SCIENCES DES ALIMENTS, 21(2001) 65-70
NOTE
Some compounds detected for the first time in oak wood extracts by GC/MS
María Dolores GUILLÉN *, María José MANZANOS
RÉSUMÉ Quelques composés détectés pour la première fois dans les extraits de bois de chêne par GC/MS.
Les constituants mineurs de la sciure de bois de chêne ont été extraits par le dichlorométhane et analysés par couplage chromatographie en phase gazeuse/
spectrométrie de masse. Parmi les nombreux composés extraits, le sualène, la vitamine E, les α- et β-amyrines, la β-sitostenone, la 5α-stigmast-3-one et la friedeline (ou des isomères de ces deux derniers) sont identifiés pour la premiè- re fois comme des constituants du bois de chêne. La présence de l’acide o-tri- méthylellagique, et celle d’un groupe de composés provisoirement considérés comme des alkylferulates, dont nous donnons les spectres de masse, est aussi à signaler. La présence de beaucoup de ces composés dans le bois de chêne est d’un grand intérêt pour certains procédés de l’industrie alimentaire en raison de leurs propriétés.
Mots clés : bois de chêne, constituants chimiques, activité antioxydante, GC/
MS, spectrométrie de masse.
SUMMARY
The minor components of oak wood sawdust, extracted with dichloromethane, were studied by means of gas chromatography/mass spectrometry. Among the high number of compounds found, squalene, vitamin E, α- and β-amyrin, β-si- tostenone, 5α-stigmast-3-one and friedelin, or isomers of the two latter, are re- ported here as oak components for the first time. The presence of o- trimethylellagic acid and a group of compounds, tentatively considered as alkyl ferulates, whose mass spectra are given, is also noteworthy. The occurrence of many of these compounds in oak is of great interest in the processing of some foods because of their properties.
Key-words:oak wood, components, dichloromethane extract, antioxidant acti- vity, GC/MS, mass spectral data.
Tecnología de los Alimentos, Facultad de Farmacia, Universidad del País Vasco, Paseo de la Universidad 7, E-01006 Vitoria, Spain.
* Correspondance.
1 - INTRODUCTION
Oak wood has received much attention because it is used to make casks for the ageing of high quality alcoholic beverages. It is well known that this wood is a source of flavour precursors which improve the organoleptic properties of the alcoholic beverages during the maturation process.
Oak wood has been also used for the traditional smoking of foods in many European countries. It has been proved that this wood confers appreciable orga- noleptic properties to the smoked foods, because smoke composition depends, among other factors, on the wood nature (GUILLÉN and IBARGOITIA, 1996; GUILLÉN and MANZANOS, 1999).
Several methods have been used for the study and characterization of oak.
Some authors have studied oak extracts using different analytical techniques and the number and nature of compounds detected is very different in each case. These compounds depend on the nature of the wood subject of study, on its species, geographic origin, tree localization and treatment, but also on the solvent and technique used for the extraction, and on the analytical technique used to study the extract. In most of the studies on uncharred or charred oak extracts using high performance liquid chromatography (HPLC) (MOUTOUNETet al., 1989; FERNÁNDEZ DESIMÓN et al., 1996) only a restricted number of com- pounds have been detected or identified. However, in studies using gas chroma- tography/mass spectrometry (GC/MS) the number of detected compounds is considerable (WEIBMANNet al., 1989; PÉREZ-COELLO et al.,1998).
This paper reports some of the detected components in the dichloromethane extract of a French oak wood studied by GC/MS. This study can contribute to understand the influence of wood composition on the development of functional properties and flavour in some processed foods in which oak wood is involved.
2 - MATERIAL AND METHODS
2.1 Sample and extraction
The studied sample was French oak (Quercus sp.) sawdust, of unknown ori- gin and species. The sawdust was air-dried and sieved to yield a particle size
≤ 2 mm. Extraction of between 1-2 g of the sawdust was carried out with 15 mL of dichloromethane in an ultrasonic bath for 1 h, to ensure an exhaustive extrac- tion, as in previous studies (GUILLÉN and MANZANOS, 1998; 1999). The soluble fraction was separated by filtration. Multiple oak sample extraction experiments were performed.
2.2 GC/MS analysis
The GC/MS study was performed using a Hewlett-Packard chromatograph, model 6890 series II, equipped with a mass spectrometer selective detector
Components of oak extracts 67
5973, and a Hewlett-Packard Vectra Pentium computer. A fused-silica capillary column (60 m long, 0.25 mm i.d., 0.25 µm thickness), coated with a nonpolar sta- tionary phase (Hewlett-Packard 5, cross-linked 5% phenyl methyl silicone) was used. The temperature program began at 50ºC (0.5 min) with an increase of 5ºC min–1 up to 300ºC (10 min). Helium was used as the gas carrier with a rate of 26 cm·s–1. Injector and detector temperatures were 250 and 280ºC respecti- vely. The injection technique used was splitless, and the injection volume was 1 µL. Mass spectra were recorded at an ionization energy of 70 eV. Replicate chromatographic analyses of the extract were performed.
