The effects of sample preparation and gas chromatograph injection techniques on the accuracy of measuring guaiacol, 4-methylguaiacol and other volatile oak compounds in oak extracts by stable isotope dilution analyses

The deuterium-labeled standards [(2)H(3)]-guaiacol and [(2)H(3)]-4-methylguaiacol were synthesized and utilized in a method employing gas chromatography-mass spectrometry to determine the concentration of guaiacol and 4-methylguaiacol in wine or extracts of oak shavings. The method was combined with previously published methods for 4-ethylphenol, 4-ethylguaiacol, cis- and trans-oak lactone and vanillin, so that all these compounds could be quantified in a single analysis. The method can employ either liquid-liquid extraction or headspace solid-phase microextraction (SPME) and is rapid, robust, precise, and accurate. Under certain conditions, there was artifactual generation, to varying degrees, of guaiacol, 4-methylguaiacol, cis-oak lactone, and vanillin during the analysis of oak extracts, especially when diethyl ether extraction and injector block temperatures at or above 225 degrees C were employed. The most substantial effects were observed for guaiacol, in which results could be exaggerated by over 10 times. These artifacts could be avoided by using headspace SPME or by preparing liquid-liquid extracts with pentane or pentane/diethyl ether (2:1) injected at 200 degrees C providing spot checks using headspace SPME were performed. Data obtained for previously published quantitative determination of guaiacol in oak extracts should be reexamined carefully, with special attention paid to their respective methods of sample preparation and analysis.     

Rates of formation of cis- and trans-oak lactone from 3-methyl-4-hydroxyoctanoic acid

The rates of formation of both cis- and trans-oak lactone from the corresponding isomers of 3-methyl-4-hydroxyoctanoic acid have been measured in model wine at room temperature for a range of pH values. The half-life for formation of the trans-isomer at pH 2.9 was calculated to be 3.1 h, whereas that for the cis-isomer, at the same pH, was calculated to be 40.5 h. The k(trans)/k(cis) ratio in model wine was found to 12.86 +/- 1.34 over the range of pH values employed. A reason for the more facile formation of the trans-isomer, based on conformational reasons, has been proposed. In acidic aqueous media the equilibrium between the oak lactones and their corresponding ring-opened analogues was found to favor the former entirely, with no evidence for the latter being found. Implications of the present study for the future analysis of oak samples, as well as for the interpretation of existing data, are discussed.     

Identification of a precursor of beta-methyl-gamma-octalactone in the wood of sessile oak (Quercus petraea (Matt.) Liebl.)

The 6'-O-gallate derivative of (3S, 4S)-4-beta-D-glucopyranosyloxy-3-methyloctanoic acid was isolated for the first time from the wood of Sessile oak (Quercus petraea (Matt.) Liebl.) and shown to be a precursor of cis-beta-methyl-gamma-octalactone. The structure of this precursor was determined by HRFAB-MS, NMR, LCMS, and chiral analysis of the liberated (3S,4S)-beta-methyl-gamma-octalactone on a chiral fused silica capillary column. Optical rotation was shown to be identical to that of the same compound previously isolated from the wood of Platycarya strobilacea Sieb. et Zucc. by Tanaka and Kouno in 1996. Moreover, the 6'-O-gallate derivative of a threo-4-beta-D-glucopyranosyloxy-3-methyloctanoic acid was tentatively identified as a minor precursor of trans-beta-methyl-gamma-octalactone in the same wood of Sessile oak.     

Insecticidal, anti-juvenile hormone, and fungicidal activities of organic extracts from different Penicillium species and their isolated active components.

