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Hindawi Publishing Corporation Journal of Analytical Methods in Chemistry Volume 2013, Article ID 572896, 9 pages http://dx.doi.org/10.1155/2013/572896 Research Article Evaluating the Polyphenol Profile in Three Segregating Grape (Vitis vinifera L.) Populations Alberto Hernández-Jiménez, 1 Rocío Gil-Muñoz, 2 Yolanda Ruiz-García, 1 Jose María López-Roca, 1 Adrián Martinez-Cutillas, 2 and Encarna Gómez-Plaza 1 1 Food Science and Technology Department, Faculty of Veterinary Science, University of Murcia, Campus de Espinardo, 30071 Murcia, Spain 2 Instituto Murciano de Investigaci´ on y Desarrollo Agroalimentario, Carretera La Alberca s/n, 30150 Murcia, Spain Correspondence should be addressed to Encarna G´ omez-Plaza; [email protected] Received 20 May 2013; Accepted 17 July 2013 Academic Editor: Shixin Deng Copyright © 2013 Alberto Hern´ andez-Jim´ enez et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. is paper explores the characteristics of the anthocyanin and flavonol composition and content in grapes from plants resulting from intraspecific crosses of Vitis vinifera varieties Monastrell × Cabernet Sauvignon, Monastrell × Syrah, and Monastrell × Barbera, in order to acquire information for future breeding programs. e anthocyanin and flavonol compositions of twenty-seven hybrids bearing red grapes and 15 hybrids bearing white grapes from Monastrell × Syrah, 32 red and 6 white from Monastrell × Cabernet Sauvignon, and 13 red from Monastrell × Barbera have been studied. Among the intraspecific crosses, plants with grapes presenting very high concentrations of anthocyanins and flavonols were found, indicating a transgressive segregation for this character, and this could lead to highly colored wines with an increased benefits for human health. As regards the qualitative composition of anthocyanins and flavonols, the hydroxylation pattern of the hybrids that also may influence wine color hue and stability presented intermediate values to those of the parentals, indicating that values higher than that showed by the best parental in this respect will be difficult to obtain. e results presented here can be helpful to acquire information for future breeding efforts, aimed at improving fruit quality through the effects of flavonoids. 1. Introduction Anthocyanins are responsible for the color of red grape varieties and the wines produced from them. Flavonols are also important because they participate both in stabilizing anthocyanins in young red wines through copigmentation and in increasing the health-related properties of wine [1, 2]. Grape anthocyanins and flavonols are final products arising from the flavonoid biosynthetic pathway (Figure 1). Vitis vinifera varieties are characterized by the presence of 3-O- glucosides of delphinidin, peonidin, petunidin, cyanidin, and malvidin, together with their acylated derivatives [3]. e 3-O-glucosides of kaempferol, quercetin, and myricetin are the major flavonols in grapes as first reported by Cheynier and Rigaud [4] and recently confirmed by Castillo-Mu˜ noz et al. [5], with quercetin glycosides usually being dominant [5], although a high presence of quercetin-3-O-glucuronide has also been observed in some varieties such as Petit Verdot [5, 6]. When studying grapes for winemaking, it is not only the quantity of anthocyanins that is important. e hydrox- ylation pattern of the B-ring of anthocyanins is one of the main structural features of flavonoids and is an important determinant of their coloration, stability, and antioxidant capacity. Trihydroxylated anthocyanins (delphinidin, petuni- din, and malvidin-3-glucosides) are more stable in wines than dihydroxylated ones (cyanidin and peonidin-3-glucosides) [7]. ose with orthodiphenolic groups (cyanidin, delphini- din, and petunidin) have an enhanced susceptibility to oxidation. e methoxylated anthocyanins are also more stable. e same applies to acylated anthocyanins, since their esterification of anthocyanins promotes intramolecular aggregation or stacking, which protects the oxonium ion from decomposition [8].
