Determination of stilbenes in hop pellets from different cultivars

About 30% of the polyphenols in wort and beer derive from hop, but little is yet known about their nutritional impact. The recent discovery of trans-resveratrol and piceid isomers in hop opens new doors to understanding beer health benefits. In the present work, resveratrol was quantified by HPLC-APCI-MS/MS in pellets from 9 different cultivars. Concentrations ranging from 4 to 9 mg/kg trans-piceid, from 2 to 6 mg/kg cis-piceid, and up to 1 mg/kg trans-resveratrol were detected. As previously shown for total polyphenols and flavonoids, the lower the alpha-acid content, the higher the total stilbene content.     

Hop as an interesting source of resveratrol for brewers: optimization of the extraction and quantitative study by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

Nowadays, hop is used almost exclusively by brewers for bitterness and flavor. Although hop polyphenols have been widely studied in the past decade for their antioxidant activity in the boiling kettle, very little is known about their real impact on health. The discovery of resveratrol in hop pellets highlights the potential health-promoting effect of moderate beer consumption. Here, we have optimized a quantitative extraction procedure for resveratrol in hop pellets. Preliminary removal of hydrophobic bitter compounds with toluene and cyclohexane at room temperature allows 99% trans-resveratrol recovery by ethanol:water (75:25, v/v) solid/liquid extraction at 60 degrees C. Reverse phase liquid chromatography proves an excellent means of separating isomers. In addition, we have compared two mass spectrometry ionization methods-atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI)-in both the positive and the negative modes. On the basis of standard additions applied with the optimized extraction procedure and reverse phase high-performance liquid chromatography-APCI(+)-tandem mass spectrometry, it appears that Tomahawk hop pellets (T90, harvest 2002) contain 0.5 ppm trans-resveratrol, 2 ppm trans-piceid, no cis-resveratrol, and 0.9 ppm cis-piceid.     

Occurrence of resveratrol and piceid in American and European hop cones

The recent discovery in hops of trans-resveratrol and its glucoside, trans-piceid, both famous in wine for their potential role in the "French paradox", opens new doors to understanding hop health benefits. In the present work, trans-resveratrol and trans-piceid were quantified by HPLC-APCI-MS/MS in 40 American and European hop cone samples from harvests of 2004, 2005, and 2006. Their content varies greatly, in the range 0.5-12 mg/kg. All German varieties revealed low amounts of stilbenes. The highest concentrations were found in Cascade (2004) and Willamette (2004 and 2006), two American low-alpha-acid varieties. Yet, probably because trans-resveratrol is a phytoalexin, a strong influence of harvest year was also observed.     

Cellular uptake and efflux of trans-piceid and its aglycone trans-resveratrol on the apical membrane of human intestinal Caco-2 cells

Two stilbenes (trans-piceid and its aglycone trans-resveratrol) were investigated in the uptake across the apical membrane of the human intestinal cell line Caco-2 in order to determine their mechanisms of transport. The uptake was quantified using a reverse phase high-performance liquid chromatography method with fluorescence detection. The rate of cellular accumulation in the cells was found to be higher for trans-resveratrol than for trans-piceid. In addition, trans-resveratrol uses passive transport to cross the apical membrane of the cells, whereas the transport of trans-piceid is likely active. With regard to the mechanisms of transport, the involvement of the active transporter SGLT1 in the absorption of trans-piceid was deduced using various inhibitors directly or indirectly exploiting the activity of this transporter (glucose, phlorizin, and ouabain). Moreover, we investigated the involvement of the multidrug-related protein 2 (MRP2), an efflux pump present on the apical membrane, in stilbene efflux by Caco-2 cells. The effect of MK-571 (an MRP inhibitor) seems to implicate MRP2 as responsible for apical efflux of trans-piceid and trans-resveratrol.     

