Journal of Food Composition and Analysis 23 (2010) 699–705

Original Article

Flavonol profiles of Vitis vinifera white grape cultivars

Noelia Castillo-Munoz a, Sergio Gomez-Alonso b, Esteban Garcıa-Romero b, Isidro Hermosın-Gutierrez a,*a Instituto Regional de Investigacion Cientıfica Aplicada, Escuela Universitaria de Ingenierıa Tecnica Agrıcola, Universidad de Castilla-La Mancha, Ronda de Calatrava 7,

13071 Ciudad Real, Spainb Instituto de la Vid y el Vino de Castilla-La Mancha, Carretera de Albacete s/n, 13700 Tomelloso, Spain

A R T I C L E I N F O

Article history:

Received 7 October 2009

Received in revised form 25 January 2010

Accepted 22 March 2010

Keywords:

Cultivar authenticity

Cultivar differentiation

Flavonol profile

Isorhamnetin

Kaempferol

Quercetin

Vitis vinifera

White grape

Biodiversity and nutrient

Food analysis

Food composition

A B S T R A C T

The flavonol profiles of a wide set of Vitis vinifera white grape cultivars were determined by HPLC–DAD–

ESI-MSn in order to confirm the presence of isorhamnetin-type flavonols and also to evaluate their ability

for cultivar authenticity and differentiation. Only 3-O-glycoside derivatives of kaempferol, quercetin and

isorhamnetin were identified as grape flavonols. The 3-O-glucosides and 3-O-galactosides of kaempferol,

quercetin, and isorhamnetin, and also the 3-O-glucuronides of kaempferol and quercetin, were detected

in all the grape varieties. In addition, traces of isorhamnetin 3-O-glucuronide and rutin (quercetin 3-O-

(600-rhamnosyl)-glucoside) were detected in some cases. Flavonol profiles of white grapes are dominated

by quercetin-type flavonols. However, the preliminary results have shown remarkable differences

(Principal Component Analysis) in the contribution of kaempferol-type flavonols to the characteristic

white grape flavonol profiles, which could suggest their use for cultivar authenticity and differentiation

purposes. In addition, some white grape cultivars (e.g., Pedro Ximenez, Gewurztraminer, Verdejo, Albillo,

and Riesling) were characterised by relatively high and significantly different proportions of the newly

reported, usually very minor, isorhamnetin-type flavonols.

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1. Introduction

Flavonols are a class of flavonoid compounds located in Vitis

vinifera grape berry skins, where they are involved in UV screening(Haselgrove et al., 2000; Price et al., 1995), with their biosynthesisbeing light-dependent (Downey et al., 2004). Regarding the colourof the wines, flavonols are yellow pigments that contribute directlyto the colour of white wines, but which in red wines flavonols aremasked by anthocyanins, the red pigments. However, flavonolsaffect red wine colour by means of copigmentation (Boulton, 2001;Schwarz et al., 2005). In addition, flavonols have been identified asone of the best phenolics with antioxidant activity in wine,especially in white wines (Burda & Olezsek, 2001; De Beer et al.,2005; Montoro et al., 2005).

Phenolic compound biosynthesis in V. vinifera grapes is undergenetic control, and the differences among grape cultivars aresometimes significant enough to make it possible to use grapephenolic composition as a tool for cultivar authenticity anddifferentiation. In the case of red wine grape cultivars, theanthocyanin profiles have been widely used for cultivar authen-ticity purposes. Flavonol profiles have also demonstrated some

* Corresponding author. Tel.: +34 926 295300; fax: +34 926 295351.

E-mail address: isidro.hermosin@uclm.es (I. Hermosın-Gutierrez).

0889-1575/$ – see front matter � 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.jfca.2010.03.017

potential usefulness for cultivar differentiation for both red andwhite wine grape cultivars, although the results have not alwaysled to consistent conclusions (Andrade et al., 2001; Cantos et al.,2002; Castillo-Munoz et al., 2007; Di Stefano et al., 1993;Gomez-Alonso et al., 2007; Mattivi et al., 2006; RodrıguezMontealegre et al., 2006). Perhaps one of the reasons for theinconsistency shown by grape flavonol profiles has been linked tothe limited knowledge about these grape phenolics, in contrast tothe very well-known grape anthocyanins.