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grape polyphenols
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Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2013 Article ID 572896 9 pageshttpdxdoiorg1011552013572896
Research ArticleEvaluating the Polyphenol Profile in Three SegregatingGrape (Vitis vinifera L) Populations
Alberto Hernaacutendez-Jimeacutenez1 Rociacuteo Gil-Muntildeoz2 Yolanda Ruiz-Garciacutea1
Jose Mariacutea Loacutepez-Roca1 Adriaacuten Martinez-Cutillas2 and Encarna Goacutemez-Plaza1
1 Food Science and Technology Department Faculty of Veterinary Science University of Murcia Campus de Espinardo30071 Murcia Spain
2 Instituto Murciano de Investigacion y Desarrollo Agroalimentario Carretera La Alberca sn 30150 Murcia Spain
Correspondence should be addressed to Encarna Gomez-Plaza encarnagomezumes
Received 20 May 2013 Accepted 17 July 2013
Academic Editor Shixin Deng
Copyright copy 2013 Alberto Hernandez-Jimenez et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
This paper explores the characteristics of the anthocyanin and flavonol composition and content in grapes from plants resultingfrom intraspecific crosses ofVitis vinifera varietiesMonastrelltimesCabernet SauvignonMonastrelltimes Syrah andMonastrelltimesBarberain order to acquire information for future breeding programsThe anthocyanin and flavonol compositions of twenty-seven hybridsbearing red grapes and 15 hybrids bearing white grapes from Monastrell times Syrah 32 red and 6 white from Monastrell times CabernetSauvignon and 13 red fromMonastrell times Barbera have been studied Among the intraspecific crosses plants with grapes presentingvery high concentrations of anthocyanins and flavonols were found indicating a transgressive segregation for this character andthis could lead to highly colored wines with an increased benefits for human health As regards the qualitative composition ofanthocyanins and flavonols the hydroxylation pattern of the hybrids that also may influence wine color hue and stability presentedintermediate values to those of the parentals indicating that values higher than that showed by the best parental in this respectwill be difficult to obtain The results presented here can be helpful to acquire information for future breeding efforts aimed atimproving fruit quality through the effects of flavonoids
1 Introduction
Anthocyanins are responsible for the color of red grapevarieties and the wines produced from them Flavonols arealso important because they participate both in stabilizinganthocyanins in young red wines through copigmentationand in increasing the health-related properties of wine [1 2]Grape anthocyanins and flavonols are final products arisingfrom the flavonoid biosynthetic pathway (Figure 1) Vitisvinifera varieties are characterized by the presence of 3-O-glucosides of delphinidin peonidin petunidin cyanidin andmalvidin together with their acylated derivatives [3] The3-O-glucosides of kaempferol quercetin and myricetin arethe major flavonols in grapes as first reported by Cheynierand Rigaud [4] and recently confirmed by Castillo-Munozet al [5] with quercetin glycosides usually being dominant[5] although a high presence of quercetin-3-O-glucuronide
has also been observed in some varieties such as Petit Verdot[5 6]
When studying grapes for winemaking it is not onlythe quantity of anthocyanins that is important The hydrox-ylation pattern of the B-ring of anthocyanins is one of themain structural features of flavonoids and is an importantdeterminant of their coloration stability and antioxidantcapacity Trihydroxylated anthocyanins (delphinidin petuni-din andmalvidin-3-glucosides) aremore stable inwines thandihydroxylated ones (cyanidin and peonidin-3-glucosides)[7] Those with orthodiphenolic groups (cyanidin delphini-din and petunidin) have an enhanced susceptibility tooxidation The methoxylated anthocyanins are also morestable The same applies to acylated anthocyanins sincetheir esterification of anthocyanins promotes intramolecularaggregation or stacking which protects the oxonium ionfrom decomposition [8]
2 Journal of Analytical Methods in Chemistry
Naringenin
Dihydroquercetin Dihydromyricetin
Leucocyanidin
DFR
ANS
UFGT
Phenylalanine
DFR
ANS
UFGT
Eriodictyol
Leucodelphinidin
DelphinidinCyanidin
MTMT
FLS
Quercetin
FLS
Myricetin
GT GT GT
Quercetinglucoside glycoside
Myricetinglycoside
MT
Laricitringlycoside
glycoside
MT
glucosideMT
Cyanidin-3-glucoside
Peonidin-3-glucoside Petunidin-3-glucoside Malvidin-3-glucoside
MT
Delphinidin-3-glucoside
F39984005998400H
F39984005998400H
F3998400H
F3998400H
F3998400H
F3998400H Dihydrokaempferol
Pentahydroxyflavone
Kaempferol
Kaempferol
Isorhamnetin
Syringetin
Figure 1 Flavonoid biosynthetic pathway PAL phenyl ammonia lyase F31015840H flavonoid-31015840-hydroxylase F3101584051015840H flavonoid-3101584051015840-hydroxylaseUFGT UDP-glucose flavonoid 3-O-glucosyltransferase MT methyl transferase DFR dihydroxyflavanol-4-reductase ANS anthocyaninsynthase and FLS flavonol synthase
As hydroxylation of the B-ring is carried out by flavon-oid-31015840-hydroxylase (F31015840H) and flavonoid-3101584051015840-hydroxylase(F3101584051015840H) enzymes the composition of anthocyanins in grapeskins will be determined by the relative activities of theseenzymes At the same time methyl transferase (MT) activitywill determine the different methoxylation patterns of B ringand acyl transferase the presence of acyl derivatives Most ofthese enzymes also control the synthesis of flavonols