Determination of stilbenes (delta-viniferin, trans-astringin, trans-piceid, cis- and trans-resveratrol, epsilon-viniferin) in Brazilian wines

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Stilbenes have been shown to protect lipoproteins from oxidative damage and to have cancer chemopreventive activity. We describe a method for the direct determination of stilbenes in several red wines using high-performance liquid chromatography with UV detection. In a survey of 12 commercial wines from the south of Brazil (Rio Grande del Sul), levels of delta-viniferin are reported for the first time in different varieties of red wines. Brazilian red wine contains trans-astringin, trans-piceid, trans-resveratrol, cis-resveratrol (in high quantity: 5 times more than the trans form), epsilon-viniferin, and a compound isolated for the first time in wine, trans-delta-viniferin. Isolation and identification of delta-viniferin was achieved by NMR after extraction and fractionation of red wine phenolics. delta-Viniferin contributes, as well as cis-resveratrol and trans-piceid, to a significant proportion of stilbenes in wine dietary intake, particularly with Merlot varieties containing an average level of 10 mg/L for delta-viniferin, 15 mg/L for cis-resveratrol, and 13 mg/L for trans-piceid. The total stilbene intake from wine origin was estimated for the Brazilian population as 5.3 mg/day per person (on the basis of a regular wine consumption of 160 mL/day). delta-Viniferin can contribute to around 20% of total stilbenes in wine (average of 6.4 mg/L in red Brazilian wines). It would be important in the future to investigate the origins of the differences in wine stilbene levels in relation to the vine varieties, and the bioavailability of the newly extracted stilbene delta-viniferin in plasma after consumption of different types of wines.     

Potentiality of red sorghum for producing stilbenoid-enriched beers with high antioxidant activity

trans-Piceid and trans-resveratrol were authenticated for the first time by high-resoution mass spectrometry in red sorghum grains. A 0.4-1 mg/kg amount of trans-piceid and up to 0.2 mg/kg trans-resveratrol were quantified by reversed phase high-performance liquid chromatography-atmospheric pressure chemical ionization(+)-tandem mass spectrometry. The white sorghum samples contained only traces of trans-piceid (up to 0.1 mg/kg), and trans-resveratrol was absent. In much lower amounts than procyanidins, stilbenoids are not able to contribute significantly to the exceptional antioxidant activity of red sorghum (ORAC, 83-147 μmol TE/g; AAPH, 0.61-1.79 min/mg kg(-1)). More than 10 mg/kg of total stilbenoids have been reported in some hop varieties. Yet, as hop is a minor wort ingredient as compared to cereals, red sorghum could be the main source of trans-piceid in beer. Hop remains, however, the single source of cis-piceid.     

Constitutive expression of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its derivatives in substantial quantities

Resveratrol, a well-known phytoalexin and antioxidant, is produced by the action of stilbene synthase (STS) in some plant species. Hop (Humulus lupulus L.) plants of the Tettnang variety were transformed with a gene encoding for STS from grapevine. Under the control of the constitutive 35S cauliflower mosaic virus promoter, expression of the transgene resulted in accumulation of resveratrol and high levels of its glycosylated derivatives in leaves and inflorescences. Piceid, the predominant derivative, reached a concentration of up to 560 microg/g of fresh weight (f.w.) in hop cones, whereas no stilbenes were detected in nontransformed controls (wild-type). In transgenic plants the amounts of alpha- and beta-acids, naringenin chalcone, and prenylated flavonoids did not change significantly when compared with nontransformed plants. Transgenic plants showed normal morphology and flower development as did the nontransformed controls. The results clearly show that in hop constitutive expression of sts interferes neither with plant development nor with the biosynthesis of secondary metabolites relevant for the brewing industry. Since resveratrol is a well-known phytoalexin and antioxidant, sts transgenic hop plants could display enhanced pathogen resistance against microbial pathogens, exhibit new beneficial properties for health, and open new venues for metabolic engineering.     

