ESI–MS

Food Chemistry 125 (2011) 1398–1405

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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 b

Centro de Investigação em Química, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal 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 2009 Received in revised form 14 June 2010 Accepted 3 October 2010

Keywords: Cork Ellagitannins Flavanoellagitannins LC-DAD/ESI-MS Phenolic compounds Quercus suber L.

a b s t r a c t The aim of the present work was to identify the extractable phenolic compounds present in cork from Quercus suber L. The structures of thirty three compounds were tentatively identified by liquid chromatography coupled to electrospray ionisation mass spectrometry (HPLC–DAD/ESI–MS). The majority of those compounds were gallic acid derivatives, in the form of either galloyl esters of glucose (gallotannins), combinations of galloyl and hexahydroxydiphenoyl esters of glucose (ellagitannins), dehydrated tergallic-C-glucosides or ellagic acid derivatives. Others were found to correspond to low molecular weight phenolic compounds, like acids and aldehydes. Mongolicain, a flavanoellagitannin in which hydrolysable tannin and flavan-3-ol moieties are connected through a carbon–carbon linkage, was also detected in cork from Q. suber L. The results illustrate the rich array of phenolic compounds present in cork. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Cork is the suberized cellular tissue that is continuously produced by the phellogen of the cork oak tree (Quercus suber L.), a native species of the Mediterranean region. The unique properties of this raw material, which include low density, low permeability to liquids, ability to adhere to a glass surface, compressibility, resilience, elasticity, chemical inertness and resistance to microbial growth, have promoted its use in a variety of sectors, but its most visible and profitable products are the stoppers for wine bottling (Casey, 1994; Jung & Hamatscheck, 1992). Natural cork is composed essentially of suberin, the main cork component, lignin, polysaccharides (hemicelluloses and cellulose) and extractable components (Pereira, 1988). The relative abundance of these fractions is extremely variable, even among trees of the same forest. Chemical composition can be influenced by geographical origin, climate and soil conditions, genetic origin, dimension and age of the tree (virgin or reproduction) or even the different 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 to the cork cell wall, i.e., the extractable material, is mainly composed of aliphatic, triterpenic and phenolic compounds, exhibiting only ca. 2% of carbohydrates (Rocha, Coimbra, & Delgadillo, 2004; Rocha, Ganito, Barros, Carapuça, & Delgadillo, 2005) and it can be eas-

⇑ Corresponding author. Tel.: +351 220402558; fax: +351 220402658. E-mail address: [email protected] (V. de Freitas). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.10.016

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 important due to their contribution to sensory properties – colour, flavour, astringency and bitterness (Mazzoleni, Caldentey, Careri, Mangia, & Colagrande, 1994). Phenolic acids do not have a direct influence on the organoleptic characters of wines but some of them are precursors of volatile phenols (especially vinyl and ethyl derivatives of phenols), substances that influence wine aroma (Chatonnet, Boidron, & Pons, 1990; Singleton, 1995). Ellagitannins play an important role in wine oxidation processes as they rapidly react with dissolved oxygen and facilitate the hydroperoxidation of wine constituents (Vivas & Glories, 1996). These compounds affect the condensation rate of proanthocyanidins as well as condensed tannin precipitation 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 an important role in the ageing of wines (Pocock, Sefton, & Williams, 1994). In addition to these properties, polyphenolic compounds have been shown to be responsible for many health benefits, including antibacterial, antiviral, anticarcinogenic, anti-inflammatory and antiallergic activity (Laranjinha & Cadenas, 1999; Santos-Buelga & Scalbert, 2000).