Plant adaptation to different environments and centuriesof selection by humans has produced numerous genotypes inwhich the intensity of the red coloration varies extensivelyA mixture of variation in anthocyanin content and therelative proportion of different anthocyanins can producedifferent phenotypes for skin pigmentation with conse-quent technological and nutritional differences [9] Alsothese differences could be a very useful chemotaxonomicaltool [3]
At present cross-breeding and bud mutation are still themost common way for developing new wine grape cultivars[10]The determination of themechanisms of the inheritanceof the flavonoid composition could help in the election ofthe parentals in the breeding programs The objective ofthis study therefore was to explore the characteristics ofthe anthocyanin and flavonol composition and content inintraspecific hybrids of Monastrell times Syrah Monastrell timesCabernet Sauvignon and Monastrell times Barbera in orderto acquire information for future breeding efforts aimed atimproving fruit quality through the effects of flavonoids andto provide an insight into the mechanisms that control theinheritance of flavonoid characteristics among hybrids
2 Material and Methods
A collection of plants arising from crosses from Monastrelltimes Syrah Monastrell times Cabernet Sauvignon and Monastrelltimes Barbera was used in this study The study was conductedin an experimental vineyard of 1 ha located in Bullas (Mur-cia SE Spain) The parentals (Monastrell Syrah CabernetSauvignon and Barbera) were planted in 1997 whereas theseeds for the interspecific hybrids were planted in 2000 Thetraining system was a bilateral cordon trellised to a three-wire vertical system and drip irrigation was applied Plantingdensity was 25m between rows and 125m between vinesTwo two-bud spurs (4 nodes) were left at pruning timeGrapes were sampled in 2007
The plants derived from Monastrell self-pollination andfrom pollen donors other than Syrah Cabernet Sauvignonor Barberawere identified genetically and discarded using themicrosatellite (SSR Simple Sequence Repeat) loci segregating1 1 1 1 according to Bayo-Canha et al [11] Total DNA wasextracted from approximately 20mg of young frozen leavesusing a DNeasy Plant Mini Kit (Qiagen Valencia CA USA)following the manufacturerrsquos protocol Genotyping was car-ried out as described in Adam-Blondon et al [12] PCR prod-ucts were separated by capillary electrophoresis performedon an ABI Prism 3100 genetic analyzer (Applied BiosystemsCarlsbad CA USA) and the fragments were sized usingGeneMapper software (Applied Biosystems Carlsbad CAUSA)
Grapes were harvested at a total of soluble solids contentbetween 23 and 27∘Brix The sampling was randomly madeby picking berries from the top central and bottom parts of
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several clusters of each hybrid vine The size of the samplewas around 300 berries which were bulked and separated in3 subsamples of approximately 100 berries to run triplicateanalyses Grape samples were kept frozen (minus20∘C) untilextraction and analysis
21 AnthocyaninMonoglycosides and Flavonols in Berry SkinsGrapes were peeled with the help of a scalpel Samples (2 g)were immersed in methanol (40mL) in hermetically closedtubes and placed on a stirring plate at 150 rpm and 25∘CAfter 2 hours the methanolic extracts were acidified with 5formic acid (1 2 vv) filtered through 02120583m PTFE filtersand analysed by HPLC
22 Identification and Quantification of Anthocyanins TheHPLC analyses were performed on a Waters 2695 liquidchromatograph (Waters PA USA) equipped with a Waters2996 diode array detector and a Primesep B2 column (SielcIllinois USA) 25 times 04 cm 5120583m particle size using assolvents water plus 5 formic acid (solvent A) and HPLCgrade acetonitrile (solvent B) at a flow rate of 08mLminminus1Elution was performed with a gradient starting with 5 Bto reach 9 B at 28min 13 B at 30min 21 B at 52min24 B at 65min and 70 B at 75min maintaining thisgradient for 5 minutes Chromatograms were recorded at520 nm (anthocyanins) and 360 nm (flavonols)
Identification of the compounds was carried out bycomparing their UV spectra recorded with the diode arraydetector and those reported in the literature Also an HPLC-MS analysis was made to confirm the identity of each peakAn LC-MSD-Trap VL-01036 liquid chromatograph-ion trapmass detector (Agilent Technologies Waldbronn Germany)equipped with an electrospray ionization (ESI) system wasused Elutionwas performed in theHPLC analysis conditionsdescribed previously with a flow rate of 08mL minminus1 Theheated capillary and voltage were maintained at 350∘C and4 kV respectively Mass scans (MS) were measured frommz100 up tomz 800
Anthocyanins were quantified at 520 nm as malvidin-3-glucoside using malvidin-3-glucoside chloride as externalstandard (Extrasynthese Genay France) Flavonols werequantified at 360 nm as quercetin-3-glucoside using thiscompound as external standard (Sigma Missouri USA)
23 Statistical Data Treatment All the analyses were per-formed with the statistical package Statgraphics 51
3 Results and Discussion