Some compounds were identified from retention times and mass spectra of standard compounds (all those compounds are asterisked in table 1), as in pre- vious studies (GUILLÉN and MANZANOS, 1998; 1999); and others were tentatively identified from their mass spectra by comparison with a commercial database (Wiley 138k Mass Spectral Database, 1990). Compounds used as standards for identification were available from Aldrich, Fluka and Sigma. Components with volatility higher than or similar to that of CH2Cl2 were not analyzed.
The semi-quantification of the components of the oak extract was carried out by integrating the chromatographic peaks.
3 - RESULTS AND DISCUSSION
The extraction yield of the oak sawdust was 0.3% on dry basis. Table 1 shows the 101 tentatively identified components.
Among phenolic derivatives, guaiacol, syringol and their alkyl and carbonyl derivatives were present in low proportions. These components, with well known organoleptic and antioxidant properties, have also been found before in oak wood at trace levels (FERNÁNDEZ DE SIMÓN et al., 1996; PÉREZ-COELLO et al., 1998), in small proportions in high quality alcoholic beverages (MOUTOUNET et al., 1989) and all of them, in significant concentrations, in smoke and smoke flavou- rings (GUILLÉN and IBARGOITIA, 1996; GUILLÉN and MANZANOS, 1999).
This group also includes di-tert-butyl-hydroxytoluene or BHT, at trace levels, and octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propionate in a significant proportion (2.1%). The presence of these two known synthetic antioxidants, not detected in oak wood before, is not due to possible contamination by the solvent used. Nevertheless both have structures related to those mentioned above.
In addition, this group includes o-trimethylellagic acid, a compound not pre- viously described as an oak component; and several compounds, supposedly alkyl ferulates (AF), whose mass spectral data are also given in table 1. All these latter compounds contain as base peak m/z 194 and as fragments m/z 177, 150 and 137; these ions are characteristic of mass spectra of ferulic acid. In addition, the difference between the molecular ion peaks of these mass spectra and the base peaks agrees with the molecular weight of long chain linear hydrocarbons.
For this reason, it could be inferred that these compounds could have the struc- tures of ferulates having alkyl chains of 18, 20 and 22 carbon atoms. Mass spec-
Table 1
Components in the dichloromethane extract of oak wood sawdust, together with the proportions of some components (in parenthesis)
Phenolic derivatives (4.1%):phenol* (tr); 2-methoxyphenol (guaiacol)* (tr); 4-methyl-2- methoxyphenol* (tr); 4-ethyl-2-methoxyphenol* (tr); 2,6-dimethoxyphenol (syringol)* (tr);
4-(2-propenyl)-2-methoxyphenol* (tr); 4-propyl-2-methoxyphenol* (tr); 4-hydroxy-3- methoxybenzaldehyde* (0.1%); 4-methyl-2,6-dimethoxyphenol* (tr); 4-(1-propenyl)-2- methoxyphenol (or isomer)* (tr); 1-(4-hydroxy-3-methoxyphenyl)-ethanal (tr); 4-(2-prope- nyl)-2,6-dimethoxyphenol* (tr); 4-hydroxy-3,5-dimethoxybenzaldehyde* (tr); methyl 2,4- dihydroxy-3,6-dimethylbenzoate (or isomer) (tr); 6-methyl-2,4-di-tert-butyl-phenol (or iso- mer) (tr); di-tert-butyl-hydroxytoluene (BHT)* (tr); 4-hydroxy-3,5-di-tert-butylbenzaldehyde (0.1%);octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propionate*(2.1%);o-trimethylel- lagic acid (1.2%); AF [446(59), 194(100), 177(30)] (tr); AF [446(65), 194(100), 177(53), 150(23), 145(16), 137(25)] (tr); AF [474(57), 194(100), 177(53)] (tr); AF [474(67), 194(100), 177(60), 150(23), 137(26)] (0.4%); AF[502(82), 194(100), 177(70)] (tr);AF [502(73), 194(100), 177(68), 150(26), 145(16)] (0.2%)
Aromatic hydrocarbons (traces): (1-pentylhexyl)-benzene (tr); (1-butylheptyl)-benzene (tr);
(1-propyloctyl)-benzene (tr); (1-pentylheptyl)-benzene (tr); (1-butyloctyl)-benzene (tr); (1- propylnonyl)-benzene (tr); (1-ethyldecyl)-benzene (nq); (1-methylundecyl)-benzene (tr); (1- pentyloctyl)-benzene (tr); (1-butylnonyl)-benzene (tr); (1-propyldecyl)-benzene (nq); (1- methyldodecyl)-benzene (tr); 2-methylnapthalene* (tr); 1-methylnapthalene* (tr); dimethyl- napthalene (tr); phenanthrene* (tr)
Oxygen aromatic derivatives (traces): benzenethanol (tr); 2-phenyl ethyl acetate (tr);
methyl 2-methoxybenzoate (tr); methyl 3-phenyl-2-propenoate* (tr); 4-(2-propenyl)-1,2-di- methoxybenzene* (tr); ethyl 4-ethoxybenzoate (tr); 5-methoxy-6,7-dimethylbenzofuran (tr);
diphenylmethanone (tr)