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

A number of low molecular weight phenolic compounds were isolated and identified from reproduction cork of Spanish Q. suber, including ellagic acid as the main compound, with detectable quantities of gallic, protocatechuic, vanillic, caffeic and ferulic acids. Protocatechuic and sinapic aldehydes, vanillin, coniferaldehyde and coumarins like aesculetin and scopoletin were also identified (Conde, Cadahía, García-Vallejo, Fernández de Simón, & González Adrados, 1997). Although the inner bark of the cork oak tree is an important tannin source, very little information is available on tannins and related polyphenols present in cork. The quantitative evaluations of the different tannin groups in cork have revealed small amounts of tannins that are extracted with MeOH/H2O, being the ellagitannins the most representative compounds of this polyphenolic extract (Cadahía, Conde, Fernández de Simón, & García-Vallejo, 1996). Several molecular structures of ellagitannins with wide distribution in the wood and bark of species of Quercus have already been identified (Hervé Du Penhoat et 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 castalagin and vescalagin, the pentosylated monomers roburin E and grandinin and the dimer roburin A, besides other ellagitannins with related structure and other ellagic acid derivatives (Cadahía et al., 1996). A number of other hydrolysable and complex tannins were recently isolated from the leaves of Algerian Q. suber, including cocciferins D2, D3 and T2, pedunculagin, acutissimin B, tellimagrandin I, castavaloninic acid, isocastavaloninic acid, mongolicain A, and desgalloylstachyurin (Ito et al., 2002). In the acorns of Q. suber a series of gallotannins and ellagitannins have also been detected (Cantos et al., 2003), but to our knowledge there is no information regarding the presence of this group of compounds in cork from Q. suber. Therefore, the aim of this work was the isolation and identification of the different groups of polyphenolic compounds present in cork 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 from Sigma–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. suber L trees free of outer corkback was obtained by grinding and sieving Portuguese cork (by-product of cork stoppers industry), kindly supplied by Amorim and Irmãos (Mozelos, Portugal). Samples of grounded cork (60.0 g) were extracted with 1.0 L of model wine solution (12% ethanol, 5.0 gL 1 tartaric acid buffered to pH 3.2) for 72 h at room temperature with occasional agitation. The suspension was filtered on a Büchner funnel and ethanol was removed by vacuum distillation. The aqueous residue was then spray dried on a Büchi Mini Spray Drier B-290Ò and the powder obtained was redissolved in water and extracted three times with ethyl acetate. The organic fractions were combined and after drying with anhydrous sodium sulphate they were evaporated to dryness under vacuum. The residue was dissolved in H2O/MeOH (9:1; v/v) and freeze-dried.

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2.3. Column chromatography Fractionation of cork phenolic compounds was carried out according to the method described elsewhere (de Freitas, Glories, Bourgeois, & Vitry, 1998). A portion of the above freeze-dried residue was dissolved in methanol and chromatographed on a TSK Toyopearl HW-40(S) prepared column (250  16mm i.d.) using methanol as the eluent, at 0.8 mLmin 1. Phenolic compounds were separated based on molecular weight. Fractions were collected upon detection with a Gilson 115 UV Detector and a SP4290 integrator from Spectra-Physics at 280 nm and each fraction obtained (I–VIII) was freeze-dried after eliminating the solvent with a rotatory evaporator under reduced pressure at 30°. The resulting solids were analysed by HPLC–DAD and LC–DAD/ESI–MS. 2.4. HPLC–DAD analysis The samples were analysed by HPLC (Merck-Hitachi Elite Lachrom) on a 150  4.6 mm i.d. (5 lm pore size) reversed-phase C18 column (Merck) thermostated at 25° C (Merck-Hitachi Column Oven L-2300), according to an adaptation of a method described elsewhere (Peña-Neira et al., 1999). Detection was carried out at 280 nm using a diode array detector (Merck-Hitachi Diode Array Detector L-2455). Two solvents were applied for elution: Awater/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 (isocratic flow) at a flow rate of 0.3 mLmin 1. The sample injection volume was 20 lL. The chromatographic column was washed with 100% B for 10 min and then stabilized with the initial conditions for another 10 min. 2.5. LC–DAD/ESI–MS analysis A Finnigan Surveyor series liquid chromatograph, equipped with the same column as mentioned earlier and thermostated at 25° C was used. The samples were analysed using the same solvents, gradients, injection volume, and flow rate referred above for the HPLC analysis. Double-online detection was done by a photodiode spectrophotometer and mass spectrometry. The mass detector was a Finnigan LCQ DECA XP MAX (Finnigan Corp., San Jose, CA) quadrupole ion trap equipped with atmospheric pressure ionisation (API) source, using electrospray ionisation (ESI) interface. The vaporiser and the capillary voltages were 5 kV and 4 kV, respectively. The capillary temperature was set at 325° C. Nitrogen was used as both sheath and auxiliary gas at flow rates of 80 and 30, respectively (in arbitrary units). Spectra was recorded in the negative ion mode between m/z 120 and 2000. The mass spectrometer was programmed to do a series of three scans: a full mass, a zoom scan of the most intense ion in the first scan, and a MS–MS of the most intense ion using relative collision energies of 30 and 60. 3. Results and discussion The aqueous extract of cork was chromatographed on TSK Toyopearl gel, yielding eight fractions, as shown in Fig. 1. Each fraction was analysed by HPLC/ESI–MS and some structures were postulated by the UV–Vis spectrum and MS fragmentation pattern. The MS fragmentation provides important information about the structure which is particularly useful in the case of gallic and ellagic tannins. Thus, the losses of 152 and 170 mass units from the [M H] quasi-molecular ion indicates the presence of galloyl groups and the loss of 302 from the [M H] quasi-molecular ion and the presence of an ion at m/z 301 indicates the presence of HHDP groups. Loss of 44 mass units is characteristic of a free