For this study 27 hybrids bearing red grapes and 15 hybridsbearing white grapes from Monastrell times Syrah 32 red and6 white from Monastrell times Cabernet Sauvignon and 13red from Monastrell times Barbera were studied The presenceof white hybrids in Monastrell times Cabernet Sauvignon andMonastrell times Syrah indicates the heterozygous nature ofthe parentals in regard to genes controlling anthocyaninsynthesis whereas Barbera being homozygous [13] does notproduce hybrids bearing white grapes Boss et al [14] and
Kobayashi et al [15] showed that expression of the UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) gene iscritical for anthocyanin biosynthesis in grape Experimentswith the berry skins of white and red cultivars revealed thatthe UFGT gene was expressed in all the red cultivars butnot in the white ones whereas the other genes involved inanthocyanin biosynthesis (Figure 1) are expressed in bothwhite and red cultivars The presence or absence of theenzymeUFGT is controlled byMyb-related regulatory genesand the insertion of the retroelement Gret1 in the promoterregion of VvmybA1 gene appears to be associated withwhite-fruited cultivars when present in a homozygous statePigmented cultivars possess at least one allele at theVvmybA1locus not containing this large retroelement [13 16] as is thecase of parentals of this study
Table 1 shows the results of the anthocyanin analysisfor the studied grapes and Figure 2 the range of concen-trations among the hybrids Syrah grapes contained higherconcentration of anthocyanins than Monastrell grapes Themean concentration in their hybrid grapes (1631mgg freshskin) was slightly higher than in Syrah (1506mgg freshskin) and the maximum value reached by the grapes ofone seedling (3949mgg fresh skin) was twice the valuefound in Syrah Cabernet Sauvignon and Barbera grapesalso showed higher concentration of anthocyanins thanMonastrell and were similar to that of Syrah grapes Themean values of anthocyanin content in the grapes of theirseedlings were slightly lower than in Cabernet Sauvignonand Barbera (1442 and 1208mgg fresh skin respectively)and higher than that shown by Monastrell grapes (740mggfresh skin) and again the maximum value found in theirseedlings was double that of Cabernet Sauvignon and Bar-bera grapes Many hybrids presented grapes with muchhigher concentration than their parentals as can be seenin Figure 2 The appearance of a large number of hybridsin which the anthocyanin concentration is not within therange of concentration of their parental phenotypes is calledtransgressive segregation frequent in intraspecific crossesand in domesticated populations The occurrence of thesegregation of a given traitmanifestedmainly in one direction(as happens in our case most of the hybrids showing highervalues of anthocyanin concentration than the parentals) mayimply that the trait has undergone fairly constant directionalselection or a certain overdominance of the genes controllingphenolic synthesis [17 18] Similar results were found byLiang et al [10] in grapes but our findings differ from thoseof Ju et al [19] in apples that found that crossing between twored-fruited apple cultivars produced less colored progenyA previous work exploring the proanthocyanidin content ofMonastrell times Syrah hybrid grapes also reported this type ofsegregation for this character [20]
As stated previously the presence of the enzyme UFGTis necessary for anthocyanin biosynthesis However thebiosynthesis of the different anthocyanin precursors is drivenupstream of the enzyme UFGT by the activity of F31015840H andF3101584051015840H enzymes which add either a single hydroxyl groupor two to dihydrokaempferol (Figure 1) Once convertedto dihydroquercetin or dihydromyricetin these intermedi-ates flow through common downstream enzymes to form
4 Journal of Analytical Methods in Chemistry
Table 1 Mean values (119899 = 3) of the anthocyanin profile and total anthocyanin content (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids
del cyan pet peon malv nonacylated acylated dihydrox trihydrox Total (mgg)Monastrell
Mean 125 137 100 200 397 552 448 377 623 740Syrah
Mean 72 36 81 109 681 496 503 167 833 1506CS
Mean 96 52 101 74 677 459 541 126 874 1507Barbera
Mean 98 31 139 47 686 656 343 77 923 1402Mon times Sy (27)a
Mean 100 71 86 154 548 432 568 265 734 1631Minimum 57 32 64 65 295 462 297 132 462 386Maximum 243 154 139 375 727 702 713 537 868 3949
Mon times CS (32)b
Mean 124 60 133 105 578 525 472 165 835 1444Minimum 75 35 94 56 444 352 313 95 689 487Maximum 234 121 203 215 685 686 647 311 905 2867
Mon times Bar (13)c
Mean 109 77 133 162 519 720 280 239 761 1208Minimum 41 11 90 44 317 415 101 67 552 161Maximum 170 146 197 364 779 898 585 447 933 2788
abcThe number in parenthesis represents the number of hybrids for each crossing del percentage of delphinidin derivatives cyan percentage of cyanidin derivatives pet percentage of petunidin derivatives peon percentage ofpeonidin derivatives malv percentage of malvidin derivatives nonacylated percentage of nonacylated anthocyanins acylated percentage of acylatedanthocyanins dihydrox percentage of dihydroxylated anthocyanins trihydrox percentage of trihydroxylated anthocyanins and Total (mgg) totalanthocyanin content (mg per g of skin)