Organic extracts from mycelium and culture broth of 21 Penicillium isolates have been tested for insecticidal, insect anti-juvenile hormone (anti-JH), and antifungal activities. Culture broth extracts were the most active, mainly against insects; nearly 25% of them have shown high entomotoxicity (100% mortality at 100 microg/cm(2)). A strong in vivo anti-JH activity against Oncopeltus fasciatus Dallas was detected in the culture broth extracts from P. brevicompactum P79 and P88 isolates. The two new natural products isolated from P79, N-(2-methyl-3-oxodec-8-enoyl)-2-pyrroline (1) and 2-hept-5-enyl-3-methyl-4-oxo-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b]-1, 3-oxazine (2), possessed anti-JH and insecticidal activity, respectively, against O. fasciatus. Synthesized natural compound 1 has shown an ED(50) of 0.7 microg/nymph when assayed on newly molted fourth-instar nymphs of O. fasciatus. Promising biological activities have also been detected in the synthetic precursors.     

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.     

Maturing wines in oak barrels. Effects of origin,volume, and age of the barrel on the wine volatile composition

The influence of the oak origin, volume, and age of the barrel on the volatile composition of a red wine after six months of maturation have been studied. Major volatile compounds in wine were determined using liquid-liquid extraction with dichloromethane, and those volatiles being due to the oak were determined by extraction with pentane-ether using deuterated analogues as internal standards. The results show that there is little difference between the volatile composition of wines matured in French oak barrels and those matured in American oak barrels. The concentration of cis-oak lactone was the most significant difference. The greatest instrumental and sensory differences were found between new and used barrels, with important decreases in lactones and vanillin concentration in used barrels. When studying the effect of the volume of the barrel, the characteristics of the wine matured in 220-L new barrels versus those matured in 1000-L new barrels were very different. In new barrels, the larger the volume of the barrel, the lower the concentrations of those compounds responsible for the oaky aroma. These differences were less evident when comparing wines matured in used barrels with different capacities.     

Acyl-homoserine lactones produced by Pantoea sp. isolated from the "maize white spot" foliar disease

The "maize white spot" foliar disease is a problem of increasing importance to Brazilian maize crops. A bacterium isolated from water-soaked lesions from infected maize leaves was pathogenic in biological assays in vivo. It was identified as a Gram-negative, nonsporulating, facultative anaerobic bacterium, belonging to the genus Pantoea. Chemical study of the extracts from bacterial cultivation media allowed the identification of (S)-(-)-N-butanoyl-homoserine lactone and trace amounts of N-hexanoyl-homoserine lactone, widely recognized quorum-sensing signaling substances employed in cell-to-cell communication systems. The absolute configuration of natural (S)-(-)-N-butanoyl-homoserine lactone was determined by gas chromatography-flame ionization detection with a chiral stationary phase and by comparison of circular dichroism spectroscopic data with enantiopure synthetic substances. Biological evaluations with reporter Agrobacterium tumefaciens NTL4(pZLR4) were carried out with synthetic and natural products and also with extracts from maize leaves contaminated with the isolated bacterium, as well as from healthy leaves.     

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.     

Influence of oak wood on the aromatic composition and quality of wines with different tannin contents

The aromatic compounds associated with aging in wood have been studied in Monastrell wines containing different quantities of tannin. Analyses were carried out before and after a six month period in wooden barrels (225 L) of American and French (Allier) medium-toasted oak. Three classes of wine were made: one from a free-run must; one to which enological tannin was added; and one to which was added the wine obtained by pressing the grape pomaces. The aromas were determined by GC-MS, and the quality was evaluated by a group of expert tasters. The aromatic compounds, some of which came from the wines themselves and others which came from the wood, all increased in concentration by the end of the aging process. The ratio between cis- and trans-whiskylactone contents in wines reflected the different types of oak wood used. The note "vanilla" used by the tasters is probably not totally due to the presence of vanillin. The wines of best quality were those with a natural tannin content which had been aged in American oak, whereas the wines to which enological tannin had been added but aged in the same wood were considered the worst. An equalizing effect on the sensorial wines qualities was seen to result from the use of French oak. The furfural, eugenol, and cis-, and in some cases trans-, whiskylactone contents identified the wines from the different classes of wood. The wood from which the barrels were made had a greater effect on wines differentiation than the tannin content.     