Ultraviolet-C and induced stilbenes control ochratoxigenic Aspergillus in grapes

This study investigated the efficacy of ultraviolet-C (254 nm) and induced stilbenes to inhibit Aspergillus carbonarius and Aspergillus tubingensis and control ochratoxin A production in grapes. In addition, the stilbene synthesis as a response to UV-C treatment and to infection of ochratoxigenic Aspergillus was compared. The initial microbial inactivation by a previously optimized UV-C illumination protocol for increasing trans-resveratrol content in grapes (50 W/m (2), 40 cm, 60 s) was similar on undamaged and damaged grapes, achieving 1.2 and 1.3 log conidia/100 g reductions, respectively. After 5 days of storage at 22 degrees C, UV-C treatment and the stilbenes induced by UV-C inhibited ochratoxigenic Aspergillus growth in undamaged grapes. UV-C elicited the biosynthesis of trans-resveratrol, while microbial infection and tissue damage triggered the biosynthesis of trans-piceid. trans-Resveratrol was not synthesized as a consequence of ochratoxigenic Aspergillus contamination. However, when trans-resveratrol was synthesized by UV-C, it contributed to inhibiting the development of ochratoxin A producing aspergilli. Furthermore, UV-C treatment also contributed to decrease ochratoxin A production by ochratoxigenic aspergilli. Therefore, UV-C is a promising emerging technology either for reducing the potential ochratoxigenic risk in grapes, which is of particular interest to the wine industry, and also for increasing trans-resveratrol content of grapes, which would provide an added value to the wine.     

Formation and accumulation of alpha-acids,beta-acids,desmethylxanthohumol, and xanthohumol during flowering of hops (Humulus lupulus L.)

Important secondary metabolites, present in hops (Humulus lupulus L.), include alpha-acids and beta-acids, which are essential for the brewing of beer, as well as the prenylated chalcones, desmethylxanthohumol, and xanthohumol, which exhibit interesting bioactive properties. Their formation and accumulation in five selected hop varieties, Wye Challenger, Wye Target, Golding, Admiral, and Whitbread Golding Variety, were quantitatively monitored by high-performance liquid chromatography using UV detection. All target compounds were present from the onset of flowering, not only in female hop cones but also in male inflorescences, albeit in low concentrations. During development from female inflorescences to cones, levels of alpha-acids, beta-acids, desmethylxanthohumol, and xanthohumol gradually increased, while each hop variety exhibited individual accumulation rates. Furthermore, these compounds were present in leaves of fully grown hops as well. The study demonstrated that key compounds for flavor and potential beneficial health effects associated with beer not only reside in the glandular lupulin structures but also are distributed over various parts of the hop plant.     

Use of GC-olfactometry to identify the hop aromatic compounds in beer

This paper describes a sensorial aroma extract dilution analysis (AEDA) approach to the analysis of beer aromas derived from hops. To obtain an extract with an odor representative of the original product, the XAD extraction procedure was applied and the experimental conditions were optimized. The aromagrams of three beers were compared: one brewed without hops, one brewed with Saaz hop pellets, and one brewed with Challenger hop pellets. One spicy/hoppy compound, unmodified from hop to beer, proved responsible for the most intense odor in both hopped beer extracts. Another flavoring compound in hops, linalool, also survives through the process to the final beer. Other compounds such as gamma-nonalactone and humuladienone, although not found in our extracts of hop, significantly modify beer aromagrams after hopping. Sulfur compounds characteristic of Challenger hops proved to be at least partially responsible for the unpleasant flavor found in the corresponding beer.     

