Food Chemistry 125 (2011) 1398–1405

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Food Chemistry

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Analytical Methods

Analysis of phenolic compounds in cork from Quercus suber L. by HPLC–DAD/ESI–MS

Ana Fernandes a, André Sousa a, Nuno Mateus a, Miguel Cabral b, Victor de Freitas a,⇑a Centro de Investigação em Química, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugalb Amorim and Irmãos, S.A., Rua de Meladas 380, 4536-902 Mozelos VFR, Portugal

a r t i c l e i n f o

Article history:Received 11 August 2009Received in revised form 14 June 2010Accepted 3 October 2010

Keywords:CorkEllagitanninsFlavanoellagitanninsLC-DAD/ESI-MSPhenolic compoundsQuercus suber L.

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.10.016

⇑ Corresponding author. Tel.: +351 220402558; faxE-mail address: vfreitas@fc.up.pt (V. de Freitas).

a b s t r a c t

The aim of the present work was to identify the extractable phenolic compounds present in cork fromQuercus suber L. The structures of thirty three compounds were tentatively identified by liquid chroma-tography coupled to electrospray ionisation mass spectrometry (HPLC–DAD/ESI–MS). The majority ofthose compounds were gallic acid derivatives, in the form of either galloyl esters of glucose (gallotan-nins), combinations of galloyl and hexahydroxydiphenoyl esters of glucose (ellagitannins), dehydratedtergallic-C-glucosides or ellagic acid derivatives. Others were found to correspond to low molecularweight phenolic compounds, like acids and aldehydes. Mongolicain, a flavanoellagitannin in whichhydrolysable tannin and flavan-3-ol moieties are connected through a carbon–carbon linkage, was alsodetected in cork from Q. suber L. The results illustrate the rich array of phenolic compounds present incork.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Cork is the suberized cellular tissue that is continuously pro-duced by the phellogen of the cork oak tree (Quercus suber L.), a na-tive species of the Mediterranean region. The unique properties ofthis raw material, which include low density, low permeability toliquids, ability to adhere to a glass surface, compressibility, resil-ience, elasticity, chemical inertness and resistance to microbialgrowth, have promoted its use in a variety of sectors, but its mostvisible and profitable products are the stoppers for wine bottling(Casey, 1994; Jung & Hamatscheck, 1992).

Natural cork is composed essentially of suberin, the main corkcomponent, lignin, polysaccharides (hemicelluloses and cellulose)and extractable components (Pereira, 1988). The relative abun-dance of these fractions is extremely variable, even among treesof the same forest. Chemical composition can be influenced by geo-graphical origin, climate and soil conditions, genetic origin, dimen-sion and age of the tree (virgin or reproduction) or even thedifferent parts of the tree from which the cork was obtained(Conde, Cadahía, García-Vallejo, & Fernández de Simón, 1998;Pereira, 1988). The material unbounded or loosely bounded tothe cork cell wall, i.e., the extractable material, is mainly composedof aliphatic, triterpenic and phenolic compounds, exhibiting onlyca. 2% of carbohydrates (Rocha, Coimbra, & Delgadillo, 2004; Ro-cha, Ganito, Barros, Carapuça, & Delgadillo, 2005) and it can be eas-

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ily extracted with solvents (Snakkers, Nepveu, Guilley, & Cantagrel,2000). In fact, if cork is in direct contact with ethanolic solution,some cork components can migrate into the wine after bottling,thus affecting wine quality (Varea, García-Vallejo, Cadahía, &Fernández De Simón, 2001). Volatile and non-volatile compounds,soluble in ethanol/water, such as hydrocarbons, alcohols, ketones,phenolic compounds and tannins are oenologically importantdue to their contribution to sensory properties – colour, flavour,astringency and bitterness (Mazzoleni, Caldentey, Careri, Mangia,& Colagrande, 1994). Phenolic acids do not have a direct influenceon the organoleptic characters of wines but some of them are pre-cursors of volatile phenols (especially vinyl and ethyl derivatives ofphenols), substances that influence wine aroma (Chatonnet, Boi-dron, & Pons, 1990; Singleton, 1995). Ellagitannins play an impor-tant role in wine oxidation processes as they rapidly react withdissolved oxygen and facilitate the hydroperoxidation of wine con-stituents (Vivas & Glories, 1996). These compounds affect the con-densation rate of proanthocyanidins as well as condensed tanninprecipitation and anthocyanidins destruction (Vivas & Glories,1993) and they can undergo numerous chemical transformations,e.g. reacting with flavanols (Saucier, Jourdes, Glories, & Quideau,2006). On the other hand, their taste properties grant them animportant role in the ageing of wines (Pocock, Sefton, & Williams,1994).