Our group has recently demonstrated (Castillo-Munoz et al.,2009) that the flavonols occurring in red grape cultivars belong tothree glycosylated series (3-O-glucosyl, 3-O-galactosyl, and 3-O-glucuronyl) of the six possible flavonoid structures (kaempferol,quercetin, isorhamnetin, myricetin, laricitrin, and syringetin). Withregard to white grape cultivars, the presence of isorhamnetin-typeflavonols in the skin of white grapes has been suggested on the basisof hydrolysis treatment and subsequent detection of the freeaglycone isorhamnetin (Mattivi et al., 2006) and also by thecoincidence of the UV–vis spectra and chromatographic retentiontime with a true standard of isorhamnetin 3-O-glucoside (RodrıguezMontealegre et al., 2006). However, the occurrence of isorhamnetin-type flavonols and also myricetin-type flavonols has been consid-ered exclusive to red grape cultivars (Cheynier et al., 2003).

The aim of this work has been the determination of theoccurring flavonol in V. vinifera white grape cultivars by means of

N. Castillo-Munoz et al. / Journal of Food Composition and Analysis 23 (2010) 699–705700

the application of the previously developed HPLC–DAD–MS/MSanalytical method for red grape flavonols (Castillo-Munoz et al.,2009). The study has been extended to a wide set of grape cultivars,comprising both Spanish autochthonous and white wine cultivarsfound worldwide, which have been analysed to establish theircharacteristic skin flavonol profiles. We have paid special attentionto confirm the occurrence of the suggested isorhamnetinderivatives and also the evaluation of the possibility of usingflavonol profiles as chemical markers for cultivar authenticity anddifferentiation of white grape cultivars. Although grape cultivarauthenticity can be provided by more accurate genetic methods(e.g., PCR methods), such methods are not currently available forwhite wines; in this case, the determination of the characteristicflavonol profiles of white grape cultivars can constitute the basisfor the use of flavonol profiles for cultivar authenticity anddifferentiation purposes in white wines.

2. Materials and methods

2.1. Chemicals and grape samples

All solvents were of HPLC quality and all chemicals were ofanalytical grade (>99%). Water was of Milli-Q quality. Commercialstandards of flavonol 3-O-glycosides: 3-O-glucosides of quercetin,kaempferol and isorhamnetin, and 3-O-galactoside of quercetin(Extrasynthese, Genay, France). Samples of quercetin and kaemp-ferol 3-O-glucuronides, non-commercial flavonol standards, werea gift from Dr. Ullrich Engelhardt (Institute of Food Chemistry,Technical University of Braunschweig, Germany).

Twenty-two white grape cultivars were analysed (Table 1),comprising both autochthonous and foreign, minor and wide-spread, grape cultivars that are currently grown in the warmclimate Spanish region of La Mancha (south-central Spain).Healthy white wine grapes cultivated under the same conditionsusing drip-irrigation and bilateral Royat cordon trellis, in theexperimental vineyard of the Instituto de la Vid y el Vino deCastilla-La Mancha (IVICAM, Tomelloso, Spain) were collected at

Table 1Individual flavonol profiles (molar percentages of each individual flavonol over the tota

flavonols with the same flavonoid structure) profiles of white grape cultivars.

White grape

cultivar

Origin Q-3-gal Q-3-glcU Q-3-glc Rutina K-3-gal K-

Airen S 4.17 42.33 33.99 0.54 3.55 1.7

Albillo S 6.83 21.76 45.56 0.18 4.61 1.4

Chardonnay F 4.99 32.62 35.94 0.25 4.68 2.4

Chelva S 4.73 37.85 34.04 ND 4.42 1.5

Gewurztraminer F 2.73 46.60 30.46 1.04 1.98 3.8

Jaen S 5.46 22.85 32.20 0.33 8.25 2.7

Listan Huelva S 2.14 61.82 26.53 0.24 1.31 1.6

Macabeo S 3.65 47.04 28.95 0.62 3.23 2.5

Malvar S 5.17 37.60 38.46 0.39 3.37 1.3

Mantuo S 6.75 21.52 41.50 0.17 5.59 1.2

Merseguera S 4.48 33.90 37.12 0.11 3.18 9.3

Moscatel Alejandrıa F 3.09 42.58 22.77 1.20 5.48 3.0

Moscatel grano menudo S 4.15 29.94 27.97 0.14 6.95 1.8

Pardillo S 2.58 50.93 32.13 0.46 1.83 1.5

Parellada S 5.81 41.78 37.99 0.74 2.65 1.0

Pedro Ximenez S 4.62 32.14 38.46 ND 3.62 1.5

Riesling F 4.68 40.15 34.52 1.42 3.32 1.3

Sauvignon blanc F 3.24 48.54 30.42 0.51 2.24 3.5

Torrontes S 5.46 29.00 42.33 0.17 3.36 7.5

Ugni blanc F 4.96 37.55 37.31 1.11 3.32 2.3

Verdejo S 4.16 33.86 34.77 0.32 3.90 2.4

Viognier F 5.71 33.14 38.82 ND 4.36 1.0

Abbreviations: S, Spanish grape cultivar; F, foreign or non-Spanish grape cultivar; Q, que