Hopeaphenol: the first resveratrol tetramer in wines from North Africa

Grapes and wines are now known to constitute a rich source of phenolics such as stilbenes and flavonoids. These compounds have been shown to have cancer chemopreventive activity and potential beneficial effects on cardiovascular diseases thanks to their antioxidant and antiplatelet properties. However, because little is known about African wines and their phenolic compositions, we investigated wine samples from North Africa. A three-step method was used for the fractionation of the Merlot variety wine: column chromatography followed by centrifugal partition chromatography and reversed-phase semipreparative high-performance liquid chromatography (HPLC). Six polyphenolic compounds of the Merlot variety (from Algeria) were isolated and identified by NMR spectroscopy, five of which are known (trans-resveratrol, trans-piceid, trans-epsilon-viniferin, pallidol, and astilbin) and one that is reported for the first time in wine, (+)-hopeaphenol, a stilbene tetramer. Furthermore, these molecules were quantified in 10 commercial wines from North Africa by means of an analytical HPLC system coupled with diode array detection. Differences in concentrations were found ranging in mg/L from 4.6 to 45 (trans-piceid), 0.66 to 3.45 (trans-resveratrol), 0.2 to 1.2 (trans-epsilon-viniferin), 0.2 to 9.2 (pallidol), 0.3 to 3.8 (hopeaphenol), and 10.8 to 24.22 (astilbin). Such a high level of pallidol and astilbin has never been recorded in wine. North African wines may contribute to a significant proportion of dietary intake of stilbene and astilbin, which may have health benefits.     

Method for the quantitative extraction of resveratrol and piceid isomers in grape berry skins. Effect of powdery mildew on the stilbene content

A simple method for the quantitative extraction of resveratrol and its glycosides from grape berry skins has been developed. Optimal conditions for the extraction were investigated. Type of solvent, time, and temperature assayed influenced resveratrol and piceid yield. Adequate extraction was attained with ethanol/water (80:20 v/v) maintained at 60 degrees C for 30 min. Recovery (>96%) and reproducibility (6.83-15.13%) were satisfactory. After extraction, resveratrol and piceid isomers were quantified by high-performance liquid chromatography coupled to a ultraviolet-visible diode array detector. The amounts detected in 13 samples of 7 different varieties analyzed were, on average, 92.33 microg/g of dry skin for cis-piceid, 42.19 microg/g of dry skin for trans-piceid, and 24.06 microg/g of dry skin for trans-resveratrol. cis-Resveratrol was not detected in any sample. In grape berries infected by powdery mildew the contentw of these compounds were considerably increased and the degree of infection was positively related to their stilbene content.     

Comparison of 4-Mercapto-4-methylpentan-2-one contents in hop cultivars from different growing regions

Contributions of hop-derived thiols were examined. Extremely strong fruity, black currant-like aromas were detected in beers hopped with some U.S. cultivars. 4-Mercapto-4-methylpentan-2-one (4MMP) was supposed to be the main contributor to the fruity aroma, and the contents between cultivars were investigated. In hop pellets, a negative correlation between 4MMP concentration and copper ion content in hops was observed. 4MMP was detected only in U.S., Australian, and New Zealand cultivars, but no European ones, which are treated with copper-containing fungicides (Bordeaux mixture) and therefore have a high content of copper ions. The 4MMP content was highest in Simcoe cultivars, followed by Summit, Apollo, Topaz, Cascade pellets, and also differed between crop years. It was indicated that most 4MMP exists freely in wort or in hop pellets with only small amounts formed from precursors and that the amounts increased during the fermentation process.     

Postharvest stilbene-enrichment of red and white table grape varieties using UV-C irradiation pulses

The red table grape varieties Flame, Red Globe, Crimson, and Napoleon, as well as the white varieties Superior, Dominga, and Moscatel Italica, were irradiated with a previously optimized UV-C postharvest irradiation protocol (510 W, 40 cm, 60 s). The induction kinetics of the stilbenes trans-resveratrol, trans-piceid, trans-piceatannol, trans-astringin, and viniferins was followed by using HPLC-DAD/MS/MS. The most inducible stilbenes were trans-resveratrol, trans-piceatannol, and viniferins. Both quantitative and qualitative differences were observed in both the stilbene induction kinetics and stilbene content in the seven table grapes analyzed here. The total resveratrol content ranged from 0.69 mg/100 g fw (Dominga) to 2.3 mg/100 g fw (Red Globe). The net resveratrol induction ranged from 3.4-fold (Flame) to 2315-fold (Red Globe). The highest viniferins content was observed in the variety Flame (0.73 mg/100 g fw), although the variety Red Globe presented the highest viniferins induction (175-fold). The highest content and induction of piceatannol (0.17 mg/100 g fw and 173-fold, respectively) was observed in the variety Flame. It should be stressed that taking into account the health-beneficial effects claimed for stilbenes, the UV-C irradiated table grapes can be considered as new functional fruits that can supply (a serving of unpeeled 200 g table grapes) the resveratrol content (depending on the variety) equivalent to more than seven glasses of red wine ( approximately 1.5 L) with high resveratrol content.     

