Evolution of ellagitannins in Spanish, French, and American oak woods during natural seasoning and toasting

The evolution of tannins in Spanish oak heartwood of Quercus robur L., Quercus petraea Liebl.,Quercus pyrenaica Wild., and Quercus faginea Lam. was studied in relation to the processing of wood in barrel cooperage. Their evolution was compared with that of French oak of Q. robur (Limousin, France) and Q. petraea (Allier, France) and American oak of Quercus alba L. (Missouri), which are habitually used in cooperage. Two stages of process were researched: the seasoning of woods during 3 years in natural conditions and toasting. Total phenol and total ellagitannin contents and optical density at 420 nm of wood extracts were determined. The ellagitannins roburins A-E, grandinin, vescalagin, and castalagin were identified and quantified by HPLC, and the molecular weight distribution of ellagitannins was calculated by GPC. During the seasoning process the different ellagitannin concentrations decreased according to the duration of this process and in the same way as those in French and American woods. The toasting process also had an important influence on the ellagitannin composition of wood. Roburins A-E, grandinin, vescalagin, and castalagin decreased during this process in the Spanish wood species, in the same proportion as in the French and American ones. Also, the seasoning and toasting processes lead to qualitative variations in the structure of ellagitannins, especially in the molecular weight distribution, as was evidenced by GPC analysis of their acetylated derivatives.     

Oak ellagitannins suppress the phosphorylation of the epidermal growth factor receptor in human colon carcinoma cells

The ellagitannins castalagin and vescalagin, and the C-glycosides grandinin and roburin E as well as ellagic acid were found to potently inhibit the growth of human colon carcinoma cells (HT29) in vitro. In a cell-free system these compounds were identified as potent inhibitors of the protein tyrosine kinase activity of the epidermal growth factor receptor (EGFR) with IC 50 values in the low nanomolar range. To address the question of whether the interference with the activity of the isolated EGFR also plays a role within intact cells, effects on the phosphorylation status of the EGFR, as a measure for its activity, were determined in HT29 cells. As exemplified for castalagin and grandinin, both the nonglycosylated and the glycosylated ellagitannins effectively suppressed EGFR phosphorylation, but only at concentrations > or =10 microM, thus, in a concentration range where growth inhibition was observed. These results indicate that the suppression of EGFR-mediated signaling might contribute to the growth inhibitory effects of these compounds present in oak-matured wines and spirits such as whiskey. In contrast, despite substantial growth inhibitory properties, ellagic acid did not significantly affect EGFR phosphorylation in HT29 cells up to 100 microM.     

Identification and sensory evaluation of dehydro- and deoxy-ellagitannins formed upon toasting of oak wood (Quercus alba L.)

Traditionally, spirits such as whiskey are matured in toasted wood barrels to improve the sensory quality of the final beverage. In order to gain first insight into the puzzling road map of thermal ellagitannin transformation chemistry and provide evidence for the changes in sensory active nonvolatiles in oak wood during toasting, the purified oak ellagitannins castalagin and vescalagin, their corresponding dimers roburin A and roburin D, and 33-carboxy-33-deoxyvescalagin were thermally treated in model experiments. Besides mouth-coating and golden-brown colored melanoidin-type polymers, individual major reaction products were produced as transient intermediates which were identified for the first time by means of LC-MS/MS and 1D/2D-NMR spectroscopy. Depending strongly on the stereochemistry, castalagin is oxidized to the previously unreported dehydrocastalagin, whereas its diastereomer vescalagin, differing only in the stereochemistry at carbon C-1, is most surprisingly converted into deoxyvescalagin. Comparative model experiments with 33-carboxy-33-deoxyvescalagin revealed castalagin, vescalagin, dehydrocastalagin, and deoxyvescalagin as typical reaction products, thus indicating decarboxylation as a key step in the thermal degradation of that ellagitannin. Similar to the ellagitannin monomers, LC-MS/MS analyses gave strong evidence that the corresponding dimer roburin A, containing the vescalagin configuration at C-1, was converted into the deoxyroburin A, whereas roburin D, exhibiting the castalagin configuration at C-1, was oxidized to give the dehydroroburin D. Human sensory experiments revealed that the ellagitannin derivatives imparted an astringent mouth-coating sensation with threshold concentrations ranging from 1.1 to 126.0 micromol/L, depending strongly on their chemical structure.     