glucuronide.a The molar percentages of these two compounds have been estimated by means of t

glcU) because they coeluted with major compounds and their quantification by DAD-c

optimum ripeness for harvesting (estimated alcoholic strength ofaround 12%, v/v). The sampling was done randomly by pickingberries from the top, central and bottom parts of the cluster,following a zigzag path between two marked rows of ten vines. Wesampled berries from both exposed and shaded clusters by pickingberries of 4–5 clusters per vine. The size of the sample was around200 berries, which were bulked and separated into 2 sub-samplesof approximately 100 berries and were analysed in duplicate. Theauthenticity of each grape cultivar was provided by DNA-microsatellite analysis previously performed at the IVICAM(Fernandez-Gonzalez et al., 2008).

2.2. Extraction of flavonols from grape skin

A quantity of 100 g of healthy grapes was finger pressed toremove the pulp and the seeds. The remaining skins were washedthree times in water (Milli-Q) and softly dried twice by pattingthem between sheets of filter paper. Dried skins were successivelyextracted twice using 100 mL of a mixture 50:48.5:1.5 (v/v/v) ofCH3OH/H2O/HCOOH in each extraction step. The extraction washelped by homogenisation (Heidolph DIAX 900) for 2 min andsubsequent centrifugation at 2500 � g at 5 8C for 15 min. Theresulting flavonol solutions obtained after mixing both extracts,which can be directly injected for HPLC, were stored at�18 8C untilchromatographic analysis. Previous studies (data not shown) haveproved that two successive extractions are enough for a quantita-tive extraction of the grape skin flavonols (>99%), since the third,fourth and fifth extracts up to five contained no detectablequantities of flavonols by HPLC.

2.3. HPLC–DAD–ESI-MSn analysis of flavonols

HPLC separation, identification and quantification of flavonolswere performed on a Agilent 1100 Series system (Waldbronn,Germany), equipped with DAD (G1315B) and LC/MSD Trap VL(G2445C VL) electrospray ionisation mass spectrometry (ESI-MSn)system, and coupled to an Agilent Chem Station (version B.01.03)

l molar content) and flavonol-type (summation of the molar percentages of all the

3-glcU K-3-glc I-3-gal I-3-glc I-3-glcUa Q-type K-type I-type mmol/kg

7 13.15 0.13 0.38 ND 81.02 18.47 0.51 99.8

4 17.09 0.36 2.13 0.03 74.34 23.15 2.52 109.3

2 18.50 0.20 0.40 ND 73.81 25.60 0.60 74.3

1 16.24 0.20 1.02 ND 76.62 22.17 1.22 82.2

5 9.40 0.36 3.58 ND 80.83 15.23 3.94 50.7

1 27.38 0.19 0.63 ND 60.84 38.34 0.82 153.3

4 5.88 0.00 0.45 ND 90.73 8.82 0.45 46.8

2 12.52 0.20 1.26 ND 80.27 18.27 1.46 78.6

5 12.82 0.15 0.70 ND 81.61 17.54 0.85 160.4

1 22.85 0.15 0.27 ND 69.94 29.64 0.42 72.2

5 10.60 0.15 1.11 Traces 75.61 23.14 1.25 73.5

9 20.77 0.17 0.84 ND 69.64 29.34 1.02 65.1

3 28.15 0.24 0.63 ND 62.20 36.92 0.87 154.5

1 8.91 0.10 1.54 ND 86.10 12.26 1.64 75.7

9 9.42 0.00 0.51 ND 86.33 13.16 0.51 30.8

5 14.76 0.00 4.84 ND 75.22 19.94 4.84 7.9

4 12.46 0.32 1.80 Traces 80.77 17.11 2.12 72.7

9 9.46 0.00 1.91 0.10 82.70 15.29 2.01 41.5

7 11.36 0.11 0.64 ND 76.96 22.29 0.75 83.9

3 12.87 0.13 0.41 ND 80.94 18.52 0.54 84.7

5 17.27 0.33 2.95 Traces 73.10 23.62 3.29 97.9

1 16.12 0.16 0.69 ND 77.67 21.48 0.85 124.8

rcetin; K, kaempferol; I, isorhamnetin; gal, O-galactoside; glc, O-glucoside; glcU, O-

heir respective extracted ion chromatograms (m/z = 611 for rutin; m/z = 493 for I-3-

hromatograms was not possible.