Fatty acids and esters (5.4%):dodecanoic acid* (tr); tetradecanoic acid* (0.1%), isopropyl tetradecanoate (tr); methyl hexadecanoate (tr); hexadecanoic acid* (1.4%); 1-methylethyl hexadecanoate (0.1%); 9,12-octadecadienoic acid (1.2%); 9-octadecenoic acid* (1.2%); oc- tadecanoic acid (0.5%); eicosanoic acid (0.1%); 1-heneicosyl formiate (0.1%); docosanoic acid (0.2%); tetracosanoic acid (0.5%)
Aldehydes (0.2%):nonanal (tr); cis,trans-2,4-decadienal* (tr); trans,trans-2,4-decadienal*
(tr); eicosanal (0.1%); docosanal (0.1%)
Linear hydrocarbons (1.2%): pentadecane* (tr); hexadecane* (0.1%); heptadecane* (tr); 1- octadecene (tr); octadecane* (0.1%); nonadecane* (tr); 1-eicosene (nq); eicosane* (0.1%);
1-heneicosene (0.1%); heneicosane* (0.1%); docosane* (0.1%); 1-tricosene (0.1%); trico- sane* (0.1%), 1-pentacosene (0.1%); pentacosane* (tr); hexacosane* (0.1%); octacosane*
(0.2%)
Triterpenoids and related compounds (35.1%):squalene* (1.1%); cholesterol (0.6%); vi- tamin E* (0.9%); campesterol* (0.6%); stigmasterol* (1.5%); β-sitosterol* (11.1%); 23- ethylcholestanol (or isomer) (2.4%); β-amyrin (3.7%); 5α-stigmast-3-one (or isomer) (2.0%); α-amyrin (2.6%); β-sitostenone (5.5%); friedelin (or isomer) (3.1%)
Other compounds (0.2%): cis-caryophyllene (or isomer) (tr); 4-isopropenyl-3-carene (tr);
2,6-bis-(1,1-dimethylethyl)-2,5-cyclohexadiene-1,4-dione* (tr); 5,6,7,7a-tetrahydro-4,4,7a- trimethyl-2(4H)-benzofuranone (tr); 6,10,14-trimethyl-2-pentadecanone (0.2%)
Asterisked compounds were acquired commercially and used as reference compounds for the identi- fication. AF: Supposed alkyl ferulate; mass spectral data are given in brackets.
tr, Compounds in very small proportion.
nq, Compound not quantified because its separation was not adequate.
Components of oak extracts 69
tral data of alkyl ferulates have not been published before. Some authors have described the presence of a mixture of ferulic acid esters in oak extracts, of which tetracosyl ferulate is the major component (CHEN, 1970); more recently the feruloyl ester of docosanol has been identified as a suberin unit (GRAÇA and PEREIRA, 1999) and also some of the alkyl ferulates, mentioned in this study, have been found in stems of Thymus vulgaris L. (GUILLÉN and MANZANOS, 1998). The presence of these latter compounds in oak wood and their possible transfer to processed foods is important, not only in relation to foods preservation but also in relation to human health, because of their known antioxidant ability.
In addition to the compounds above mentioned, some alkyl aromatic hydro- carbons, not previously described as oak components, were also detected at trace levels. Likewise some polycyclic aromatic hydrocarbons, not harmful to health, together with several oxygen aromatic derivatives (alcohols, esters, ethers and ketones) were found at trace levels in this oak wood. Compounds of these two latter groups have been found in oak wood before (NISHIMURA et al., 1983; PÉREZ-COELLO et al., 1998).
As in previous reports (NISHIMURA et al., 1983; WEIBMANNet al., 1989; PÉREZ- COELLO et al.,1998), fatty acids and esters (5.4%), a numerous group of linear saturated and unsaturated hydrocarbons of a large number of carbon atoms (1.2%) and linear aldehydes (0.2%) were found. The presence of the two 2,4- decadienal isomers, could be due to the oxidation of fatty acids.
In addition, a numerous group of triterpenoids and related compounds, some of them described as oak components (WEIBMANNet al., 1989), were found in a large proportion (35%). Worthy of note is the presence of squalene, vitamin E, α- and β-amyrin, β-sitostenone, 5α-stigmast-3-one and friedelin, or isomers of the two latter, not described before as oak components. The presence of some of these latter compounds is also very important in food technology because of their well known antioxidant activity.
Finally, other compounds of various natures, in low proportions, were also detected, such as the nor-isoprenoid 5,6,7,7a-tetrahydro-4,4,7a-trimethyl- 2(4H)-benzofuranone, possibly coming from β-carotene decomposition, and some terpene derivatives.
In conclusion, the number of minor components detected was very high.
Extraction with dichloromethane in an ultrasonic bath has allowed the detection of new components, some of which have very interesting properties for food technology and human health.
ACKNOWLEDGEMENTS
This work has been supported by the Comisión Interministerial de Ciencia y Tecnología (CICYT, ALI97-1095) and Universidad del País Vasco (UPV 101.123- EB087/99).
Receveid 5 June 2000, accepted 17 October 2000.
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