Determination of ellagitannins in extracts of oak wood and in distilled beverages matured in oak barrels

A method is described for determination of ellagitannins in ethanol-water extracts of oak wood and in distilled alcoholic beverages matured in oak barrels. It is based on the combined ellagic acid content according to ellagitannin structure. Hydrolysis was carried out in the presence of hydrochloric acid under reflux in a 100 degrees C oil bath for 3 h. Total ellagic acid was thus determined by liquid chromatography (LC), and the free ellagic acid content present in the ethanol-water media was subtracted, the difference being the combined ellagic acid content corresponding to ellagitannins. A 5 micron C18 column was used with detection at 254 nm. The method is specific for ellagitannins, which is an advantage over other analytical techniques for overall evaluation of these substances extracted from wood. Results for spirits distilled from wine, grain, and sugarcane were highly variable.     

Effect of toasting intensity at cooperage on phenolic compounds in acacia (Robinia pseudoacacia) heartwood

The phenolic composition of heartwood from Robinia pseudoacacia, commonly known as false acacia, before and after toasting in cooperage was studied by HPLC-DAD and HPLC-DAD/ESI-MS/MS. A total of 41 flavonoid and nonflavonoid compounds were identified, some tentatively, and quantified. Seasoned acacia wood showed high concentrations of flavonoid and low levels of nonflavonoid compounds, the main compounds being the dihydroflavonols dihydrorobinetin, fustin, tetrahydroxy, and trihydroxymethoxy dihydroflavonol, the flavonol robinetin, the flavanones robtin and butin, and a leucorobinetinidin, none of which are found in oak wood. The low molecular weight (LMW) phenolic compounds present also differed from those found in oak, since compounds with a β-resorcylic structure, gallic related compounds, protocatechuic aldehyde, and some hydroxycinnamic compounds are included, but only a little gallic and ellagic acid. Toasting changed the chromatographic profiles of extracts spectacularly. Thus, the toasted acacia wood contributed flavonoids and condensed tannins (prorobinetin type) in inverse proportion to toasting intensity, while LMW phenolic compounds were directly proportional to toasting intensity, except for gallic and ellagic acid and related compounds. Even though toasting reduced differences between oak and acacia, particular characteristics of this wood must be taken into account when considering its use in cooperage: the presence of flavonoids and compounds with β-resorcylic structure and the absence of hydrolyzable tannins.     

Sensory-directed identification of taste-active ellagitannins in American (Quercus alba L.) and European oak wood (Quercus robur L.) and quantitative analysis in bourbon whiskey and oak-matured red wines

Aimed at increasing our knowledge on the sensory-active nonvolatiles migrating from oak wood into alcoholic beverages upon cooperaging, an aqueous ethanolic extract prepared from oak wood chips (Quercus alba L.) was screened for its key taste compounds by application of the taste dilution analysis. Purification of the compounds perceived with the highest sensory impacts, followed by liquid chromatography/mass spectrometry as well as one-dimensional and two-dimensional NMR experiments, revealed the ellagitannins vescalagin, castalagin, and grandinin, the roburins A-E, and 33-deoxy-33-carboxyvescalagin as the key molecules imparting an astringent oral sensation. To the best of our knowledge, 33-deoxy-33-carboxyvescalagin has as yet not been reported as a phytochemical in Q. alba L. In addition, the sensory activity of these ellagitannins was determined for the first time on the basis of their human threshold concentrations and dose/response functions. Furthermore, the ellagitannins have been quantitatively determined in extracts prepared from Q. alba L. and Quercus robur L., respectively, as well as in bourbon whiskey and oak-matured red wines, and the sensory contribution of the individual compounds has been evaluated for the first time on the basis of dose/activity considerations.     