Liquid chromatographic determination of scopoletin in hydroalcoholic extract of oak wood and in matured distilled alcoholic beverages

A liquid chromatographic (LC) method is described for determination of the coumarins esculin, umbelliferone, scopoletin, and 4-methyl umbelliferone in hydroalcoholic extracts of oak wood and in matured distilled alcoholic beverages. Samples were injected directly into the LC column (30 cm, 5 micron C18) and detected by fluorescence detector. Under these experimental conditions, only scopoletin (detection limit, 200 pg) was found in hydroalcoholic oak wood extracts and in spirits matured in oak wood. Applications of this method to spirits distilled from wine, grain, and sugar cane aged in oak barrels showed that amounts varied from 0.026 to 1.57 ppm.     

Extraction and formation dynamic of oak-related volatile compounds from different volume barrels to wine and their behavior during bottle storage

The extraction rate of furfuryl aldehydes, guaiacol, and 4-methylguaiacol, cis- and trans-oak lactone, and vanillin and the formation rate of furfuryl alcohol and the volatile phenols 4-ethylguaiacol and 4-ethylphenol have been studied in wines matured in different capacity oak barrels (220, 500, and 1000 L). Also, the behavior of these compounds during 1 year of wine bottle storage was followed. The lactones were extracted at a linear rate with large differences that depended on barrel volume. Those compounds related to oak toasting (guaiacol, 4-methylguaiacol, furfuryl aldehydes, and vanillin) seemed to be extracted faster during the first days of oak maturation except for vanillin, which required at least 3 months to accumulate in the wine. The volatile phenols, 4-ethylphenol and 4-ethylguaiacol, were formed in large quantities after the first 90 days of oak maturation, coinciding with the end of spring and beginning of summer. Wines matured in 1000-L oak barrels resulted in the lowest levels of volatile compound accumulation. During bottle storage, some compounds decreased in their concentration (5-methylfurfural, vanillin), others experienced increases in their levels (lactones, furfural, 4-ethylguaiacol, 4-ethylphenol), and the concentration of other compounds hardly changed (guaiacol, furfuryl alcohol).     

Effect of roasting on the formation of chlorogenic acid lactones in coffee

Of all plant constituents, coffee has one of the highest concentrations of chlorogenic acids. When roasting coffee, some of these are transformed into chlorogenic acid lactones (CGL). We have studied the formation of CGL during the roasting of coffee beans in Coffea arabica cv. Bourbon; C. arabicacv. Longberry; and C. canephora cv. Robusta. Individual CGL levels were determined by comparison of HPLC peaks with those of synthetic CGL standards. Seven CGL were identified: 3-caffeoylquinic-1,5-lactone (3-CQL), 4- caffeoylquinic-1,5-lactone (4-CQL), 3-coumaroylquinic-1,5-lactone (3-pCoQL), 4-coumaroylquinic-1,5-lactone (4-pCoQL), 3-feruloylquinic-1,5-lactone (3-FQL), 4-feruloylquinic-1,5-lactone (4-FQL), and 3,4-dicaffeoylquinic-1,5-lactone (3,4-diCQL). 3-CQL was the most abundant lactone in C. arabica and C. canephora, reaching peak values of 230 +/- 9 and 254 +/- 4 mg/100 g (dry weight), respectively, at light medium roast ( approximately 14% weight loss). 4-CQL was the second most abundant lactone (116 +/- 3 and 139 +/- 2 mg/100 g, respectively. The maximum amount of CGL represents approximately 30% of the available precursors. The relative levels of 3-CQL and 4-CQL in roasted coffee were reverse to those of their precursors in green coffee. This suggests that roasting causes isomerization of chlorogenic acids prior to the formation of lactones and that the levels of lactones in roasted coffee do not reflect the levels of precursors in green coffee.     

Identifying new volatile compounds in toasted oak

Toasting wood to be used in barrels for aging wine produces a great number of volatile and odiferous compounds. Three new volatile odorous compounds in toasted oak were identified. Analysis by high-performance gas chromatography of toasted oak extracts, combined with olfactory detection, enabled various chromatographic peaks with these specific aromas to be isolated. These same odors were simultaneously studied by heating glucose both with and without proline and phenylalanine. Aromatic compounds of interest were identified thanks to a combination of gas chromatography and both mass and infrared spectrometry. An analysis RMN was also used. Hydroxymaltol, 2,5-furanedicarbaldehyde, and furylhydroxymethyl ketone have been detected in extract of toasted oak wood. These molecules may be formed by direct pyrolysis of sugar or Maillard reactions. The acetylformoine was not detected in extract of toasted oak wood, whereas it was detected in heated extracts of various sugars and sugars mixtures with amino acids.     