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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 in these fractions showed that they could be arranged into two groups: those showing a spectrum data in agreement with those of ellagic acid with two maximum wavelengths of absorption (kmax) at 255 nm and 367 nm and those showing a characteristic spectrum of gallic acid with a single kmax at 271 nm. The first group includes compounds that present an ellagic acid residue in their molecular structure and the second group includes all the galloyl and hexahydroxydiphenoyl derivatives (Cantos et al., 2003). 3.1. Fraction I 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).

carboxyl group and losses of 18 (H2O) from the [M H] ion is characteristic of C-glucosidic ellagitannins. When molecular weights of hydrolysable tannins differ by two units, it can be related to the difference between either an HHDP group or two galloyl groups (Barry, Davies, & Mohammed, 2001). Fig. 2 shows the HPLC-DAD chromatogram of four representative fractions obtained. Table 1 shows UV–Vis and ESI–MS charac-

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

800 800

II

I 12 2

7

600

600

mAU

mAU

6 3

400

400 8

1

200

200 4

12

5

15

10

20

30

40

50

60

70

80

90

0 100 110 120

10

20

30

15

40

27 17 14

50

Time (min)

60

70

80

90

Time (min)

2200

800

VIII

V 24

1800

600

25 21

mAU

mAU

0 100 110 120

1400

400 1000 32 28

600

200 23

19

200

10

20

30

40

50

60

70

Time (min)

80

90

0

100 110 120

10

20

30

40

50

60

70

80

90

0

100 110 120

Time (min)

Fig. 2. HPLC–DAD chromatograms of four isolated fractions (I, II, V, VII). Identification of the numbers present in the chromatograms is shown in Tables 1 and 2.

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A. Fernandes et al. / Food Chemistry 125 (2011) 1398–1405 Table 1 HPLC/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 Vanillin Protocatechuic acid Gallic acid Conyferaldehyde Caffeic acid Ferulic acid Ellagic acid Ellagic acid-pentose Ellagic acid-deoxyhexose Ellagic acid-hexose Valoneic acid dilactone HHDP-glucose Valoneic acid Dehydrated tergallic-C-glucoside HHDP-galloyl-glucose Trigalloy-glucose Di-HHDP-glucose HHDP-digalloyl-glucose Tetragalloyl-glucose Castalagin/Vescalagin Di-HHDP-galloyl-glucose Trigalloyl-HHDP-glucose Pentagalloyl-glucose Mongolicain A/B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

137 151 153 169 177 179 193 301 433 447 463 469 481 505 613 633 635 783 785 787 933 935 937 939 1175