Evolution of phenolic compounds of spanish oak wood during natural seasoning. First results

Low molecular weight polyphenols and ellagitannins were analyzed by HPLC, and the molecular weight distribution of ellagitannins was calculated by GPC, in oak heartwood of Quercus robur L., Quercus petraea Liebl., Quercus pyrenaica Wild., and Quercus faginea Lam., grown in Spain, before and after 1 year of seasoning, in Bordeaux, France. During this process, the concentrations of low molecular weight polyphenols (acids and aldehydes, benzoic and cinnamic, and coumarins) increased, and those of ellagitannins (castalagin, vescalagin, and roburins A-E) decreased. A similar behavior for the A and B compounds in all species was not found. This modification in the chemical composition was similar in the four Spanish species of Quercus studied and allowed the differentiation between the unseasoned wood and the wood after the first year of seasoning.     

Concentration and mean degree of polymerization of Rubus ellagitannins evaluated by optimized acid methanolysis

Ellagitannins are a major class of phenolics largely responsible for the astringent and antioxidant properties of raspberries and blackberries. The Rubus ellagitannins constitute a complex mixture of monomeric and oligomeric tannins. Rubus oligomeric ellagitannins contain, beside the well-known ellagic acid and gallic acid moieties, the sanguisorboyl linking ester group. When exposed to acids or bases, ester bonds are hydrolyzed and the hexahydroxydiphenic acid spontaneously cyclizes into ellagic acid. This study describes a new, rapid procedure for the acid hydrolysis of Rubus ellagitannins in methanol, which results in maximal yield and enables the quantification of all the major reaction products. Additionally, the method provides the rationale for estimating the mean degree of polymerization of Rubus ellagitannins.     

Evolution of castalagin and vescalagin in ethanol solutions. Identification of new derivatives.

Brandies, cognac, armagnac, whiskeys, and rums are aged in oak barrels to improve their organoleptic properties. During this period, numerous compounds such as ellagitannins are extracted from the wood and can subsequently be transformed into new derivatives by chemical reactions. Model solutions of castalagin and vescalagin have been studied to determine the behavior of polyphenols in ethanol-water. Upon prolonged exposure to 40 and 70% (v/v) ethanol at room temperature, hemiketal derivatives containing ethoxy groups have been characterized by LC/MS and NMR. These compounds further evolve to afford the corresponding ketals. They have also been detected in the extracts of oak wood stored under similar conditions.     

Validation of a mass spectrometry method to quantify oak ellagitannins in wine samples

Detection and individual quantification of oak wood ellagitannins in oak barrel aged red wine samples are difficult mainly due to their low levels and the similarity between their structures. In this work, a quantification method using mass spectrometry has been developed and validated to quantify wine ellagitannins after sample fractionation with a previously reported method. The use of an internal standard is a requirement to correct mass signal variability. (-)-Gallocatechin, among the different tested compounds, was the only one that proved to be a suitable internal standard making possible the accurate and individual quantification of the main oak wood ellagitannins. The developed methodology has been used to detect and quantify these ellagitannins in different Spanish commercial wines, proving its usefulness.     

Impact of concentration of ellagitannins in oak wood on their levels and organoleptic influence in red wine

Some wood substances such as ellagitannins can be extracted during wine aging in oak barrels. The level of these hydrolyzable tannins in wine depends of some parameters of oak wood. Their impact on the organoleptic perception of red wine is poorly known. In our research, oak staves were classified in three different groups according to their level of ellagitannins estimated by NIRS (near infrared spectroscopy) online procedure (Oakscan). First, the ellagitannin level and composition were determine for each classified stave and an excellent correlation between the NIRS classification (low, medium and high potential level of ellagitannin) and the ellagitannin content estimated by HPLC-UV was found. Each different group of NIRS classified staves was then added to red wine during its aging in a stainless tank, and the extraction and evolution of the ellagitannins were monitored. A good correlation between the NIRS classification and the concentration of ellagitannins in red wine aging in contact with the classified staves was observed. The influence of levels of ellagitannins on the resulting wine perception was estimated by a trained judge's panel, and it reveals that the level of ellagitannins in wine has an impact on the roundness and amplitude of the red wine.     

Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC-ESI-MS

Ellagic acid, ellagic acid glycosides, and ellagitannins found in various fruits and nuts, including muscadine grape, are reported to have potential health-promoting benefits and antioxidant properties. This study isolated and identified several ellagic acid derivatives present in muscadine grapes and determined their relative antioxidant properties (AOX). Compounds were extracted from grape skins and pulp using methanol, and the solvent was evaporated. Isolates were dissolved in citric acid buffer (pH 3.5) and absorbed onto C18 cartridges. Nonretained polyphenolics were collected separately and again partitioned from Sephadex LH-20, whereas retained polyphenolics were first eluted with ethyl acetate followed by methanol. Ellagic acid derivatives were identified on the basis of UV and mass spectra, and the presence of ellagitannins was confirmed by a significant increase in free ellagic acid with HPLC followed by acid hydrolysis. Muscadine grapes contained phenolic acids, flavonols, anthocyanins, ellagic acid, and numerous ellagic acid derivatives. AOX varied in the order ethyl acetate > methanol > C18 nonretained fractions; each correlated to both total phenolics (r = 0.90) and total ellagic acid (r = 0.99) contents. Results of this study revealed previously unidentified ellagic acid derivatives in muscadine grapes.     

Analysis by high-performance liquid chromatography diode array detection mass spectrometry of phenolic compounds in fruit of Eucalyptus globulus cultivated in Algeria

A method based on high-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) following fractionation by chromatography on a Sephadex LH-20 column has been developed to determine the phenolic composition of fruit of Eucalyptus globulus growing in Algeria. The presence of 18 gallotannins, 26 ellagitannins, and 2 flavonols was established. Tentative identification is provided for these compounds on the basis of UV-visible spectra and mass spectrometry data. Most compounds described in this study have not previously detected in fruit of E. globulus. Moreover, this is the first report of methyl digalloyl diglucose, 3,3'-O-dimethylellagic acid 4-O-β-glucopyranoside, ellagic acid hexose, methyl ellagic acid pentose, methyltetragalloylglucose, and valoneic acid isomers (sanguisorbic, flavogallic acid dilactone) in the genus Eucalyptus. Quantitatively, ellagic acid and its derivatives, including ellagitannins, are largely predominant.     

Effect of the seasoning method on the chemical composition of oak heartwood to cooperage

The effect of various oak wood seasoning methods, natural seasoning in open air, artificial seasoning in a kiln, and a mixed method that combines open air and kiln drying, on the chemical composition in American ( Quercus alba) and French ( Quercus petraea) oak was studied. The results confirm the evolution of ellagitannins in wood, low molecular polyphenols, and volatile compounds in relation to oak species and seasoning method. Natural seasoning in open air can be considered a superior method to mixed or artificial drying for wood seasoning in cooperage. It was more effective in reducing the excess of ellagitannins, especially in French oak wood. Moreover, the evolution of wood volatile compounds was more positive in the natural seasoning than mixed or artificial drying, since it led to woods with higher aromatic potential (larger concentrations of compounds such as volatile phenols, phenolic aldehydes, furanic compounds, and cis- and trans-beta-methyl-gamma-octalactones) than mixed or artificial methods. However, the three seasoning methods showed similar effectiveness regarding the desired reduction of off-flavors compounds.     