N. Castillo-Munoz et al. / Journal of Food Composition and Analysis 23 (2010) 699–705 701

data-processing station. The mass spectra data was processedwith the Agilent LC/MS Trap software (version 5.3). The samples,after filtration (0.20 mm, polyester membrane, ChromafilPET 20/25, Machery-Nagel, Duren, Germany) were injected(50 mL) onto a reversed-phase column Zorbax Eclipse XDB-C18(4.6 mm � 250 mm; 5 mm particle; Agilent, Germany), thermo-statised at 40 8C. The chromatographic conditions were developedin a previous work (Castillo-Munoz et al., 2009). The solvents were:solvent A (acetonitrile/water/formic acid, 3:88.5:8.5, v/v/v),solvent B (acetonitrile/water/formic acid, 50:41.5: 8.5, v/v/v),and solvent C (methanol/water/formic acid, 90:1.5:8.5, v/v/v). Theflow rate was 0.63 mL/min. The linear solvents gradient was: 96%A, 4% B, and 0% C maintained for 7 min; 70% A, 17% B, and 13% Cafter 31 min; 50% A, 30% B, and 20% C after 14 min; 30% A, 40% B,and 30% C after 0.5 min; 0% A, 50% B, and 50% C after 4.5 min andmaintained for 1 min; finally they were returned to initialconditions after 7 min. Quantification was carried out using theDAD-chromatograms obtained at 360 nm, by means of externalstandard calibration curves (due to the lack of certain standards,the 3-O-galactosides of kaempferol and isorhamnetin and the 3-O-glucuronide of isorhamnetin were quantified as their respective3-O-glucosides). For identification, ESI-MSn was used setting thefollowing parameters: positive ion mode; dry gas, N2, 11 mL/min;drying temperature, 350 8C; nebulizer, 65 psi; capillary, �2500 V;capillary exit offset, 70 V; skimmer 1, 20 V; skimmer 2, 6 V; scanrange, 50–1200 m/z.

2.4. Statistical data analysis

The grape flavonol profiles data was submitted to PrincipalComponents Analysis (SPSS version 17.0, SPSS Inc.) in order to testthe possibilities of differentiation and classification of the white

[(Fig._1)TD$FIG]

Fig. 1. Flavonol 3-O-glycosides found in Vitis vinifera white grapes: a) flavonol 3-O-gluco

(600-rhamnosyl)-glucoside (also known as rutin). Flavonoid structures (flavonol aglycon

grape cultivars paying attention to their characteristic flavonolprofiles.

3. Results and discussion

3.1. Identification of white grape flavonols

Flavonols detected in the studied white grapes occurred only asthe expected 3-O-glycosides, and they comprised the series of 3-O-glucosides, 3-O-galactosides, and 3-O-glucuronides of kaempferoland quercetin, together with the neutral glycosides (3-O-glucosideand 3-O-galactoside) of isorhamnetin for all the grape samplesanalysed. In addition, the 3-O-(600-rahmnosyl)-glucoside of quer-cetin (the so-called rutin) was detected for the most of the grapesamples, and also isorhamnetin 3-O-glucuronide was detected as atrace compound in some of the grape samples (Fig. 1). Fig. 2 showsa characteristic DAD-chromatogram of white grape flavonols(detection at 360 nm) together with the ESI-MS and ESI-MS2

extracted ion chromatograms (EIC) used for the identification ofevery series of flavonol-type 3-O-glycosides. The chromatographicand spectral (UV–vis and ESI-MSn) data of all the identifiedflavonols were in accordance with those of their availablestandards and also with reported data (Castillo-Munoz et al.,2007, 2009).

