement in the absorption of flavonoids from the gastro-intestinal tract and their subsequent tissue distribution. A focus on the mammalian bilirubin transporter bilitranslocase (TCDB 2.A.65.1.1) provides further insight into flavonoid bioavailability and its relationship with plasma bilirubin (an endogenous antioxidant). The general function of bilitranslocase as a flavonoid membrane transporter is further demonstrated by the occurrence of a plant homologue in organs (petals, berries) where flavonoid biosynthesis is most active. Bilitranslocase appears associated with sub-cellular membrane compartments and operates as a flavonoid membrane transporter.

Phenolics in Slovenian Bilberries (Vaccinium myrtillus L.) and Blueberries (Vaccinium corymbosum L.)

Phenolics from bilberries ( Vaccinium myrtillus L.) sampled from seven different locations and highbush blueberries ( Vaccinium corymbosum L.) from one location in Slovenia were analyzed. In samples of both species 15 anthocyanins were identified by LC-MS/MS. Their contents were expressed as cyanidin 3-glucoside equivalents (C3GE); bilberries contained 1210.3 ± 111.5 mg C3GE/100 g fw and blueberries 212.4 ± 14.1 mg C3GE/100 g fw. Glycosides of delphinidin and cyanidin were predominant (488.5 vs 363.6 mg C3GE/100 g fw) in the bilberries and glycosides of malvidin (108.0 vs 100.8 mg C3GE/100 g fw) in the blueberries, whereas the contents of peonidin were lowest (74.5 vs 4.8 mg C3GE/100 g fw) in both berries. The contents of flavanols, flavonols, phenolic acids, and stilbenes were determined by LC-MS. For the first time, rutin was identified (bilberries, 0.2 ± 0.0 mg/100 g fw; blueberries, 3.1 ± 0.1 mg/100 g fw). Chlorogenic acid (as 3-caffeoylquinic acid) was the most abundant among the phenolic acids (23.1 ± 1.0 mg/100 g fw in bilberries and 70.0 ± 3.4 mg/100 g fw in blueberries). Statistical analysis shows that the content of 27 individual flavonoids, phenolic acids, and stilbenes can be used to identify the picking region of these Slovenian bilberries.

Bilberry and blueberry anthocyanins act as powerful intracellular antioxidants in mammalian cells

Berry anthocyanins have pronounced health effects, even though they have a low bioavailability. The common mechanism underlying health protection is believed to relate to antioxidant activity. Berry extracts, chemically characterised for their phenolic content, were prepared from bilberries (Vaccinium myrtillusL.) and blueberries (Vaccinium corymbosumL.); the bilberry extract was further purified to obtain the anthocyanin fraction. The antioxidant activity of each extract was examined at the cellular level. For this purpose a specific assay, known as cellular antioxidant activity assay (CAA), was implemented in different cell lines: human colon cancer (Caco-2), human hepatocarcinoma (HepG2), human endothelial (EA.hy926) and rat vascular smooth muscle (A7r5). Here we show for the first time that anthocyanins had intracellular antioxidant activity if applied at very low concentrations (<1 μg/l; nM range), thereby providing a long-sought rationale for their health protecting effects in spite of their unfavorable pharmacokinetic properties.

Determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection

A modified preparation of sample was developed for the determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection, using on-line pre-column derivatization. Ice-cold deoxygenated methanol was used to deactivate the oxidation enzymes in juices or wines and keep the glutathione stable. The optimum recovery of glutathione content in grape juice and wine was obtained when either the sample of grape juice or wine was mixed in ice-cold deoxygenated methanol in the ratio 10:90 (v:v) and further diluted in sodium acetate buffer in the ratio 1:1 (v:v). The optimized method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation data the method is appropriate for the determination of glutathione content in grape juice and wine. Glutathione contents in grape juices made from White Muscat grapes and Sauvignon Blanc wines were analysed. The average glutathione content in 28 young Sauvignon Blanc wines was 12.5 mg L(-1).

EXPRESSION OF BILITRANSLOCASE IN THE VASCULAR ENDOTHELIUM AND ITS FUNCTION AS A FLAVONOID TRANSPORTER

AIMS Ingestion of flavonoid-rich beverages acutely affects endothelial function, causing vasodilation. This effect might be dependent on flavonoid transport into the endothelium. We investigated flavonoid uptake into vascular endothelial cells and whether this was mediated by bilitranslocase (TC 2.A.65.1.1), a bilirubin-specific membrane carrier that also transports various dietary flavonoids. METHODS AND RESULTS Human and rat aortic primary endothelial cells as well as Ea.hy 926 cells were found to express bilitranslocase, as assessed by immunocytochemistry and immunoblotting analysis using anti-sequence bilitranslocase antibodies targeting two distinct extracellular epitopes of the carrier. Bilitranslocase function was tested by measuring the rate of bromosulfophthalein (a standard bilitranslocase transport substrate) uptake into endothelial cells and was inhibited not only by bilitranslocase antibodies but also by quercetin (a flavonol). Similarly, uptake of both quercetin and malvidin 3-glucoside (an anthocyanin) were also found to be antibody-inhibited. Quercetin uptake into cells was inhibited by bilirubin, suggesting flavonoid uptake via a membrane pathway shared with bilirubin. CONCLUSION The uptake of some flavonoids into the vascular endothelium occurs via the bilirubin-specific membrane transporter bilitranslocase. This offers new insights into the vascular effects of both flavonoids and bilirubin.