Metabolism of oak leaf ellagitannins and urolithin production in beef cattle

Oak leaves have a high concentration of ellagitannins. These phytochemicals can be beneficial or poisonous to animals. Beef cattle are often intoxicated by oak leaf consumption, particularly after suffering feed restriction. The severity of the poisoning has recently been associated with the ruminal microbiota, as different bacterial populations were found in animals that tolerated oak leaves and in those that showed clinical and pathological signs of toxicity. Intoxication has previously been linked to the production of phenolic metabolites, particularly catechol, phloroglucinol, and resorcinol. This suggested that the microbial metabolism of ellagitannins could also be associated with its tolerance or intoxication in different animals. Therefore, it is essential to understand the metabolism of ellagitannins in cattle. Here we show that ellagitannins are metabolized in the cattle rumen to urolithins. Different urolithins were detected in ruminal fluid, feces, urine, and plasma. Oak leaf ellagitannins declined as they were converted to urolithins, mainly isourolithin A and urolithin B, by the ruminal and fecal microbiota. Urolithin aglycons were observed in rumen and feces, and glucuronide and sulfate derivatives were detected in plasma and urine. Sulfate derivatives were the main metabolites detected in plasma, while glucuronide derivatives were the main ones in urine. The main urolithins produced in cattle were isourolithin A and urolithin B. This is a relevant difference from the monogastric mammals studied previously in which urolithin A was the main metabolite produced. Low molecular weight phenolics of the benzoic, phenylacetic, and phenylpropionic groups and metabolites such as catechol, resorcinol, and related compounds were also detected. There was a large variability in the kinetics of production of these metabolites in individual animals, although they produced similar metabolites in all cases. This large variability could be associated with the large variability in the rumen and intestine microbiota that has previously been observed. Further studies are needed to demonstrate if the efficiency in the metabolism of ellagitannins by the microbiota could explain the differences observed in susceptibility to intoxication by the different animals.     

Influence of geographical origin and botanical species on the content of extractives in American, French, and East European oak woods

The chemical composition of East European (Republic of Moldova, Ukraine, and Romania) oaks was investigated profoundly for the first time in the present study and compared with American and French counterparts. Taking into account the high natural variability of oak extractives contents, the wide-ranging sampling was performed for all oak origins: 276 French oaks, 102 East European oaks of both species (Quercus robur L. and Quercus petraea Liebl.), and 56 American oaks (Quercus alba). These oaks were compared with great attention paid to the extractives, which are most important for sensorial impact in wine or spirit maturation, such as ellagitannins and principal odorant substances (aromatic aldehydes, lactones and phenols). The substances in question were studied by application of HPLC and GC-MS techniques. The pattern of all studied extractive contents allowed adequate separation of oak samples according to their geographical origin or botanical species. The highest separation rate was for American and French oaks, whereas East European samples could be partially misclassified in two sets mentioned above. The most important variables for species discrimination were whiskey lactone related variables and ellagitannins, whereas the most important features for distinguishing the origin were eugenol, 2-phenylethanol, vanillin, and syringaldehyde. These substances allowed the distinction of French and East European woods of the same species. With regard to chemical composition, East European wood held the intermediary place between American and French oaks according to their ellagitannin and whiskey lactone levels; nevertheless, it was characterized by specific high values of eugenol, aromatic aldehydes, and 2-phenylethanol.     

Antioxidant activity of isolated ellagitannins from red raspberries and cloudberries

Ellagitannins from red raspberries (Rubus idaeus) and cloudberries (Rubus chamaemorus) were isolated by using column chromatography and preparative HPLC. The berry phenolic isolates consisted of 80% (cloudberry) and of 60% (raspberry) of ellagitannins, with raspberries also containing anthocyanins. The main ellagitannins of both raspberries and cloudberries were identified by ESI-MS to consist of the dimeric sanguiin H-6 and the trimeric lambertianin C. Monomeric ellagitannins such as casuarictin in raspberries and pedunculagin in cloudberries were also found. The antioxidant activity of the berry phenolic isolate, ellagitannin isolate (mixture), ellagitannin main fraction (dimer and trimer), and ellagic acid was studied in bulk and emulsified methyl linoleate, in human low-density lipoprotein in vitro, and the radical scavenging activity was studied in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test. Cloudberry and red raspberry ellagitannins were highly effective as radical scavengers. Berry ellagitannins also showed significant antioxidant activity toward oxidation of both human LDL and methyl linoleate emulsions. However, only weak or moderate antioxidant activity was exhibited by ellagitannins toward oxidation of bulk oil. Thus, ellagitannins contribute significantly to the antioxidant capacity of cloudberries and red raspberries in lipoprotein and lipid emulsion environments, the latter being more relevant for food applications.     