In addition to these properties, polyphenolic compounds havebeen shown to be responsible for many health benefits, includingantibacterial, antiviral, anticarcinogenic, anti-inflammatory andantiallergic activity (Laranjinha & Cadenas, 1999; Santos-Buelga& Scalbert, 2000).

A. Fernandes et al. / Food Chemistry 125 (2011) 1398–1405 1399

A number of low molecular weight phenolic compounds wereisolated and identified from reproduction cork of Spanish Q. suber,including ellagic acid as the main compound, with detectablequantities of gallic, protocatechuic, vanillic, caffeic and ferulicacids. Protocatechuic and sinapic aldehydes, vanillin, coniferalde-hyde and coumarins like aesculetin and scopoletin were also iden-tified (Conde, Cadahía, García-Vallejo, Fernández de Simón, &González Adrados, 1997). Although the inner bark of the cork oaktree is an important tannin source, very little information is avail-able on tannins and related polyphenols present in cork. The quan-titative evaluations of the different tannin groups in cork haverevealed small amounts of tannins that are extracted withMeOH/H2O, being the ellagitannins the most representative com-pounds of this polyphenolic extract (Cadahía, Conde, Fernándezde Simón, & García-Vallejo, 1996). Several molecular structuresof ellagitannins with wide distribution in the wood and bark ofspecies of Quercus have already been identified (Hervé Du Penhoatet al., 1991; Mayer, Gabler, Riester, & Korger, 1967; Mayer, Seitz, &Jochims, 1969; Mayer, Seitz, Jochims, Schauerte, & Schilling, 1971;Nonaka, Ishimaru, Azuma, Ishimatsu, & Nishioka, 1989; Scalbert,Monties, & Janin, 1989). These include the monomers castalaginand vescalagin, the pentosylated monomers roburin E and grandi-nin and the dimer roburin A, besides other ellagitannins with re-lated structure and other ellagic acid derivatives (Cadahía et al.,1996).

A number of other hydrolysable and complex tannins were re-cently isolated from the leaves of Algerian Q. suber, including coc-ciferins D2, D3 and T2, pedunculagin, acutissimin B, tellimagrandinI, castavaloninic acid, isocastavaloninic acid, mongolicain A, anddesgalloylstachyurin (Ito et al., 2002). In the acorns of Q. suber aseries of gallotannins and ellagitannins have also been detected(Cantos et al., 2003), but to our knowledge there is no informationregarding the presence of this group of compounds in cork from Q.suber.

Therefore, the aim of this work was the isolation and identifica-tion of the different groups of polyphenolic compounds present incork from Q. suber L.

2. Material and methods

2.1. Chemicals

TSK Toyopearl gel HW-40 (S) was purchased from Tosoh(Tokyo, Japan). Gallic and ellagic acid were purchased fromSigma–Aldrich (Madrid, Spain) and Fluka (Buchs, Switzerland),respectively.

2.2. Plant material and extraction

Grounded cork (0.5–1 mm particle size) from different Q. suberL trees free of outer corkback was obtained by grinding and sievingPortuguese cork (by-product of cork stoppers industry), kindlysupplied by Amorim and Irmãos (Mozelos, Portugal).