As found for other grape samples (Cantos et al., 2002; Castillo-Munoz et al., 2009; Giovanelli & Brenna, 2007), rutin was alsodetected in many of the white grape cultivars analysed in thiswork. The detection of rutin was only possible by extracting theESI-MS ion chromatogram at m/z 611 (Fig. 2c), the expected m/zvalue for its pseudomolecular ion ([M+H]+), because rutin almostcompletely coeluted with quercetin 3-O-glucoside and didnot show a differentiable peak (or peak-shoulder) in the

sides; b) flavonol 3-O-galactosides; c) flavonol 3-O-glucuronides; d) quercetin 3-O-

e): R = H, kaempferol; R = OH, quercetin; R = OCH3, isorhamnetin.

[(Fig._2)TD$FIG]

Fig. 2. Characteristic HPLC flavonol profile of white grapes: a) DAD-chromatogram

(detection at 360 nm); b) MS-extracted ion chromatogram at m/z 303 (quercetin-

type flavonols); c) MS-extracted ion chromatogram at m/z 611 (rutin); d) MS-

extracted ion chromatogram at m/z 287 (kaempferol-type flavonols); e) MS-

extracted ion chromatogram at m/z 317 (isorhamnetin-type flavonols); f) MS-

extracted ion chromatogram at m/z 493 (isorhamnetin 3-O-glucuronide). Peak

assignation: Q, quercetin; K, kaempferol; I, isorhamnetin; gal, 3-O-galactoside; glc,

3-O-glucoside; glcU, 3-O-glucuronide.

N. Castillo-Munoz et al. / Journal of Food Composition and Analysis 23 (2010) 699–705702

DAD-chromatogram. The ESI-MS detector also provided anestimation of the contribution of rutin to the DAD-peak in whichcoeluted with quercetin 3-O-glucoside. The integration of thedifferent peaks obtained for the two latter coeluting flavonols intheir respective EIC at their characteristic pseudomolecular ion m/zvalues (465 for quercetin 3-O-glucoside and 611 for rutin) allowedus to estimate that rutin accounted for no more than 5% of the areaof the DAD-peak mainly attributable to quercetin 3-O-glucoside.This value was rather similar to that estimated in red grapes(Castillo-Munoz et al., 2009), and rutin was not detected at all inthree of the studied samples. Therefore, rutin was a very minorflavonol in white grapes, accounting for estimated molarpercentages of 0.45 � 0.41% (Table 1).

The occurrence of isorhamnetin-type flavonols in white grapeshas been previously suggested by indirect evidence afterhydrolysis (detection of free isorhamnetin) and coincidence witha true standard of isorhamnetin 3-O-glucoside (Mattivi et al., 2006;Rodrıguez Montealegre et al., 2006). In this paper we reportunambiguous ESI-MSn data for their occurrence: the suspectedisorhamnetin 3-O-glucoside generated a pseudomolecular ion([M+H]+) at m/z 479 that further suffered the loss of a fragment of162 amu in the ion trap to give rise to a product ion ([(M-glc)+H]+)at m/z 317, which corresponds to the protonated isorhamnetinaglycone moiety. A second peak eluting before isorhamnetin 3-O-glucoside showed the same ESI-MS spectra and fragmentationpattern (ESI-MS2) and it was assigned the structure of isorhamne-tin 3-O-galactoside (Fig. 2e), as has been also reported for red

grapes (Castillo-Munoz et al., 2009). The occurrence of isorham-netin 3-O-glucuronide could also be expected, as for red grapes,which coelutes with isorhamnetin 3-O-glucoside under thechromatographic conditions used here. In fact, a signal was foundwhen the ion chromatogram was extracted at m/z = 493 (Fig. 2f),the expected m/z value for the pseudomolecular ion ([M+H]+) ofisorhamnetin 3-O-glucuronide. However, only two of the grapesamples gave a signal intense enough for integration (less than 5%of the DAD-peak area mainly attributable to isorhamnetin 3-O-glucoside, as estimated by EIC at m/z = 493). Moreover, for most ofthe grape samples, this isorhamnetin derivative was detected as atrace or was not detected at all. To the best of our knowledge, thereare no previous reports on the occurrence of isorhamnetin 3-O-galactoside and 3-O-glucuronide in white grapes.

Other low intense peaks were found in the DAD-chromatogramat 360 nm (Fig. 2a) which could be assigned to flavonols. However,no signals were obtained in the ion chromatograms extracted at m/z values of 319, 333, and 347, the expected values for the prtonatedproduct ions ([(M-gly)+H]+) of flavonols based on the aglyconesmyricetin, laricitrin, and syringetin respectively. In addition, noneof these DAD-peaks showed the characteristic UV–vis spectra offlavonols (data not shown). In summary, all the aforementionedresults suggest that white grape cultivars have characteristicflavonol profiles which only contain flavonol 3-O-glycosides basedon kaempferol, quercetin and isorhamnetin flavonoid structures, inagreement with previous findings (Mattivi et al., 2006). The lattersuggestion could be applied to the differentiation of white winesmade with white grapes from those elaborated with red grapes(the so-called ‘‘blancs de noirs’’).