disubstituted and trisubstituted anthocyanins when UFGTis expressed and to form other polyphenols (flavanolsflavonols) at different developmental stages All the studiedhybrids bearing red grapes synthesised all five anthocyanins(the dihydroxylated cyaniding peonidin-3-glucosides thetrihydroxylated delphinidin petunidin and malvidin-3-glucosides) together with their acylated derivatives Thismeans that all the parentals and the hybrids expressed func-tional F31015840HandF3101584051015840Hgenes for the synthesis of 3101584041015840-OHand310158404101584051015840-OH anthocyanins as well as methyltransferases (MT)for the methylation of primary anthocyanins As regardsthe percentage of the different anthocyanins in the differentparentals Monastrell grapes were characterized by a highpercentage of cyanidin suggesting a lower F3101584051015840H activitythan in the other varieties A low expression of F3101584051015840H hasbeen associated with cyanidin-based anthocyanins in grapeleafs [21] The percentages of malvidin-based anthocyaninsin Monastrell did not exceed 40 so the total percentageof trihydroxylated anthocyanins was low The percentage oftrihydroxylated anthocyanins reached 83 in Syrah grapes874 in Cabernet Sauvignon grapes and 923 in Barberagrapes The percentage of trihydroxylated anthocyanins waseven higher in some Monastrell times Cabernet Sauvignonand Monastrell times Barbera hybrids reaching values as highas 905 and 93 respectively The mean value of thepercentage of trihydroxylated anthocyanins in the hybridgrapes is close to the mean value between both parentals
The segregation can be fitted to a normal distribution and avery low number of hybrids presented higher or lower valuesthan in the parentals as can be seen in Figure 3These resultswere similar to those obtained during the first screening ofthe anthocyanin profile in Monastrell times Cabernet Sauvignongrapes [22] In spite of the presence of all anthocyaninbiosynthetic enzymes in all the investigated hybrids (sinceall the possible structures were found) a genotype-specificregulation of the structural genes along the core pathway andat the main branching points is presumed to underlie theobservedmethoxylation and hydroxylation variations amongthe grapes from the parentals and those of their hybrid plants
With regard to the percentage of anthocyanin acylationBarbera grapes showed the lowest percentage of acylation(343) followed by Monastrell (448) and CabernetSauvignon showed the highest percentages (54) The meanvalues of the percentage of acylated anthocyanins for Monas-trell times Cabernet Sauvignon hybrids were 47 568 forMonastrell times Syrah (higher than the percentage found inSyrah) while the hybrid grapes from Monastrell times Barberashowed the lowest percentages of acylation (28) As inthe case of the anthocyanin concentration data there was atendency towards higher values of acylation in the hybridsand no hybrid contained only nonacylated anthocyanins
As regards flavonols (Table 2) these flavonoids werepresent in both white and red grapes We could iden-tify mono- (kaempferol) di- (quercetin and isorhamnetin)
Journal of Analytical Methods in Chemistry 5
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Figure 2 Concentration of anthocyanins in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c)hybrid grapes (each bar represents the mean value of three samples)
6 Journal of Analytical Methods in Chemistry
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Journal of Analytical Methods in Chemistry 7
Table 2 Mean values of the flavonol profile and total flavonolcontent (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids (119899 = 3)
monohydr dihydr trihydrox TotalMonastrell
Mean 109 707 184 056Syrah
Mean 134 442 424 073CS
Mean 147 446 407 019Barbera
Mean 43 533 424 049Red hybrids
Mon times SyMean 104 524 372 073Minimum 53 363 165 018Maximum 160 726 550 183
Mon times CSMean 113 511 376 037Minimum 50 352 203 011Maximum 209 650 583 078
Mon times BarMean 94 619 287 042Minimum 47 429 87 012Maximum 136 839 434 139
White hybridsMon times Sy
Mean 134 841 24 035Minimum 41 746 06 008Maximum 237 947 72 128
Mon times CSMean 200 748 51 008Minimum 168 689 09 002Maximum 259 784 127 018
monohydrox percentage of monohydroxylated flavonols dihydroxpercentage of dihydroxylated flavonols trihydrox percentage of trihy-droxylated flavonols Total total flavonol content (mg per g of skin)
and trihydroxylated (myricetin laricitrin and syringetin)flavonol glycosides (glucosides glucoronides and smallquantities of galactosides) In red grapes the monohydrox-ylated flavonols represented the lowest percentage especiallyin Barbera grapes (43) Monastrell grapes presented a veryhigh percentage of dihydroxylated flavonols (707) muchhigher than in the other varieties and therefore a lowerpercentage of trihydroxylated flavonols whereas BarberaSyrah and Cabernet Sauvignon grapes reached a percentageof trihydroxylated flavonols of around 45ndash50 This factindicates lower F3101584051015840H activity in Monastrell grapes as alsoobserved for the anthocyanins
As regards the flavonol content Cabernet Sauvignongrapes showed low concentrations of flavonols and Monas-trell and Syrah much higher concentrations while the highestvalue was found in red grapes from a hybrid plant fromMonastrell times Syrah (Table 2 Figure 4) reaching values as