Samples of grounded cork (60.0 g) were extracted with 1.0 L ofmodel wine solution (12% ethanol, 5.0 gL�1 tartaric acid buffered topH 3.2) for 72 h at room temperature with occasional agitation.The suspension was filtered on a Büchner funnel and ethanolwas removed by vacuum distillation. The aqueous residue wasthen spray dried on a Büchi Mini Spray Drier B-290� and the pow-der obtained was redissolved in water and extracted three timeswith ethyl acetate. The organic fractions were combined and afterdrying with anhydrous sodium sulphate they were evaporated todryness under vacuum. The residue was dissolved in H2O/MeOH(9:1; v/v) and freeze-dried.

2.3. Column chromatography

Fractionation of cork phenolic compounds was carried outaccording to the method described elsewhere (de Freitas, Glories,Bourgeois, & Vitry, 1998). A portion of the above freeze-dried res-idue was dissolved in methanol and chromatographed on a TSKToyopearl HW-40(S) prepared column (250 � 16mm i.d.) usingmethanol as the eluent, at 0.8 mLmin�1. Phenolic compounds wereseparated based on molecular weight. Fractions were collectedupon detection with a Gilson 115 UV Detector and a SP4290 inte-grator from Spectra-Physics at 280 nm and each fraction obtained(I–VIII) was freeze-dried after eliminating the solvent with a rota-tory evaporator under reduced pressure at 30�. The resulting solidswere analysed by HPLC–DAD and LC–DAD/ESI–MS.

2.4. HPLC–DAD analysis

The samples were analysed by HPLC (Merck-Hitachi Elite La-chrom) on a 150 � 4.6 mm i.d. (5 lm pore size) reversed-phaseC18 column (Merck) thermostated at 25� C (Merck-Hitachi ColumnOven L-2300), according to an adaptation of a method describedelsewhere (Peña-Neira et al., 1999). Detection was carried out at280 nm using a diode array detector (Merck-Hitachi Diode ArrayDetector L-2455). Two solvents were applied for elution: A-water/acetic acid (99:1; v/v) and B-water/acetonitrile/acetic acid(79:20:1; v/v/v). The gradient profile was: 0–55 min, 80–20% A,55–70 min, 20–10% A, 70–90 min, 10–0% A, 90–120 min, 0% A (iso-cratic flow) at a flow rate of 0.3 mLmin�1. The sample injection vol-ume was 20 lL. The chromatographic column was washed with100% B for 10 min and then stabilized with the initial conditionsfor another 10 min.

2.5. LC–DAD/ESI–MS analysis

A Finnigan Surveyor series liquid chromatograph, equippedwith the same column as mentioned earlier and thermostated at25� C was used. The samples were analysed using the same sol-vents, gradients, injection volume, and flow rate referred abovefor the HPLC analysis. Double-online detection was done by a pho-todiode spectrophotometer and mass spectrometry. The massdetector was a Finnigan LCQ DECA XP MAX (Finnigan Corp., SanJose, CA) quadrupole ion trap equipped with atmospheric pressureionisation (API) source, using electrospray ionisation (ESI) inter-face. The vaporiser and the capillary voltages were 5 kV and 4 kV,respectively. The capillary temperature was set at 325� C. Nitrogenwas used as both sheath and auxiliary gas at flow rates of 80and 30, respectively (in arbitrary units). Spectra was recorded inthe negative ion mode between m/z 120 and 2000. The massspectrometer was programmed to do a series of three scans: a fullmass, a zoom scan of the most intense ion in the first scan, and aMS–MS of the most intense ion using relative collision energiesof 30 and 60.

3. Results and discussion

The aqueous extract of cork was chromatographed on TSK Toyo-pearl gel, yielding eight fractions, as shown in Fig. 1. Each fractionwas analysed by HPLC/ESI–MS and some structures were postu-lated by the UV–Vis spectrum and MS fragmentation pattern.

The MS fragmentation provides important information aboutthe structure which is particularly useful in the case of gallic andellagic tannins. Thus, the losses of 152 and 170 mass units fromthe [M�H]� quasi-molecular ion indicates the presence of galloylgroups and the loss of 302 from the [M�H]� quasi-molecular ionand the presence of an ion at m/z 301 indicates the presence ofHHDP groups. Loss of 44 mass units is characteristic of a free

Fig. 1. TSK Toyopearl gel fractionation of phenolic compounds from cork extract(250 � 16 mm i.d.; MeOH as eluent; flow rate at 0.8 mL min�1).