Characterization of phenolic compounds in strawberry (Fragaria x ananassa) fruits by different HPLC detectors and contribution of individual compounds to total antioxidant capacity

Phenolic compounds in strawberry (Fragaria x ananassa) fruits were identified and characterized by using the complementary information from different high-performance liquid chromatography detectors: diode array, mass spectrometer in positive and negative mode, and coulometric array. Electrochemical profiles obtained from the coulometric array detector contributed to the structural elucidation suggested from the UV-vis and mass spectra. About 40 phenolic compounds including glycosides of quercetin, kaempferol, cyanidin, pelargonidin, and ellagic acid, together with flavanols, derivatives of p-coumaric acid, and ellagitannins, were described, providing a more complete identification of phenolic compounds in strawberry fruits. Quercetin-3-malonylhexoside and a deoxyhexoside of ellagic acid were reported for the first time. Antioxidative properties of individual components in strawberries were estimated by their electrochemical responses. Ascorbic acid was the single most important contributor to electrochemical response in strawberries (24%), whereas the ellagitannins and the anthocyanins were the groups of polyphenols with the highest contributions, 19 and 13% at 400 mV, respectively.     

Protein binding and astringent taste of a polymeric procyanidin, 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose, castalagin, and grandinin

The objective of the present investigation was to examine the oral astringency and protein-binding activity of four structurally well-defined tannins, namely, procyanidin [epicatechin16(4-->8)catechin], pentagalloyl glucose (1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose), castalagin, and grandinin, representing the three main structural categories of tannins, the proanthocyanidins, the gallotannins, and the ellagitannins. Astringency threshold and dos/response were determined by the half-tongue test using a trained human panel. Protein-binding stoichiometry and relative affinity were determined using radioiodinated bovine serum albumin in precipitation or competitive binding assays. Procyanidin and pentagalloyl glucose were perceived as highly astringent compounds and had relatively steep dose/response curves, but castalagin and grandinin had a lower mass threshold for detection. In vitro, procyanidin was the most effective protein-precipitating agent and grandinin the least. Increasing the temperature increased protein precipitation by the hydrolyzable tannins, especially grandinin. All four polyphenols had higher relative affinities for proline-rich proteins than for bovine serum albumin.     

Benzothiadiazole induces the accumulation of phenolics and improves resistance to powdery mildew in strawberries

Benzothiadiazole (BTH) enhanced the accumulation of soluble and cell-wall-bound phenolics in strawberry leaves and also improved the resistance to powdery mildew infection under greenhouse conditions. The most pronounced change was seen in the levels of ellagitannins, which increased up to 2- to 6-fold 4 days after the BTH application, but persisted only in the inoculated plants. The induction of phenolic metabolism by BTH was also reflected in the fruits, several compounds being increased in inoculated, BTH-treated plants. Basal salicylic acid (SA) content was high in strawberry leaves, but increased in a similar fashion to other phenolics after the treatments. Several phenolic compounds were identified in strawberries for the first time. For example, ellagic acid deoxyhexose, three agrimoniin-like ellagitannins, sanguiin H-10- and lambertianin C-like ellagitannins in the leaves, ellagic acid, p-coumaric acid, gallic acid, and kaempferol hexose in the cell-wall-bound fraction of the leaves, and kaempferol malonylglucoside in the fruits. The findings show that BTH can enhance the accumulation of phenolics in strawberry plants which may then be involved in the BTH-induced resistance to powdery mildew.