high as 183mgg of skin In the hybrids bearing white grapesa lower concentration of flavonols was measured comparedwith that of the hybrids bearing red grapes Azuma et al[23 24] stated that Myb genes besides the regulation of theUFGT expression appear to enhance the expression of allthe genes involved in the anthocyanin biosynthesis pathwaybecause the transcription of all anthocyanin biosynthesisgenes appears to be slightly activated which would explainthe higher concentration of flavonols in red grapes Also inthese white skinned grapes trihydroxylated flavonols werebarely presentMattivi et al [25] studying the flavonol profileof several grape varieties did not detect trihydroxylatedflavonols in white grapes Bogs et al [26] did not findsignificant expression of F3101584051015840H and UFGT in white grapevarieties which suggests a related regulation of F3101584051015840H andUFGT during berry ripening and justifies the almost nullpresence of trihydroxylated flavonols in white grapes F3101584051015840Hwas detected in white grapes var Chardonnay but prior toveraison [26] since it is needed for flavanol biosynthesiswhich seems to be controlled differently indeed no differ-ences in the percentage of trihydroxylated flavanols wereobserved between the red and white grapes arising fromthe cross of Monastrell times Syrah [20] However other studiesstated that flavonol synthase (FLS) was not upregulated whenUFGT was expressed and that the increase in flavonols inred grapes was a consequence of an increase flux through theflavonoid pathway [27]
4 Conclusions
The study of the anthocyanin and flavonol profiles of thegrapes from the hybrid plants can be useful for a targetedinformative metabolomic analysis [28] a tool for selectingpromising grapes according to their profile andor content
Seedlings with grapes presenting very high concentra-tions of anthocyanins and flavonols can be expected fromintraspecific crosses and these resulting grapes could leadto highly colored wines with increased health-related prop-erties In this way three plants arising from Monastrell timesSyrah (hybrids 8 37 and 71) presented anthocyanin andflavonol concentrations higher than 20 and 1mgg fresh skinrespectively (Figures 2 and 4) Also four plants arising fromMonastrell timesCabernet Sauvignon (hybrids 38 59 55 and 80)and two from Monastrell times Barbera (hybrid plants 120 121)showed anthocyanin values higher than 20mgg fresh skinaccompanied with high concentration of flavonols (Figures2 and 4) The hydroxylation pattern which also influenceswine color and its stability will be strongly influenced by theparentals pattern since values higher than that shown by thebest parental in this respect will be difficult to obtain Alsoin the case of the crosses between heterozygous parentals(Monastrell Cabernet Sauvignon and Syrah) hybrids bearingwhite grapes can be obtained some of them with a highconcentration of flavonols that could be of importance in thehealth properties of this fruit and its derived products suchas the wineThe information obtained in this study should behelpful for selecting parentals for breeding programs
8 Journal of Analytical Methods in Chemistry
0
500
1000
1500
2000
(120583g
g)
B-M
timesS
30B-
Mtimes
S 1
B-M
timesS
19M
timesS
28M
timesS
31B-
Mtimes
S 10
0M
timesS
20B-
Mtimes
S 59
B-M
timesS
118
B-M
timesS
9B-
Mtimes
S 14
B-M
timesS
94B-
Mtimes
S 82
B-M
timesS
15M
timesS
26M
timesS
0M
timesS
75B-
Mtimes
S 40
Mtimes
S 38
Mtimes
S 27
Mtimes
S 66
B-M
timesS
73B-
Mtimes
S 65
Mtimes
S 21
Mtimes
S 76
Mon
astre
llM
timesS
11M
timesS
3M
timesS
114
Mtimes
S 62
Mtimes
S 34
Syra
hM
timesS
56M
timesS
117
Mtimes
S 47
Mtimes
S 97
Mtimes
S 4
Mtimes
S 42
Mtimes
S 46
Mtimes
S 71
Mtimes
S 37
B-M
timesS
91M
timesS
8M
timesS
57
(a)
0
200
400
600
800
1000
(120583g
g)
B-M
timesCS
17
B-M
timesCS
13
B-M
timesCS
93
B-M
timesCS
171
B-M
timesCS
146
Mtimes
CS 9
9M
timesCS
27
Mtimes
CS D
Mtimes
CS 1
84M
timesCS
135
Mtimes
CS 4
B-M
timesCS
98
Mtimes
CS 1
07Ca
bern
etS
Mtimes
CS 9
1M
timesCS
7M
timesCS
26
Mtimes
CS 1
36M
timesCS
163
Mtimes
CS 9
6M
timesCS
84
Mtimes
CS 1
58M
timesCS
100
Mtimes
CS 1
99M
timesCS
72
Mtimes
CS E
Mtimes
CS 4
9M
timesCS
BM
timesCS
37
Mtimes
CS 1
9M
timesCS
16
Mtimes
CS 8
0M
timesCS
55
Mon
astre
llM
timesCS
38
Mtimes
CS 9
0M
timesCS
CM
timesCS
59
Mtimes
CS 5
6M
timesCS
209
Mtimes
CS A
(b)
0
200
400
600
800
1000
1200
1400
1600
Mtimes
B 55
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 66
Mtimes
B 75
Mtimes
B 10
9
Barb
era
Mtimes
B 69
Mtimes
B 12
1
Mtimes
B 12
0
Mon
astre
ll
Mtimes
B 70
Mtimes
B 84
(120583g
g)
Mtimes
B 94
Mtimes
B 12
2
(c)
Figure 4 Concentration of flavonols in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c) hybridgrapes (yellow bars indicate white grape bearing hybrids)
Journal of Analytical Methods in Chemistry 9
Acknowledgment
This work was made possible by financial assistance of theMinisterio de Ciencia e Innovacion Project AGL2006-11019
References
[1] F Alen-Ruiz M S Garcıa-Falcon M C Perez-Lamela EMartınez-Carballo and J Simal-Gandara ldquoInfluence of majorpolyphenols on antioxidant activity in Mencia and Brancellaored grapesrdquo Food Chem vol 113 pp 53ndash60 2008
[2] S C Forester and A L Waterhouse ldquoMetabolites are key tounderstanding health effects of wine polyphenolicsrdquo Journal ofNutrition vol 139 no 9 pp 1324Sndash1831S 2009