1400 A. Fernandes et al. / Food Chemistry 125 (2011) 1398–1405

carboxyl group and losses of 18 (H2O) from the [M�H]� ion is char-acteristic of C-glucosidic ellagitannins. When molecular weights ofhydrolysable tannins differ by two units, it can be related to thedifference between either an HHDP group or two galloyl groups(Barry, Davies, & Mohammed, 2001).

Fig. 2 shows the HPLC-DAD chromatogram of four representa-tive fractions obtained. Table 1 shows UV–Vis and ESI–MS charac-

mA

U

10 20 30 40 50 60 70 80 90 100 110 120

200

600

1000

1400

1800

2200

0

V

Time (min)

32

19

28

24

Time (min) 10 20 30 40 50 60 70 80 90 100 110 120

mA

U

200

400

600

800

0

I

1

3

4

6

2 7

5

Fig. 2. HPLC–DAD chromatograms of four isolated fractions (I, II, V, VII). Identificat

teristics of the tentatively identified compounds in Q. suber L.Unidentified compounds are shown in Table 2.

The UV spectra of the different phenolic compounds present inthese fractions showed that they could be arranged into twogroups: those showing a spectrum data in agreement with thoseof ellagic acid with two maximum wavelengths of absorption(kmax) at 255 nm and 367 nm and those showing a characteristicspectrum of gallic acid with a single kmax at 271 nm. The first groupincludes compounds that present an ellagic acid residue in theirmolecular structure and the second group includes all the galloyland hexahydroxydiphenoyl derivatives (Cantos et al., 2003).

3.1. Fraction I

Fractions I was collected in the first 150 min of the chromato-graphic fractionation. This fractions revealed the presence of lowmolecular weight phenols such as phenolic acids like gallic (4),protocatechuic (3), caffeic (6), and ferulic (7) acids (Fig. 2). Identi-fication of phenolic acids was based on comparing their retentionbehaviour on the HPLC and with that of standard material an onmass spectrometric detection that gave respective [M�H]� quasi-molecular ion peaks at m/z 169, 153, 179 and 193. These fractionsalso exhibited in the MS spectra ion peaks at m/z 137, 151 and 177corresponding to protocatechuic aldehyde (1), vanillin (2) and

Time (min) 10 20 30 40 50 60 70 80 90 100 110 120

mA

U

200

400

600

800

0

1515

12

12

8

27 17

II

14

Time (min)10 20 30 40 50 60 70 80 90 100 110 120

mA

U

200

400

600

800

0

VIII

21

25

23

ion of the numbers present in the chromatograms is shown in Tables 1 and 2.

Table 1HPLC/ESI–MS data for phenolic compounds tentatively identified in cork of Quercus suber.

Postulated compounds Compound number [M�H]� m/z MS2 ions (m/z) MS3 ions (m/z)* kmáx (nm) Fraction

Protocatechuic aldehyde 1 137 109 – 280; 310 IVanillin 2 151 136 108 279; 308 IProtocatechuic acid 3 153 109 – 259; 293 IGallic acid 4 169 125 97 270 IConyferaldehyde 5 177 162 133; 120; 106 289; 340 ICaffeic acid 6 179 – – 322 IFerulic acid 7 193 149 134 319 IEllagic acid 8 301 257; 229 – 250; 373 I, IIEllagic acid-pentose 9 433 301 257; 229 253; 373 IEllagic acid-deoxyhexose 10 447 301 257; 229 253; 373 IEllagic acid-hexose 11 463 301 257; 229 253; 373 IValoneic acid dilactone 12 469 425 407; 301 253; 373 IIHHDP-glucose 13 481 301 257; 229 262; 379 IValoneic acid 14 505 313 – 253; 370 IIDehydrated tergallic-C-glucoside 15 613 593; 523; 493 465; 301; 299 253; 373 IIHHDP-galloyl-glucose 16 633 463; 301 257; 229; 185 262 IIITrigalloy-glucose 17 635 483; 465 421; 313; 169 277 IIDi-HHDP-glucose 18 783 481; 301 257; 229 271 III; IVHHDP-digalloyl-glucose 19 785 633; 483; 301 257; 229 271 III; IVTetragalloyl-glucose 20 787 635; 617 573; 465; 403 274 IIICastalagin/Vescalagin 21 933 915; 631 613; 569 247 VII; VIIIDi-HHDP-galloyl-glucose 22 935 633; 873; 783 571; 329; 299 271 VITrigalloyl-HHDP-glucose 23 937 785; 767; 635; 465 301 277 VIIIPentagalloyl-glucose 24 939 787; 769 617; 599; 447 279 VMongolicain A/B 25 1175 873; 855; 721; 677 829; 785; 767 267 VIII