3.2. Flavonol profiles of white grape cultivars

Table 1 summarizes the flavonol profiles (molar percentages ofthe contribution of each individual flavonol 3-O-glycoside to thetotal content of them) obtained for every white grape cultivar.Table 1 also shows the aglycone-type flavonol profiles obtained bythe sum total of all the molar percentages of flavonol 3-O-glycosides having the same type of aglycone (namely quercetin-type, kaempferol-type, and isorhamnetin-type flavonols). The useof aglycone-type flavonol profiles combined with flavonol 3-O-glycosides profiles allowed better results in the statisticaldifferentiation among grape cultivars. In general, the results werein agreement with the scarce data available on white grapeflavonol profiles. However, the already published data onlyconsidered the most abundant flavonols (e.g., quercetin 3-O-glucoside and 3-O-glucuronide, and the 3-O-glucosides of kaemp-ferol and isorhamnetin) which were tentatively identified only byHPLC–DAD (Rodrıguez Montealegre et al., 2006) or only consideredthe flavonols grouped by aglycone-type (Mattivi et al., 2006).

Quercetin-type flavonols dominated the flavonol profile ofwhite grapes, accounting for 60.8–90.7% of the total flavonols witha mean value of 77.2 � 7.3%. Kaempferol-type flavonols were thesecond in importance (range, 8.8–38.3%; mean value, 21.4 � 7.4%),whereas isorhamnetin-type flavonols usually occurred as very minorcompounds (mean value of 1.5 � 1.2%, although five of the whitegrape cultivars reached values of between 2% and 4.8%). Kaempferol-type flavonols have been found as minor compounds contributing tothe flavonol profiles of red grape cultivars (Mattivi et al., 2006;Castillo-Munoz et al., 2007), usually accounting for no more than 5%.However, it is remarkable that in the case of white grape cultivarsthey are important flavonols, in agreement with previously reporteddata (Mattivi et al., 2006). The 3-O-glucosides were the mainderivatives for each of the aglycone-type flavonols, with theexception of quercetin 3-O-glucuronide which was the mainquercetin-type flavonol for a half of the studied white grape cultivars,as has been also found in many red grape cultivars (Castillo-Munoz

Table 2Component (PC) Analysis of flavonol profiles of white grape cultivars.

PC Most correlated variables

with each PC

Loadings % Explained

variance

1 Quercetin-type flavonols �0.983 38.67

Kaempferol-type flavonols 0.982

Kaempferol 3-O-glucoside 0.968

Kaempferol 3-O-galactoside 0.960

2 Quercetin 3-O-glucoside 0.975 20.86

Quercetin 3-O-galactoside 0.902

3 Isorhamnetin-type flavonols 0.983 18.23

Isorhamnetin 3-O-glucoside 0.965

N. Castillo-Munoz et al. / Journal of Food Composition and Analysis 23 (2010) 699–705 703

et al., 2007). A poor correlation was found between the contents of the3-O-glucoside and 3-O-glucuronide derivatives of quercetin (R2 ofonly 0.4096), thus suggesting that glucosylation and glucuronylationdo not seem to be competitive steps in the biosynthetic pathway ofgrape flavonols. Finally, Table 1 also shows the total content offlavonols found in the analysed white grape cultivars, the concentra-tions ranging between 7.9 and 160.4 mmol/kg of fresh grape with amean value of 83.7 � 39.2 mmol/kg. Bearing in mind that the flavonolcontent of the grape is strongly affected by the degree of illuminationof the grape cluster (Downey et al., 2004; Haselgrove et al., 2000;Price et al., 1995), the total content of flavonols cannot be consideredto be characteristic of a grape cultivar. However, all the analysedgrape cultivars were grown in similar conditions (especially the typeof canopy management which, to a large extent, determines thedegree of illumination of the grape cluster). For this reason, the verylow content of flavonols shown in the analysed Pedro Ximenez grapescan be highlighted (only 7.9 mmol/kg) and, to a lesser extent, inParellada grapes (30.8 mmol/kg). In contrast, the analysed samples ofcultivars Jaen, Moscatel grano menudo, and Malvar, accounted for thehighest total flavonol contents (153.3–160.4 mmol/kg).