[3] A Ortega-Regules I Romero-Cascales J M Lopez-Roca J MRos-Garcıa and E Gomez-Plaza ldquoAnthocyanin fingerprint ofgrapes environmental and genetic variationsrdquo Journal of theScience of Food and Agriculture vol 86 no 10 pp 1460ndash14672006
[4] V Cheynier and J Rigaud ldquoHPLC separation and characteri-zation of flavonols in the skins of Vitis vinifera var CinsaultrdquoAmerican Journal of Enology and Viticulture vol 37 pp 248ndash252 1986
[5] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero andI Hermosın-Gutierrez ldquoFlavonol profiles of Vitis vinifera redgrapes and their single-cultivar winesrdquo Journal of Agriculturaland Food Chemistry vol 55 no 3 pp 992ndash1002 2007
[6] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero M VGomez A H Velders and I Hermosın-Gutierrez ldquoFlavonol 3-O-glycosides series of Vitis vinifera Cv Petit Verdot red winegrapesrdquo Journal of Agricultural and Food Chemistry vol 57 no1 pp 209ndash219 2009
[7] P Sarni H Fulcrand V Souillol JM Souquet andV CheynierldquoMechanisms of anthocyanin degradation in grape must likemodel solutionsrdquo Journal of the Science of Food and Agriculturevol 69 no 3 pp 385ndash391 1995
[8] C Malien-Aubert O Dangles andM J Amiot ldquoColor stabilityof commercial anthocyanin-based extracts in relation to thephenolic composition Protective effects by intra- and inter-molecular copigmentationrdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 170ndash176 2001
[9] H Chang H C Eun and S C Hyang ldquoQuantitative Structure-Activity Relationship (QSAR) of antioxidative anthocyanidinsand their glycosidesrdquo Food Science and Biotechnology vol 17 no3 pp 501ndash507 2008
[10] Z Liang C Yang J Yang et al ldquoInheritance of anthocyaninsin berries of Vitis vinifera grapesrdquo Euphytica vol 167 no 1 pp113ndash125 2009
[11] A Bayo-Canha J I Fernandez-Fernandez A Martınez-Cutillas and L Ruiz-Garcıa ldquoPhenotypic segregation andrelationships of agronomic traits in Monastrell times Syrah winegrape progenyrdquo Euphytica vol 186 pp 393ndash407 2012
[12] A F Adam-Blondon C Roux D Claux G Butterlin DMerdinoglu and P This ldquoMapping 245 SSR markers on theVitis vinifera genome a tool for grape geneticsrdquoTheoretical andApplied Genetics vol 109 no 5 pp 1017ndash1027 2004
[13] A R Walker E Lee J Bogs D A J McDavid M R Thomasand S P Robinson ldquoWhite grapes arose through the mutationof two similar and adjacent regulatory genesrdquo Plant Journal vol49 no 5 pp 772ndash785 2007
[14] P K Boss C Davies and S P Robinson ldquoAnalysis of theexpression of anthocyanin pathway genes in developing Vitis
vinifera L cv Shiraz grape berries and the implications forpathway regulationrdquo Plant Physiology vol 111 no 4 pp 1059ndash1066 1996
[15] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002
[16] D Lijavetzky L Ruiz-Garcıa J A Cabezas et al ldquoMoleculargenetics of berry colour variation in table graperdquo MolecularGenetics and Genomics vol 276 no 5 pp 427ndash435 2006
[17] M C de Vicente and S D Tanksley ldquoQTL analysis of transgres-sive segregation in an interspecific tomato crossrdquo Genetics vol134 no 2 pp 585ndash596 1993
[18] L H Rieseberg M A Archer and R K Wayne ldquoTransgressivesegregation adaptation and speciationrdquoHeredity vol 83 no 4pp 363ndash372 1999
[19] Z Ju C Liu Y Yuan YWang andG Liu ldquoColoration potentialanthocyanin accumulation and enzyme activity in fruit ofcommercial apple cultivars and their F1 progenyrdquo ScientiaHorticulturae vol 79 no 1-2 pp 39ndash50 1999
[20] A Hernandez-Jimenez E Gomez-Plaza A Martınez-Cutillasand J A Kennedy ldquoGrape skin and seed proanthocyanidinsfrom Monastrell times Syrah grapesrdquo Journal of Agricultural andFood Chemistry vol 57 no 22 pp 10798ndash10803 2009
[21] H Kobayashi S Suzuki F Tanzawa andT Takayanagispi ldquoLowexpression of flavonoid 3rsquo5rsquo-hydroxylase (F3rsquo5rsquoH) associatedwith cyanidin-based anthocyanins in grape leafrdquo AmericanJournal of Enology and Viticulture vol 60 no 3 pp 362ndash3672009
[22] E Gomez-Plaza R Gil-Munoz A Hernandez-Jimenez JM Lopez-Roca A Ortega-Regules and A Martınez-CutillasldquoStudies on the anthocyanin profile ofVitis vinifera intraspecifichybrids (Monastrell times Cabernet Sauvignon)rdquo European FoodResearch and Technology vol 227 no 2 pp 479ndash484 2008
[23] A Azuma S Kobayashi N Mitani et al ldquoGenomic and geneticanalysis ofMyb-related genes that regulate anthocyanin biosyn-thesis in grape berry skinrdquoTheoretical and Applied Genetics vol117 no 6 pp 1009ndash1019 2008
[24] A Azuma S Kobayashi H Yakushiji M Yamada N Mitaniand A Sato ldquoVvmybA1 genotype determines grape skin colorrdquoVitis vol 46 no 3 pp 154ndash155 2007
[25] FMattivi R Guzzon U VrhovsekM Stefanini and R VelascoldquoMetabolite profiling of grape flavonols and anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7692ndash7702 2006
[26] J Bogs A Ebadi D McDavid and S P Robinson ldquoIdentifi-cation of the flavonoid hydroxylases from grapevine and theirregulation dining fruit developmentrdquo Plant Physiology vol 140no 1 pp 279ndash291 2006
[27] F Quattrocchio A Baudry L Lepiniec and E GrotewoldldquoThe regulation of flavonoid biosynthesisrdquo in The Science ofFlavonoids E Grotewold Ed pp 97ndash122 Springer New YorkNY USA 2008