HHDP:hexahydroxydiphenyl.* MS3 of the MS2 bold type ion.

Table 2HPLC/ESI–MS data for unknown compounds present in cork of Quercus suber.

Unknown compounds Compound number [M�H]� m/z MS2 ions (m/z) MS3 ions (m/z)* kmáx (nm) Fraction

Compound A 26 465 447; 421; 403 403 283; 352 ICompound B 27 595 523; 505; 301 301 253; 373 IICompound C 28 807 505 355; 311; 301 – V, VICompound D 29 873 829; 785; 721 767; 683; 633 262 IIICompound E 30 915 613; 595; 523 523; 299 250; 373 IVCompound F 31 947 915; 897; 871 887; 569; 301 – VIICompound G 32 963 945; 661; 643 643; 629; 455 – VCompound H 33 977 945; 675 825; 643 – IV

HHDP: hexahydroxydiphenyl.* MS3 of the MS2 bold type ion.

A. Fernandes et al. / Food Chemistry 125 (2011) 1398–1405 1401

conyferaldehyde (5), respectively. All these compounds were pre-viously reported in cork of Q. suber (Conde et al., 1997). This frac-tion also contained ellagic acid and several ellagic acid derivatives.Ellagic acid (8) with a molecular ion at m/z 301 yielded an inten-sive product ion at m/z 257 and 229. Some of the ellagic acid deriv-atives detected were sugar conjugates. Indeed, mass spectrometricanalysis revealed a quasi-molecular anion of ellagic acid pentoseconjugate (9) at m/z 433. The MS2 spectrum of this compoundyielded an ion at m/z 301 (M-132, loss of a pentosyl unit). MS3

spectrum of the m/z 301 fragment produced two major ions atm/z 257 and 229, which matches the fragmentation pattern of el-lagic acid. Another peak at m/z 447 was identified as an ellagic aciddeoxyhexose conjugate (10) (M-146, loss of a deoxyhexosyl unit)and the peak at m/z 463 was identified as an ellagic acid hexoseconjugate (11) (M-162, loss of hexosyl residue). These ellagic acidderivatives have already been reported in Q. suber acorns and incork in previous works (Cantos et al., 2003; Conde, Cadahía, Gar-cía-Vallejo, & González-Adrados, 1998). This fraction also revealedthe presence of an ellagitannin with [M�H]� at 481 which hasbeen assigned to HHDP-glucose (13), based on molecular weightand the presence of an intense fragment at m/z 301 and anothercompound at m/z 465 (26) still unidentified. This compound differsby 18 units from the gallotannin digalloyl-glucose (m/z 483) and it

could result from the loss of water from this compound to formdehydrated digalloyl glucose (Zywicki, Reemtsma, & Jekel, 2002).

The other fractions (from II to VIII) revealed the compoundsfrom 8 to 25 (Table 1) that were tentatively identified by UV–Visspectroscopy and HPLC/ESI–MS. These were mostly gallic acidderivatives, in the form of either galloyl esters of glucose, combina-tions of galloyl or hexahydroxydiphenoyl esters of glucose, dehy-drated tergallic-C-glucosides or ellagic acid derivatives. Thepresence of mongolicain, a complex tannin was also detected(Fig. 3). The structures of compounds from 26 to 33 were not estab-lished altought some important structural evidences have been ob-tained from MS, indicating that they may be ellagic or gallictannins (Table 2).