Grape flavonol profiles were submitted to statistical analysis tocheck their ability for authentication and differentiation of whitegrape cultivars. Principal Component (PC) Analysis allowed forsome grouping of the white grape cultivars according to theirflavonol profiles, and it also provided the flavonols which could beconsidered as markers for such differences. Table 2 shows the mostcorrelated variables (loadings which have absolute values higherthan 0.9) with the first three PC explaining 77.76% of the totalvariance of the data matrix (22 grape varieties � 13 flavonol andaglycone-type profiles percentages). The main variables thatallowed the grouping of white grape cultivars were the molarpercentages of the most abundant flavonols (PC1 in Fig. 3,explaining 38.67% of the total observed variance), namelyquercetin-type and kaempferol-type flavonols (in particular the3-O-glucoside and 3-O-galactoside of kaempferol). Most of theconsidered grape cultivars (14 out of the 22 assayed) containedquercetin-type and kaempferol-type flavonols within the ranges[(Fig._3)TD$FIG]

Fig. 3. Plot of the different white grape cultivars in the space defined by the Principal Com

their characteristic flavonol profiles. The most correlated variables with PC1: Q-type (neg

glc and Q-3-gal. Abbreviations as in Table 1.

73.10–81.61% and 15.23–25.60% respectively, which can beconsidered as medium proportions for these flavonols. Theaforementioned big group of grape cultivars could be additionallydifferentiated according to their molar percentages of the 3-O-glucoside and 3-O-galactoside of quercetin (PC2 in Fig. 3, explain-ing 20.86% of the total observed variance): the cultivars Airen,Chardonnay, Chelva, Merseguera, Riesling, Ugni blanc, and Verdejoaccounted for medium proportions of both quercetin derivatives,that is within the ranges 33.99–37.31% and 4.17–4.96% respec-tively; the cultivars Gewurztraminer and Macabeo accounted forlower proportions of both flavonols; the cultivars Malvar, PedroXimenez, Torrontes, and Viognier accounted for higher proportionsof these flavonols; finally, the cultivar Albillo accounted for thehighest proportions of both quercetin 3-O-glucoside (45.56%) and3-O-galactoside (6.83%).

Other groupings of grape cultivars shown in Fig. 3 were: thecultivar Listan de Huelva which accounted for the highestproportion of quercetin-type and the lowest proportion ofkaempferol-type flavonols and it also showed low proportionsof quercetin 3-O-glucoside and 3-O-galactoside; the cultivarsPardillo, Parellada, and Sauvignon blanc accounted for medium–high proportions of quercetin-type and low–medium proportionsof kaempferol-type flavonols, Parellada cultivar accounted for

ponents 1 and 2 (PC1 and PC2) obtained by Principal Component Analysis applied to

atively), K-type, K-3-glc, and K-3-gal. The most correlated variables with PC2: Q-3-

[(Fig._4)TD$FIG]

Fig. 4. Plot of the different white grape cultivars in the space defined by the Principal Components 1 and 3 (PC1 and PC3) obtained by Principal Component Analysis applied to

their characteristic flavonol profiles. The most correlated variables with PC1: Q-type (negatively), K-type, K-3-glc, and K-3-gal. The most correlated variables with PC3: I-type

and I-3-glc. Abbreviations as in Table 1.

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medium–high proportions of quercetin 3-O-glucoside and 3-O-galactoside whereas the other two grape cultivars accounted forlow–medium contents of these two quercetin derivatives; thecultivars Mantuo and Moscatel Alejandrıa accounted for low–medium proportions of quercetin-type flavonols and medium–high proportions of kaempferol-type flavonols, but they differedconsiderably in their proportions of quercetin 3-O-glucoside and 3-O-galactoside, with Moscatel Alejandrıa reaching the lowest scoreand Mantuo the second highest score along the PC2. Finally, thelowest proportions of quercetin-type flavonols and the highest ofkaempferol-type flavonols corresponded to the cultivars Jaen andMoscatel grano menudo, but whereas the grape cultivar Jaenaccounted for medium proportions of quercetin 3-O-glucoside and3-O-galactoside, the grape cultivar Moscatel grano menudoaccounted for low–medium values.

