A simple and fast reversed-phase HPLC method using diode array detection was developed and validated for the simultaneous determination of trans-resveratrol and quercetin in Sicilian red wine from the Nero d'Avola red grape variety. Investigation was also extended to the quantitative determination of resveratrol and quercetin in grape skins and winemaking byproducts obtained from the same cultivar. Samples were eluted using a C18 narrow-bore column under isocratic conditions in less than 20 min. Quantification of trans-resveratrol and quercetin in red wine was performed without any sample pretreatment, whereas the determination of these phenolic compounds in grape skins and wine pomage required a solvent extraction procedure. Linearity was demonstrated over the 0.39-12.5 and 0.45-57.6 microg/mL range for trans-resveratrol and quercetin, respectively. Detection limits in real samples were in the low ppm level (0.07 mg/L for trans-resveratrol and 0.12 mg/L for quercetin). The HPLC-UV/DAD method was applied for the routine analyses of red wine and grape skin and winemaking byproduct extracts to evaluate their trans-resveratrol and quercetin content. In particular, a very high content of quercetin was found in wine pomace, suggesting the use of this wine byproduct as a potential source of this health-promoting phenolic compound.

In the recent years many studies on anthocyanins have revealed their strong antioxidant activity and their possible use as chemotherapeutics. The finding that sour cherries (Prunus cerasus L) (also called tart cherries) contain high levels of anthocyanins that possess strong antioxidant and anti-inflammatory properties has attracted much attention to this species. Here we report the preliminary results of the induction of anthocyanin biosynthesis in sour cherry callus cell cultures. The evaluation and characterization of the in vitro produced pigments are compared to those of the anthocyanins found in vivo in fruits of several sour cherry cultivars. Interestingly, the anthocyanin profiles found in whole fruit extracts were similar in all tested genotypes but were different with respect to the callus extract. The evaluation of antioxidant activity, performed by ORAC and TEAC assays, revealed a relatively high antioxidant capacity for the fruit extracts (from 1145 to 2592 μmol TE/100 g FW) and a lower one for the callus extract (688 μmol TE/100 g FW).

Clusters of Aleatico winegrape were picked at 18 degrees Brix and placed at 10, 20, or 30 degrees C, 45% relative humidity (RH) and 1.5 m/s of air flow to dehydrate the berries up to 40% of loss of initial fresh weight. Sampling was done at 0, 10, 20, 30, and 40% weight loss (wl). Selected polyphenols and sugar content (expressed as SSC = soluble solids content) both measured on dry weight basis, polyphenol oxidase (PPO), and phenylpropanoid pathway gene expression were analyzed. Phenolic acids increased significantly at 20% wl at 20 degrees C, while at 10 degrees C the increase was lower. Stilbenes (trans-resveratrol and trans-piceid) and catechins rose more than double to 100 mg/kg and more than 3-fold to 135 mg/kg at 20 degrees C and 10% wl. At 10 degrees C the increase of these compounds was less, but higher than initial values. At 30 degrees C, except for a significant rise at 10% wl for catechins and stilbenes, all the rest of the compounds diminished. Anthocyanins increased at 10 and 20 degrees C, but decreased at 30 degrees C. PPO rapidly increased at 20 and 30 degrees C at 10% wl and then declined, while at 10 degrees C the activity lasted longer. Relative gene expression of phenylalanine ammonia lyase (PAL), stilbene synthase (STS), chalcone isomerase (CHI), dihydroflavonol reductase (DFR) were upregulated at 10 degrees C more than at 20 degrees C, at 20% wl, while at 30 degrees C the gene expression was downregulated.

Reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array (PDA) and mass spectrometry (MS) detection was employed to study the accumulation of stilbenes and other naturally occurring polyphenol intermediates of flavonoid pathway in tomato fruits of plants genetically modified to synthesize resveratrol. The transgenic tomato fruits were obtained by overexpression of a grapevine gene encoding the enzyme stilbene synthase in tomato plants (Lycopersicon esculentum Mill.). Stilbenes and flavonoids, either glycosylated or free, were simultaneosly identified by electrospray interface (ESI)-MS in negative ionization mode and were quantified by PDA detection at the wavelength corresponding to their maximum absorbance. The two detectors were coupled online with an HPLC system utilizing a narrow-bore C18 reversed-phase column, which was eluted by a multistep gradient of increasing concentration of acetonitrile in water containing 0.5% (v/v) formic acid. The results of these analysis revealed that the genetic modification of the tomato plants originated different levels of accumulation of four stilbenes (i.e., trans- and cis-piceid and trans- and cis-resveratrol) in their fruit depending on the stages of ripening. Either at immature or at mature stages of ripening the stilbenes were preferentially accumulated in the fruit peel as the glycosylated form. The highest amount of trans-piceid and trans-resveratrol were found in the peel of fruits harvested at mature stage of ripening. The variations in the levels of rutin, naringenin, and chlorogenic acid found in the samples extracted from the fruits of transgenic tomato plants, in comparison to that determined in the control lines, seemed to be related to the genetic transformation, whose effect on the flavonoid biosynthetic pathway needs to be elucidated by additional studies.

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A straightforward semimicro separation scale RP-HPLC method was developed for the identification and quantification of phenolic acids (PAs) occurring as soluble free, soluble conjugated, and insoluble bound compounds, which were independently extracted from wholemeal of durum wheat and from its derived products coarse bran, semolina, and dried pasta. A narrow bore column and a semimicro photodiode array detector (PDA) cell, in conjunction with a single quadrupole mass spectrometer, equipped with an electrospray ionization source (ESI-MS), were employed. The method was validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability, and accuracy, which was evaluated by a recovery study. In each sample (wholemeal, coarse bran, semolina, and dried pasta), the total amounts of the three different forms of PAs were in the order bound > conjugated > free, with bound PAs accounting for 61.0-83.6% of the total PAs. Ferulic acid was the most abundant PA in both soluble free and insoluble bound forms, whereas sinapic acid predominated in the conjugated ones. The highest PA content, calculated as the sum of total PAs quantified in the three forms, was found in coarse bran, followed by wholemeal, semolina, and dried pasta.

The application of liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) was investigated for the analysis of trans-resveratrol in red wine, grape skin, grape pomace, and winemaking byproducts. Chromatographic elution performed under isocratic reversed-phase conditions using a C18 narrow-bore LC column allowed retention times lower than 12 min to be obtained. Qualitative analysis was performed in negative-ion (NI) full-scan and product-ion scan acquisition modes, whereas method validation in terms of linearity, detection limits, accuracy, and robustness was carried out under NI selected reaction monitoring conditions. The matrix-matched detection limit was calculated in the low parts per billion range (10 microg/L). Results of the application of the method to red wine, grape, and winemaking byproduct samples were compared with those obtained using an LC-UV/DAD technique. Determination of trans-resveratrol in the samples investigated showed that the highest concentration was found in red wine, whereas wine made from grape pomace contained the lowest content.

SummaryResveratrol, a plant phenolic compound, is found in grapes and red wine, but is not widely distributed in other common food sources. The pathway for resveratrol biosynthesis is well characterized. Metabolic engineering of this compound has been achieved in tomato plants capability and ascorbate content in transformed fruits were also evaluated, and a significant increase in both was found in the LoxS and 35SS lines. These results could explain the higher capability of transgenic fruits to counteract the pro-inflammatory effects of phorbol ester in monocyte-macrophages via the inhibition of induced cyclo-oxygenase-2 enzyme.