109 136 109 125 162 – 149 257; 301 301 301 425 301 313 593; 463; 483; 481; 633; 635; 915; 633; 785; 787; 873;

– 108 – 97 133; – 134 – 257; 257; 257; 407; 257; – 465; 257; 421; 257; 257; 573; 613; 571; 301 617; 829;

280; 279; 259; 270 289; 322 319 250; 253; 253; 253; 253; 262; 253; 253; 262 277 271 271 274 247 271 277 279 267

I I I I I I I I, II I I I II I II II III II III; IV III; IV III VII; VIII VI VIII V VIII

229

523; 301 465 301 483; 617 631 873; 767; 769 855;

493

301

783 635; 465 721; 677

120; 106

229 229 229 301 229 301; 229; 313; 229 229 465; 569 329;

299 185 169

403 299

599; 447 785; 767

310 308 293 340

373 373 373 373 373 379 370 373

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

*

Table 2 HPLC/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 Compound Compound Compound Compound Compound Compound Compound

26 27 28 29 30 31 32 33

465 595 807 873 915 947 963 977

447; 421; 403 523; 505; 301 505 829; 785; 721 613; 595; 523 915; 897; 871 945; 661; 643 945; 675

403 301 355; 767; 523; 887; 643; 825;

283; 352 253; 373 – 262 250; 373 – – –

I II V, VI III IV VII V IV

A B C D E F G H

311; 683; 299 569; 629; 643

301 633 301 455

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

*

conyferaldehyde (5), respectively. All these compounds were previously reported in cork of Q. suber (Conde et al., 1997). This fraction also contained ellagic acid and several ellagic acid derivatives. Ellagic acid (8) with a molecular ion at m/z 301 yielded an intensive product ion at m/z 257 and 229. Some of the ellagic acid derivatives detected were sugar conjugates. Indeed, mass spectrometric analysis revealed a quasi-molecular anion of ellagic acid pentose conjugate (9) at m/z 433. The MS2 spectrum of this compound yielded 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 at m/z 257 and 229, which matches the fragmentation pattern of ellagic acid. Another peak at m/z 447 was identified as an ellagic acid deoxyhexose conjugate (10) (M-146, loss of a deoxyhexosyl unit) and the peak at m/z 463 was identified as an ellagic acid hexose conjugate (11) (M-162, loss of hexosyl residue). These ellagic acid derivatives have already been reported in Q. suber acorns and in cork in previous works (Cantos et al., 2003; Conde, Cadahía, García-Vallejo, & González-Adrados, 1998). This fraction also revealed the presence of an ellagitannin with [M H] at 481 which has been assigned to HHDP-glucose (13), based on molecular weight and the presence of an intense fragment at m/z 301 and another compound at m/z 465 (26) still unidentified. This compound differs by 18 units from the gallotannin digalloyl-glucose (m/z 483) and it

could result from the loss of water from this compound to form dehydrated digalloyl glucose (Zywicki, Reemtsma, & Jekel, 2002). The other fractions (from II to VIII) revealed the compounds from 8 to 25 (Table 1) that were tentatively identified by UV–Vis spectroscopy and HPLC/ESI–MS. These were mostly gallic acid derivatives, in the form of either galloyl esters of glucose, combinations of galloyl or hexahydroxydiphenoyl esters of glucose, dehydrated tergallic-C-glucosides or ellagic acid derivatives. The presence of mongolicain, a complex tannin was also detected (Fig. 3). The structures of compounds from 26 to 33 were not established altought some important structural evidences have been obtained from MS, indicating that they may be ellagic or gallic tannins (Table 2). 3.2. Fraction II One group of compounds showing a UV spectrum similar to that of ellagic acid were tentatively identified in fraction II (collected for 80 min). The mass spectrum of compound 15 with a [M H] quasi-molecular ion at m/z 613 matches with the structure of the dehydrated tergallic acid-C-glucoside described in the literature (Cantos et al., 2003). These compounds had been previously detected in the acorns of Q. suber L, but to our knowledge they have