The minor isorhamnetin-type flavonols, especially its 3-O-glucoside derivative, allowed an additional differentiation of somewhite grape cultivars along the PC3 (Fig. 4, PC3 explained 18.23% ofthe total variance). Most of the grape cultivars accounted for molarpercentages of isorhamnetin-type flavonols no higher than 2.0%,and three subgroups can be differentiated: Airen, Listan de Huelva,Mantuo, Parellada, and Ugni blanc (0.4–0.6% of isorhamnetin-typeflavonols); Albillo, Chardonnay, Jaen, Malvar, Moscatel Alejandrıa,Moscatel grano menudo, Torrontes, and Viognier (0.6–1.2% ofisorhamnetin-type flavonols); Macabeo, Merseguera, Pardillo, andSauvignon blanc (1.3–2.0% of isorhamnetin-type flavonols).However, some grape cultivars accounted for higher proportionsof isorhamnetin-type flavonols: the cultivars Riesling, Albillo, andVerdejo with increasing values of 2.1%, 2.5% and 3.3% respectivelyand, remarkably, the cultivars Gewurztraminer (3.9%) and PedroXimenez (4.8%).

In summary, some of the white grape cultivars showedremarkable differences in their flavonols that can be consideredas a cultivar characteristic. The cultivars Jaen and Moscatel degrano menudo have the highest proportions of kaempferol-typeflavonols. The cultivars Albillo and Mantuo show the highestproportions of quercetin 3-O-glucoside and 3-O-galactoside. Two

individual grape cultivars seem to show very characteristicflavonol profiles, namely Listan de Huelva (lowest proportionsof kaempferol-type flavonols and very low proportions ofquercetin 3-O-glucoside and 3-O-galactoside) and Moscatel deAlejandrıa (lowest proportions of quercetin 3-O-glucoside and 3-O-galactoside and medium–high proportions of kaempferol-typeflavonols). Finally, five grape cultivars (Rielsing, Albillo, Gewurz-traminer, Verdejo, and Pedro Ximenez) have important propor-tions of isorhamnetin-type flavonols.

However, the aforementioned results are only preliminary, andconfirmation is needed with further analysis. Although the grapesamples that we analysed were grown in the same viticulturalconditions, it is obvious that several unstudied factors (likedifferences in viticultural practices, soil, or ripening degree) couldmarkedly change the flavonol profile of a white grape cultivar. Otherauthors have obtained a good differentiation of ten autochthonouswhite grape cultivars from Galicia (north-western Spain) by PCA onthe basis of their total contents of quercetin, kaempferol, anddihydroflavonols (Masa et al., 2007), but only one of the assayedcultivars, Torrontes, coincided with that of our study; in contrast toour findings, they did not find quercetin 3-O-glucoside in thiscultivar, although this disagreement is probably due to the fact thatthey only used HPLC–DAD for flavonol identification.

4. Conclusions

The skins of white grapes contain flavonols as 3-O-glycosidederivatives of only kaempferol, quercetin and isorhamnetin. Theseries of 3-O-glucosides and 3-O-galactosides of kaempferol,quercetin, and isorhamnetin, as well the 3-O-glucuronides ofkaempferol and quercetin, were found in all the white grapecultivars studied. In addition, rutin (quercetin 3-O-(600-rhamno-syl)-glucoside) was detected as a minor compound for most whitegrape cultivars. Moreover, isorhamnetin 3-O-glucoronide wasfound as a trace compound for some of the grape samples. Theidentification by ESI-MSn of the complete isorhamnetin-typeflavonol series in white grapes has been supported for the first

N. Castillo-Munoz et al. / Journal of Food Composition and Analysis 23 (2010) 699–705 705

time. Although quercetin-type was the dominant flavonol in whitegrapes, the diverse grape cultivars showed enough characteristicflavonol profiles to allow some statistical differentiation. This wasmainly attributable to their contents in quercetin-type andkaempferol-type flavonols, but also the minor, newly reported,isorhamnetin-type flavonols contributed to this differentiation.

Acknowledgments

This research was supported by the Castilla-La Mancha RegionalGovernment (Junta de Comunidades de Castilla-La Mancha), withfunds provided by the Consejerıa de Educacion y Ciencia (ProjectPAI07-0024-4184) and the Instituto de la Vid y el Vino de Castilla-La Mancha (IVICAM; Project PREG-05-024). Author SGA thank theFondo Social Europeo and the Instituto Nacional de InvestigacionesAgrarias for co-funding his contract at the IVICAM. Author NCMthanks IVICAM for her research fellowship.

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