Phytochemical and morphological characterization of hop (Humulus lupulus L.) cones over five developmental stages using high performance liquid chromatography coupled to time-of-flight mass spectrometry, ultrahigh performance liquid chromatography photodiode array detection, and light microscopy techniques

Hop (Humulus lupulus L.) inflorescences, commonly known as "hop cones", are prized for their terpenophenolic contents, used in beer production and, more recently, in biomedical applications. In this study we investigated morphological and phytochemical characteristics of hop cones over five developmental stages, using liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF-MS), and ultrahigh performance liquid chromatography photodiode array detection (UHPLC-PDA) methods to quantitate 21 polyphenolics and seven terpenophenolics. Additionally, we used light microscopy to correlate phytochemical quantities with changes in the morphology of the cones. Significant increases in terpenophenolics, concomitant with glandular trichome development and associated gross morphological changes, were mapped over development to fluctuations in contents of polyphenolic constituents and their metabolic precursor compounds. The methods reported here can be used for targeted metabolic profiling of flavonoids, phenolic acids, and terpenophenolics in hops, and are applicable to quantitation in other crops.     

Stilbenes and tyrosol as target compounds in the assessment of antioxidant and hypolipidemic activity of Vitis vinifera red wines from southern Brazil

The contents of stilbene monomers, cis-resveratrol, trans-resveratrol, cis-piceid, trans-piceid, and tyrosol, were quantified in Vitis vinifera red wines, cvs. Cabernet Franc, Merlot, Sangiovese, and Syrah, 2006 and 2007 vintages, from the Sa?o Joaquim region, a new grape-growing region at southern Brazil. Moreover, the effect of chronic consumption of these wines on the antioxidant and hypolipidemic activities was monitored in C57BL6 LDL receptor knockout mice and treated with a hypercholesterolemic diet. Red wines from this region had substantial levels of resveratrols (the predominant forms were glycoside and trans) and tyrosol. Biomonitoring of antioxidant and hypolipidemic activities in vivo revealed that consumption of these wines increased the antioxidant capacity and reduced the hypercholesterolemia and hypertriglyceridemia promoted by the hypercholesterolemic diet. Significant correlations were found between the increase of antioxidant capacity markers, the decrease of lipid levels promoted by wine consumption, and the contents of stilbenes and tyrosol, supporting the important biological activity of these compounds.     

Resveratrol and piceid levels in natural and blended peanut butters

Peanut and its derivatives, especially peanut butter, are extensively consumed in many countries, mainly in the United States, which is also the major exporter of these products. trans-Resveratrol is present in peanuts, and recently this compound has been quantified in peanut butter. It is well-known that there are beneficial effects of trans-resveratrol and its glucoside, the piceid, in health. The absorption of trans-resveratrol has been proven in animals, and certain studies show that the absorption of some phenols is enhanced by conjugation with glucose, so that it could be possible that trans-piceid would be more absorbed than its aglycon (trans-resveratrol). In our work, we have identified the presence of trans-piceid in peanut butter with a new method to quantify trans-resveratrol and trans-piceid (3-beta-glucose of trans-resveratrol). This fact is very interesting because the glucosilated form could be more easily absorbed by the intestinal gut; in this way trans-piceid would exercise its beneficial function more efficiently than trans-resveratrol. To our knowledge, this is the first time that trans-piceid has been quantified in peanut butter. Resveratrol and piceid contents in natural peanut butters were found to be significantly higher than those in blended peanut butters.