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

HO

G=

GO O

GO GO

OH

HO

OG GO

O

Penta-galloyl-glucose (m/ z 939) HO

OH

HO HO HO

O

O

HO

HO

O

O

HO

O

O O

OH

HO

OH

HO

O

HO

HO

O

O

HO OH

HO

O O

O O GO

HO

HO di-HHDP-glucose (m/ z 783)

HO

OH

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

OH

HO HO HO

O

O O

O HO

HO

O

HO

O

O

OG

HO

HO

O

HO

HO

O

OH

HO

OH

O O O

HO

HO

OH

HO

OH OH

HO

O

O

HO

O

HO

OH HO

O

O O

HO

O

O OH

O

O O

O

OH OH

OH

HO OH

HO

O

OG GO

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

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

HO

O

O GO

HO

HO

HO

O

O

HO

HO

OH

HO

O

OH

OH

Castalagin (m/ z 933)

HO HO

O

O

O O

O O

HO OH

O OH

HO

OH

Mongolicain B (m/ z 1175)

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

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

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

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

OH

OH OH

HO OH

O COOH O

OH

OH

O

HO

OH

OH

O

O

OH

- H2O

HO O

OH

HO

OH

O

O

CO

O

OH OH

O

O

OH

OH OH

OH

m/z 631

m/z 613 (15)

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

631) (Fig. 4). This latter was already reported in the literature, present in Q. suber acorn (Cantos et al., 2003), but was not detected in this cork extract. Moreover, valoneic acid dilactone (12) was also detected at m/z 469. The MS2 fragments (m/z 425 and 301) were in agreement with those previously reported for this compound (Nawwar, Marzouk, Nigge, & Linscheid, 1997). Valoneic acid (14) ([M H] at m/z 505) and an unknown compound at m/z 595 (27) were also present in fraction II. This latter differs from dehydrated tergallic acid-Cglucoside (15) by 18 units and presents the same fragmentation pattern as this compound. Probably the compound at m/z 595 results from the loss of water from dehydrated tergallic acid-C-glucoside (15). In this fraction was also detected a quasi molecular ion at m/z 635 compatible with the structure of trigalloyl-glucose (17). The MS2 spectra, revealed the presence of a quasi-molecular ion at m/z 483 and 465 probably due to the loss of a galloyl and a gallate unit, respectively. In this fraction we were also able to detect the presence of ellagic acid (8) at m/z 301, probably due to a contamination from fraction I. In the subsequent fractions, a series of gallotannins and ellagitannins have been identified. Despite the fact that the majority of these compounds have been reported in the acorns of Q. suber (Cantos et al., 2003), to our knowledge they have not been reported in 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-glucose (16) from the MS2 evidence of an intense ion at m/z 301 due to 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 as tetragalloyl-glucose (20). The fragments at m/z 617 and 635 correspond to the loss of a gallate residue (M H-170) and a galloyl residue (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 has been assigned to HHDP-digalloyl-glucose (19). The quasi-molecular 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 been assigned to di-HHDP-glucose (18) and may result from the coupling of two adjacent galloyl groups in HHDP-digalloyl-glucose by intramolecular oxidation. In this fraction a new compound with [M H] quasi-molecular ion at m/z 873 (29) was also detected, which structure was not established. However, this compound presents the same fragmentation pattern as the flavanoellagitannin isolated in fraction VIII ([M H] at m/z 1175) and differs from this compound by 302 units, 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 at m/z 977 may be derived from the ellagitannin castalagin or vescalagin (m/z 933) from which it differs by 44 units probably corresponding to a carboxyl group. Compound at [M H] 915 (30) presents a UV spectrum similar to that of ellagic acid, which probably indicates the presence of this phenolic acid residue in its structure. On the other hand, this compound presents the same fragmentation pattern as the dehydrated tergallic acid-C-glucoside (m/z 613) from which it differs from 302 units (HHDP group). Bearing this, the compound detected at m/z 915 could be formed through the attachment of a HHDP residue to the compound at m/z 613 (Fig. 6). In the literature, the compound detected at m/z 915 is often associated to dehydrated castalagin or vescalagin (Zywicki et al., 2002) which is not the case herein based on the UV–Vis and mass spectrum. Furthermore two other compounds at m/z 785 and 783 described before, were detected in this fraction which may correspond to a contamination from the previous fraction (they present the same HPLC retention time). The MS/MS spectra of the galloyl-glucose compounds found in cork from Q. suber were characterised by the sequential loss of gallic acid residues. In fraction V, collected approximately from 460 to 520 min of elution, the breakdown of pentagalloyl-glucose at m/z 939 (24) produces a first loss of a galloyl residue (M H-152) to give a fragment at m/z 787. In addition, the loss of a gallic acid to give a fragment at m/z 769 (M H-170) and a sequential loss of another galloyl residue yielding a fragment at m/z 617 (M H-170–152) were also observed. In this fraction the presence of intensive peaks at m/z 963 (32) and 807 (28) were detected (Fig. 2). These peaks remain unidentified but they may belong to ellagitannins as illustrated by the loss of 302 units from the molecular ion to give the fragment at m/z 661 and 505, respectively. 3.4. Fractions VI–VIII The analysis of fraction VI revealed the presence of quasimolecular ion at m/z 935 (di-HHDP-galloyl-glucose) as the main constituent. The breakdown of the quasi-molecular ion [M H] at m/z 935 (22) produced a fragment at m/z 633 (loss of a HHDP group) and at m/z 783 (loss of a galloyl residue). This fraction was 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 may also belong to ellagitannins as suggested by the detection of a fragment at m/z 301. Regarding the C-glucosidic ellagitannins, it was possible to tentatively identify in fractions VII and VIII the stereoisomers castalagin and vescalagin at m/z 933 (21) (Fig. 2). These compounds differ