3.2. Fraction II

One group of compounds showing a UV spectrum similar to thatof ellagic acid were tentatively identified in fraction II (collected for80 min). The mass spectrum of compound 15 with a [M�H]� qua-si-molecular ion at m/z 613 matches with the structure of thedehydrated tergallic acid-C-glucoside described in the literature(Cantos et al., 2003). These compounds had been previously de-tected in the acorns of Q. suber L, but to our knowledge they have

GOO

OGGO

GOGO

Penta-galloyl-glucose (m/ z 939)

OHHO

HO

O

G =

OHHO

HO

OOO

OHOO

HO

HO

HO O

di-HHDP-glucose (m/z 783)

HO

HO

HO

O

O

HO

HO

OH

O

OHHO

HO

OOO

OHGOO

HO

HO

HO O

HHDP-di-galloyl-glucose (m/z 785)GO

OHHO

HO

OOO

OGO

O

HO

HO

HO O

di-HHDP-galloyl-glucose (m/z 935)

HO

HO

HO

O

O

HOHO

O

OH

OHHO

HO

OOO

OGGO

GOO

HO

HO

HO O

HHDP-tri-galloyl-glucose (m/z 937)

OHOH

OO

O

HOHO

HO

Castalagin (m/z 933)

HO

HO

OH

O

OH

O O

OH

OHHO

HO

HO

OO

OH

HO

O

O

OHOH

O

O

O

HOHO

Mongolicain B (m/ z 1175)

HO

HO

O

OH

O O

HO

HO

OO

OH

O

OHOH

OH

HO

O

HO

O

O

HO

O

OH

Fig. 3. Chemical structures of some Quercus suber phenolic compounds.

1402 A. Fernandes et al. / Food Chemistry 125 (2011) 1398–1405

not been identified in cork. Dehydrated tergallic acid-C-glucosideshows in the MS2 spectrum an ion at m/z 595, 523 and 493 corre-sponding to the loss of water (M�H-18), 90 and 120 mass units,respectively. According to literature, this MS2 fragmentation pat-tern is characteristic of C-glucoside compounds corresponding tothe fragmentation of this residue (Bakhtiar, Gleye, Moulis, & Four-

asté, 1994). The MS3 showed a fragment at m/z 301 correspondingto an ellagic residue which probably explains the kmax at �250 nmand �370 nm characteristic of ellagic acid derivatives. Dehydratedtergallic acid C-glucoside (15) probably result from an internalesterification involving the carboxyl group and one hydroxyl ofthe glucose moiety of compound tergallic acid C-glucoside (m/z

OH

OH

OH

O

O

O

O

OH

OHOHOH

CO

OOH

OH

OHO

OH

OH

OH

O

O

O

O

OH

OHOHOH

COOH

OOH

OHOH

OH

m/z 631 m/z 613 (15)

- H2O

Fig. 4. Possible formation of dehydrated tergallic acid-C-glucoside (m/z 613).

A. Fernandes et al. / Food Chemistry 125 (2011) 1398–1405 1403

631) (Fig. 4). This latter was already reported in the literature,present in Q. suber acorn (Cantos et al., 2003), but was not detectedin this cork extract.

Moreover, valoneic acid dilactone (12) was also detected at m/z469. The MS2 fragments (m/z 425 and 301) were in agreement withthose previously reported for this compound (Nawwar, Marzouk,Nigge, & Linscheid, 1997). Valoneic acid (14) ([M�H]� at m/z505) and an unknown compound at m/z 595 (27) were also presentin fraction II. This latter differs from dehydrated tergallic acid-C-glucoside (15) by 18 units and presents the same fragmentationpattern as this compound. Probably the compound at m/z 595 re-sults from the loss of water from dehydrated tergallic acid-C-gluco-side (15). In this fraction was also detected a quasi molecular ion atm/z 635 compatible with the structure of trigalloyl-glucose (17).The MS2 spectra, revealed the presence of a quasi-molecular ionat m/z 483 and 465 probably due to the loss of a galloyl and a gal-late unit, respectively. In this fraction we were also able to detectthe presence of ellagic acid (8) at m/z 301, probably due to a con-tamination from fraction I.