In vivo studies on the metabolism of the monoterpene pulegone in humans using the metabolism of ingestion-correlated amounts (MICA) approach: explanation for the toxicity differences between (S)-(-)- and (R)-(+)-pulegone

The major in vivo metabolites of (S)-(-)-pulegone in humans using a metabolism of ingestion-correlated amounts (MICA) experiment were newly identified as 2-(2-hydroxy-1-methylethyl)-5-methylcyclohexanone (8-hydroxymenthone, M1), 3-hydroxy-3-methyl-6-(1-methylethyl)cyclohexanone (1-hydroxymenthone, M2), 3-methyl-6-(1-methylethyl)cyclohexanol (menthol), and E-2-(2-hydroxy-1-methylethylidene)-5-methylcyclohexanone (10-hydroxypulegone, M4) on the basis of mass spectrometric analysis in combination with syntheses and NMR experiments. Minor metabolites were be identified as 3-methyl-6-(1-methylethyl)-2-cyclohexenone (piperitone, M5) and alpha,alpha,4-trimethyl-1-cyclohexene-1-methanol (3-p-menthen-8-ol, M6). Menthofuran was not a major metabolite of pulegone and is most probably an artifact formed during workup from known (M4) and/or unknown precursors. The differences in toxicity between (S)-(-)- and (R)-(+)-pulegone can be explained by the strongly diminished ability for enzymatic reduction of the double bond in (R)-(+)-pulegone. This might lead to further oxidative metabolism of 10-hydroxypulegone (M4) and the formation of further currently undetected metabolites that might account for the observed hepatotoxic and pneumotoxic activity in humans.     

Tetrahydro-beta-carbolines, potential neuroactive alkaloids, in chocolate and cocoa

Tetrahydro-beta-carbolines (THbetaCs), potential neuroactive alkaloids, were found in chocolate and cocoa. 6-Hydroxy-1-methyl-1,2, 3,4-tetrahydro-beta-carboline (6OHMTHbetaC), 1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (THCA), 1-methyl-1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA) in both diastereoisomers (1S,3S and 1R,3S), and 1-methyl-1,2,3, 4-tetrahydro-beta-carboline (MTHbetaC), besides serotonin and tryptamine biogenic amines, were identified and quantified in dark chocolate, milk chocolate, cocoa, and chocolate-containing cereals by RP-HPLC-fluorescence and HPLC-MS. For each THbetaC, the concentration ranges were determined: 6OHMTHbetaC (0.16-3.92 microg/g), THCA (0.01-0.85 microg/g), 1S,3S-MTCA (0.35-2 microg/g), 1R,3S-MTCA (0.14-0.88 microg/g), and MTHbetaC (nd-0.21 microg/g). The highest content was generally found in chocolates and cocoas, but cereals containing chocolate also showed an appreciable amount of THbetaCs. The possible biological implications of this novel group of alkaloids in chocolate are discussed.     

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.     

Abscisic acid related compounds and lignans in prunes (Prunus domestica L.) and their oxygen radical absorbance capacity (ORAC)