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

OH

HO

HO

OH

OH

OH

HO HO

O O

HO

O

O

OH O

HO

O

O HO

OH

HO OH

O O

O

O

HO

O

OH

O -HHDP

O O

O

O

O

OH

OH

O

HO

HO

O

O

HO

OH OH

HO OH

OH

O OH

OH

HO

m/ z 873 (29)

Mongolicain B (m/ z 1175)

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

OH OH

HO

OH

O

OH HO

CO

O

OH OH

OH

+ HHDP

O

O

CO O

O

OH OH

m/z 613 (15)

O

O

O

O

O

OH

O

HO

O

O

OH

HO

CO

OH

OH OH

OH

HO

m/z 915 (30)

OH

CO OH

OH

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

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 authors also found roburin E, grandinin and the dimer roburin A but these were not detected in the experimental conditions described herein. The MS2 spectrum of castalagin or vescalagin revealed a fragment at m/z 631 similar to the molecular ion of castalin/vescalin, resulting 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, trigalloylHHDP-glucose (23) [M H] at m/z 937 was detected. This compound 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 a galloyl 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, another compound was detected at m/z 1175 (25) with a fragment at m/z 873 (loss of HHDP group). Its structure corresponds to a flavanoellagitannin (complex tannin), in which a hydrolysable tannin moiety and flavan-3-ol moiety are linked through a carbon–carbon bond. A similar compound named mongolicain A/B (25) had already been reported in Q. suber leaves (Ito et al., 2002) but not in cork. Mongolicain was proposed to be biosynthesised by oxidation of acuttissimin, another flavanoellagitannin present in several Asian Quercus species (Ishimaru et al., 1988; Nonaka et al., 1988). 4. Conclusions A method that allows the analysis of low molecular weight phenolic compounds and hydrolysable tannins (gallotannins and ellagitannin) present in cork, under the same chromatographic conditions was developed. From the results obtained, the HPLC–DAD/ESI–MS analysis has revealed 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 when macerated in hydroalcoholic solutions. Therefore, the knowledge of the polyphenolic composition of cork should be considered when studying the cork-wine relationship and the in-bottle wine evolution.

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

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