In the subsequent fractions, a series of gallotannins and ellagit-annins have been identified. Despite the fact that the majority ofthese compounds have been reported in the acorns of Q. suber(Cantos et al., 2003), to our knowledge they have not been reportedin cork.

3.3. Fractions III–V

In fraction III, collected after approximately 230 min of elution,a [M�H]� ion at m/z 633 has been assigned as HHDP-galloyl-glu-cose (16) from the MS2 evidence of an intense ion at m/z 301 dueto the loss of a galloyl-glucose residue (M�H-152–180) and 463(loss of gallate unit). [M�H]� at m/z 787 has been assigned astetragalloyl-glucose (20). The fragments at m/z 617 and 635 corre-spond to the loss of a gallate residue (M�H-170) and a galloyl res-idue (M�H-152). In this fraction, two other compounds at [M�H]�

at m/z 785 and m/z 783 were detected. Compound at m/z 785 hasbeen assigned to HHDP-digalloyl-glucose (19). The quasi-molecu-lar ion suffered the loss of a digalloyl-glucose residue (M�H-484)to give the fragment at m/z 301 and the loss of a HHDP residue(M�H-302) to give the fragment at m/z 483. The compound at m/z 783, which differs by two mass units from the previous, has beenassigned to di-HHDP-glucose (18) and may result from the cou-pling of two adjacent galloyl groups in HHDP-digalloyl-glucoseby intramolecular oxidation.

In this fraction a new compound with [M�H]� quasi-molecularion at m/z 873 (29) was also detected, which structure was notestablished. However, this compound presents the same fragmen-tation pattern as the flavanoellagitannin isolated in fraction VIII([M�H]� at m/z 1175) and differs from this compound by 302units, probably by the loss of a HHDP residue (Fig. 5).

In fraction IV, collected approximately after 400 min of elution,two compounds with [M�H]� at m/z 915 (30) and m/z 977 (33)were detected. These peaks remain unidentified but compound atm/z 977 may be derived from the ellagitannin castalagin or vesca-lagin (m/z 933) from which it differs by 44 units probably corre-sponding to a carboxyl group. Compound at [M�H]� 915 (30)presents a UV spectrum similar to that of ellagic acid, which prob-ably indicates the presence of this phenolic acid residue in itsstructure. On the other hand, this compound presents the samefragmentation pattern as the dehydrated tergallic acid-C-glucoside(m/z 613) from which it differs from 302 units (HHDP group). Bear-ing this, the compound detected at m/z 915 could be formedthrough the attachment of a HHDP residue to the compound atm/z 613 (Fig. 6). In the literature, the compound detected at m/z915 is often associated to dehydrated castalagin or vescalagin(Zywicki et al., 2002) which is not the case herein based on theUV–Vis and mass spectrum. Furthermore two other compoundsat m/z 785 and 783 described before, were detected in this fractionwhich may correspond to a contamination from the previous frac-tion (they present the same HPLC retention time).

The MS/MS spectra of the galloyl-glucose compounds found incork from Q. suber were characterised by the sequential loss of gal-lic acid residues.

In fraction V, collected approximately from 460 to 520 min ofelution, the breakdown of pentagalloyl-glucose at m/z 939 (24)produces a first loss of a galloyl residue (M�H-152) to give a frag-ment at m/z 787. In addition, the loss of a gallic acid to give a frag-ment at m/z 769 (M�H-170) and a sequential loss of anothergalloyl residue yielding a fragment at m/z 617 (M�H-170–152)were also observed. In this fraction the presence of intensive peaksat m/z 963 (32) and 807 (28) were detected (Fig. 2). These peaksremain unidentified but they may belong to ellagitannins as illus-trated by the loss of 302 units from the molecular ion to give thefragment at m/z 661 and 505, respectively.