Four new abscisic acid related compounds (1-4), together with (+)-abscisic acid (5), (+)-beta-D-glucopyranosyl abscisate (6), (6S,9R)-roseoside (7), and two lignan glucosides ((+)-pinoresinol mono-beta-D-glucopyranoside (8) and 3-(beta-D-glucopyranosyloxymethyl)-2- (4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofuran (9)) were isolated from the antioxidative ethanol extract of prunes (Prunus domestica L.). The structures of 1-4 were elucidated on the basis of NMR and MS spectrometric data to be rel-5-(3S,8S-dihydroxy-1R,5S-dimethyl-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid (1), rel-5-(3S,8S-dihydroxy-1R,5S-dimethyl-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid 3'-O-beta-d-glucopyranoside (2), rel-5-(1R,5S-dimethyl-3R,4R,8S-trihydroxy-7-oxa-6-oxobicyclo[3,2,1]oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid (3), and rel-5-(1R,5S-dimethyl-3R,4R,8S-trihydroxy-7-oxabicyclo[3,2,1]- oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid (4). The antioxidant activities of these isolated compounds were evaluated on the basis of oxygen radical absorbance capacity (ORAC). The ORAC values of abscisic acid related compounds (1-7) were very low. Two lignans (8 and 9) were more effective antioxidants whose ORAC values were 1.09 and 2.33 micromol of Trolox equiv/micromol, respectively.     

Isolation and structure of whiskey polyphenols produced by oxidation of oak wood ellagitannins

Three new phenolic compounds named whiskey tannins A and B and carboxyl ellagic acid were isolated from commercial Japanese whiskey, along with gallic acid, ellagic acid, brevifolin carboxylic acid, three galloyl glucoses, a galloyl ester of phenolic glucoside, 2,3-(S)-hexahydroxydiphenoylglucose, and castacrenin B. Whiskey tannins A and B were oxidation products of a major oak wood ellagitannin, castalagin, in which the pyrogallol ring at the glucose C-1 position of castalagin was oxidized to a cyclopentenone moiety. These tannins originated from ellagitannins contained in the oak wood used for barrel production; however, the original oak wood ellagitannins were not detected in the whiskey. To examine whether the whiskey tannins were produced during the charring process of barrel production, pyrolysis products of castalagin were investigated. Dehydrocastalagin and a new phenolcarboxylic acid trislactone having an isocoumarin structure were isolated, along with castacrenin F and ellagic acid. However, whiskey tannins were not detected in the products.     

Synthesis and analysis of thio-, thiono-, and dithio-derivatives of whiskey lactone.

Cis- and trans-3-methyl-4-octanolide (1, whiskey lactones) were converted into their thio- (2), thiono- (3), and dithio- (4) derivatives by reaction with phosphorus pentasulfide. The reaction products were characterized by GC-mass spectrometry, (1)H NMR spectroscopy, and GC-olfactometry. Two-dimensional NOESY spectra showed that sulfur is incorporated into the ring with reversal of the absolute configuration at C-4, whereas substitution of the keto-oxygen atom by sulfur occurs with retention of ring configuration. The cis- and trans-pairs of 2, 3, and 4 were separated into enantiomers by GC on heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin and heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin as chiral stationary phases. GC-olfactometry revealed a sweet coconut-like odor for the cis-thio- and pleasant mushroom-like flavors for the cis-thiono- and trans-dithio-derivatives of whiskey lactone.     

Volatile organic sulfur-containing constituents in Poncirus trifoliata (L.) Raf. (Rutaceae)

During our screening of plant materials to find new natural fragrance and flavor ingredients, we discovered two series of 3-sulfanylalkyl alkanoates in a peel extract of fruits of wild-growing Poncirus trifoliata (L.) Raf. (Rutaceae), a species closely related to Citrus. The two series belong to alkanoates of 3-methyl-3-sulfanylbutan-1-ol and 3-sulfanylhexan-1-ol, respectively, and thus are members of a family of natural molecules having in common a 1,3-positioned O,S moiety. The alkanoate residues comprise all even-numbered saturated fatty acids from C2 (acetate) to C18 (octadecanoate). Among the 20 sulfur-containing compounds identified, 14 are described for the first time as naturally occurring in a botanical species. Several cysteine-S-conjugates were synthesized as hypothetical precursors of the new volatile sulfur-containing constituents, where after S-(3-hydroxy-1,1-dimethylpropyl)-L-cysteine, S-[3-(acetyloxy)-1,1-dimethylpropyl]-L-cysteine, and S-[1-(2-hydroxyethyl)butyl]-L-cysteine were identified in the fruit peel. No cysteine-S-conjugates were detected in the fruit juice.