Uptake of grape anthocyanins into the rat kidney and the involvement of bilitranslocase.

Anthocyanins are among the most common flavonoids in the human diet. In spite of their very low bioavailability, anthocyanins are indicated as active in preventing the progress of cardiovascular and neurodegenerative diseases, obesity, inflammation, and cancer. Any piece of knowledge concerning absorption, tissue distribution, metabolism, and excretion of dietary anthocyanins is expected to help understanding the apparent paradox between their low concentrations in cells and their bioactivity. The aim of this work was to investigate the renal uptake of dietary anthocyanins and the underlying molecular mechanism. A solution containing anthocyanins extracted from grape (Vitis vinifera) was introduced into the isolated stomach of anesthetized rats; after both 10 and 30 min, plasma, liver, and kidney were analyzed for their anthocyanin contents. While anthocyanins in the liver were at apparent equilibrium with plasma both after 10 and 30 min, kidney anthocyanins were 3- and 2.3-fold higher than in plasma, after 10 and 30 min, respectively. Since the transport activity of the bilitranslocase in kidney basolateral membrane vesicles was competitively inhibited by malvidin 3-glucoside (K(i) = 4.8 +/- 0.2 microM), the anthocyanin uptake from blood into kidney tubular cells is likely to be mediated by the kidney isoform of this organic anion membrane transporter.

Exceptionally Fast Uptake and Metabolism of Cyanidin 3-Glucoside by Rat Kidneys and Liver

To asses the hypothesis that anthocyanins are rapidly taken up from the blood into tissues, where they accumulate up to their bioactivity threshold, an intravenous dose of cyanidin 3-glucoside (1) was administered to anaesthetized rats. Cyanidin 3-glucoside (1) and its metabolites were analyzed in the plasma, kidneys, liver, urine, and bile, using last-generation mass spectrometry. Compound 1 was found to rapidly disappear from plasma (t/2=0.36 min). As soon as 15 s after its administration, both 1 and its methylation product, peonidin 3-glucoside (2), were detected in the plasma, kidneys, and liver. At 1 min, both 1 and 2 had almost disappeared from the plasma, but attained their peak concentrations in the kidneys and in the liver. Compound 2 was rapidly excreted both in the bile and in the urine. Three additional methylated metabolites were detected in traces, namely, delphinidin 3-glucoside (3), petunidin 3-glucoside (4), and malvidin 3-glucoside (5). These data contribute to solving the paradox of the high bioactivity of anthocyanins in spite of their apparent low bioavailability.

Oxygen Consumption in South African Sauvignon Blanc Wines: Role of Glutathione, Sulphur Dioxide and Certain Phenolics

The aim of this research was to investigate the interaction between sulphur dioxide, glutathione (GSH) and certain phenols in the presence of oxygen in a synthetic wine and in clarified Sauvignon blanc wine. In this study, the clarified wine, from which most of the phenols had been removed, was compared to synthetic wine solution, with both mediums being enriched with caffeic acid to investigate the effect of different levels of sulphur dioxide and GSH on oxygen consumption. Moreover, thirteen young South African Sauvignon blanc wines with different levels of sulphur dioxide were oxygenated, and the oxygen consumption and phenolic and colour changes were monitored over time. The results show that oxygen consumption was influenced greatly by the presence of sulphur dioxide and, to a lesser extent, by the presence of GSH, with both compounds decreasing during the course of the experiment. During oxidation, an increase was observed in glutathionyl caffeic acid, as well as in oxidised glutathione (GSSG); however, this did not coincide with the percentage decrease in GSH. Oxidation further led to an increase in absorbance measurements at 420 and 440 nm (yellow-orange colour), which were reduced by the presence of SO2. A large variation was also observed in the oxygen consumption of the young wines, with this rate increasing with an increase in SO2 concentration. Positive correlations were also observed between oxygen, SO2, GSH and Cu concentrations, which were again negatively correlated with absorbance at 420 and 440 nm and GSSG concentrations.

Impact of Alternative Skin Contact Procedures on the Aroma Composition of White Wine

Zelen, Ribolla Gialla and Malvasia Istriana, grapes from the Vitis vinifera (L.) varieties, were subjected to four processes involving grape skin contact. Fresh and fruity young wines were produced by adding 6% or 12% grape berries, respectively, during alcoholic fermentation, freezing the pomace (Fp) and the freezing whole grapes (Fg). Wine-free volatile aromatic compounds were determined using two extraction techniques coupled to gas chromatography (GC). These aromatic compounds and wine standard chemical parameters were compared to control wines produced without skin contact. Esters, higher alcohols, terpenes, volatile phenols and C6 alcohols proved to be the most important sensorial odorants in wines. Both the content of aromatic compounds and the odour activity values showed some positive effects from skin contact. The freezing of the grapes and addition of 12% grape berries proved most effective for Zelen and Malvasia Istriana, producing a more intensive fruity and floral odour, a less intensive solvent odour and additional freshness. The same processes resulted only in additional fruitiness or a less intensive solvent odour in Ribolla Gialla while the lowest concentrations of grape derived terpenes were achieved for this variety. The three varieties were most affected by freezing processes. During the sensorial evaluation, the wines that had been subjected to skin contact were generally preferred to the control wines.