3.4. Fractions VI–VIII

The analysis of fraction VI revealed the presence of quasi-molecular ion at m/z 935 (di-HHDP-galloyl-glucose) as the mainconstituent. The breakdown of the quasi-molecular ion [M�H]�

at m/z 935 (22) produced a fragment at m/z 633 (loss of a HHDPgroup) and at m/z 783 (loss of a galloyl residue). This fractionwas collected for 90 min.

In fraction VII, collected for 120 min, one compound at m/z 947(31) was detected but remains unidentified. This compound mayalso belong to ellagitannins as suggested by the detection of a frag-ment at m/z 301.

Regarding the C-glucosidic ellagitannins, it was possible to ten-tatively identify in fractions VII and VIII the stereoisomers castala-gin and vescalagin at m/z 933 (21) (Fig. 2). These compounds differ

OHOH

O

O

O

HO

HO

Mongolicain B (m/ z 1175)

HO

HO

O

OH

O O

HO

HO

OO

OH

O

OHOH

OH

HO

O

HO

O

O

HO

O

OH

-HHDP

OHOH

HO

HO

O

OH

O O

HO

HO

OO

OH

O

OHOH

OH

HO

O

O

O

OH

m/ z 873 (29)

Fig. 5. Proposed structures for compound D at m/z 873 (29) and mongolicain A/B at m/z 1175 present in cork from Quercus suber.

OH

OHOH

OO

O

O

OH

OHOHOH

CO

OOH

OHOHO

CO

OHOH

OH

OH

OHOH

CO

OH

OHOH

OO

O

O

OH

OO

OHOHOH

CO

OOH

O

m/z 915 (30)

+ HHDP

m/z 613 (15)

Fig. 6. Proposed structure for compound E at m/z 915 (30) present in cork from Quercus suber.

1404 A. Fernandes et al. / Food Chemistry 125 (2011) 1398–1405

only in their stereochemistry at position C6 of the glucose core.These compounds have already been reported in cork from Q. Suber(Cadahía et al., 1996). Besides these ellagitannins, some authorsalso found roburin E, grandinin and the dimer roburin A but thesewere not detected in the experimental conditions described herein.The MS2 spectrum of castalagin or vescalagin revealed a fragmentat m/z 631 similar to the molecular ion of castalin/vescalin, result-ing from the loss of the ellagic acid esterified at position C1 and C3

of the glucose core.In fraction VIII, collected after 730 min of elution, trigalloyl-

HHDP-glucose (23) [M�H]� at m/z 937 was detected. This com-pound showed in the MS2 spectrum the loss of a gallate residue(M�H-170) yielding an ion fragment at m/z 767 and the loss of agalloyl group (M�H-152) yielding an ion fragment at m/z 785.An ion at m/z 635 due to the loss of a HHDP residue (M�H-302)was also detected. Besides these two hydrolysable tannins, anothercompound was detected at m/z 1175 (25) with a fragment at m/z873 (loss of HHDP group). Its structure corresponds to a flavanoel-lagitannin (complex tannin), in which a hydrolysable tannin moi-ety and flavan-3-ol moiety are linked through a carbon–carbonbond. A similar compound named mongolicain A/B (25) had al-ready been reported in Q. suber leaves (Ito et al., 2002) but not incork. Mongolicain was proposed to be biosynthesised by oxidationof acuttissimin, another flavanoellagitannin present in severalAsian Quercus species (Ishimaru et al., 1988; Nonaka et al., 1988).

4. Conclusions

A method that allows the analysis of low molecular weightphenolic compounds and hydrolysable tannins (gallotannins andellagitannin) present in cork, under the same chromatographicconditions was developed.

From the results obtained, the HPLC–DAD/ESI–MS analysis hasrevealed that Q. suber cork is characterised by a wide variety of

low molecular weight phenols and important levels of tannins,particularly ellagitannins that can be extracted from cork whenmacerated in hydroalcoholic solutions. Therefore, the knowledgeof the polyphenolic composition of cork should be consideredwhen studying the cork-wine relationship and the in-bottle wineevolution.

Acknowledgements

This work was supported by a research project grant fundingfrom ARCP (Associação) Rede Competência em Polímeros) fromPortugal. This work was also supported by project CONC-REEQ/275/2001 (FCT, POCI 2010, FSE).

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