[28] J M Cevallos-Cevallos J I Reyes-De-Corcuera E EtxeberriaM D Danyluk and G E Rodrick ldquoMetabolomic analysis infood science a reviewrdquo Trends in Food Science and Technologyvol 20 no 11-12 pp 557ndash566 2009
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Analytical Methods in Chemistry
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Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
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Journal of
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Analytical ChemistryInternational Journal of
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Quantum Chemistry
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Organic Chemistry International
ElectrochemistryInternational Journal of
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CatalystsJournal of
2 Journal of Analytical Methods in Chemistry
Naringenin
Dihydroquercetin Dihydromyricetin
Leucocyanidin
DFR
ANS
UFGT
Phenylalanine
DFR
ANS
UFGT
Eriodictyol
Leucodelphinidin
DelphinidinCyanidin
MTMT
FLS
Quercetin
FLS
Myricetin
GT GT GT
Quercetinglucoside glycoside
Myricetinglycoside
MT
Laricitringlycoside
glycoside
MT
glucosideMT
Cyanidin-3-glucoside
Peonidin-3-glucoside Petunidin-3-glucoside Malvidin-3-glucoside
MT
Delphinidin-3-glucoside
F39984005998400H
F39984005998400H
F3998400H
F3998400H
F3998400H
F3998400H Dihydrokaempferol
Pentahydroxyflavone
Kaempferol
Kaempferol
Isorhamnetin
Syringetin
Figure 1 Flavonoid biosynthetic pathway PAL phenyl ammonia lyase F31015840H flavonoid-31015840-hydroxylase F3101584051015840H flavonoid-3101584051015840-hydroxylaseUFGT UDP-glucose flavonoid 3-O-glucosyltransferase MT methyl transferase DFR dihydroxyflavanol-4-reductase ANS anthocyaninsynthase and FLS flavonol synthase
As hydroxylation of the B-ring is carried out by flavon-oid-31015840-hydroxylase (F31015840H) and flavonoid-3101584051015840-hydroxylase(F3101584051015840H) enzymes the composition of anthocyanins in grapeskins will be determined by the relative activities of theseenzymes At the same time methyl transferase (MT) activitywill determine the different methoxylation patterns of B ringand acyl transferase the presence of acyl derivatives Most ofthese enzymes also control the synthesis of flavonols
Plant adaptation to different environments and centuriesof selection by humans has produced numerous genotypes inwhich the intensity of the red coloration varies extensivelyA mixture of variation in anthocyanin content and therelative proportion of different anthocyanins can producedifferent phenotypes for skin pigmentation with conse-quent technological and nutritional differences [9] Alsothese differences could be a very useful chemotaxonomicaltool [3]
At present cross-breeding and bud mutation are still themost common way for developing new wine grape cultivars[10]The determination of themechanisms of the inheritanceof the flavonoid composition could help in the election ofthe parentals in the breeding programs The objective ofthis study therefore was to explore the characteristics ofthe anthocyanin and flavonol composition and content inintraspecific hybrids of Monastrell times Syrah Monastrell timesCabernet Sauvignon and Monastrell times Barbera in orderto acquire information for future breeding efforts aimed atimproving fruit quality through the effects of flavonoids andto provide an insight into the mechanisms that control theinheritance of flavonoid characteristics among hybrids
2 Material and Methods
A collection of plants arising from crosses from Monastrelltimes Syrah Monastrell times Cabernet Sauvignon and Monastrelltimes Barbera was used in this study The study was conductedin an experimental vineyard of 1 ha located in Bullas (Mur-cia SE Spain) The parentals (Monastrell Syrah CabernetSauvignon and Barbera) were planted in 1997 whereas theseeds for the interspecific hybrids were planted in 2000 Thetraining system was a bilateral cordon trellised to a three-wire vertical system and drip irrigation was applied Plantingdensity was 25m between rows and 125m between vinesTwo two-bud spurs (4 nodes) were left at pruning timeGrapes were sampled in 2007
The plants derived from Monastrell self-pollination andfrom pollen donors other than Syrah Cabernet Sauvignonor Barberawere identified genetically and discarded using themicrosatellite (SSR Simple Sequence Repeat) loci segregating1 1 1 1 according to Bayo-Canha et al [11] Total DNA wasextracted from approximately 20mg of young frozen leavesusing a DNeasy Plant Mini Kit (Qiagen Valencia CA USA)following the manufacturerrsquos protocol Genotyping was car-ried out as described in Adam-Blondon et al [12] PCR prod-ucts were separated by capillary electrophoresis performedon an ABI Prism 3100 genetic analyzer (Applied BiosystemsCarlsbad CA USA) and the fragments were sized usingGeneMapper software (Applied Biosystems Carlsbad CAUSA)
Grapes were harvested at a total of soluble solids contentbetween 23 and 27∘Brix The sampling was randomly madeby picking berries from the top central and bottom parts of
Journal of Analytical Methods in Chemistry 3
several clusters of each hybrid vine The size of the samplewas around 300 berries which were bulked and separated in3 subsamples of approximately 100 berries to run triplicateanalyses Grape samples were kept frozen (minus20∘C) untilextraction and analysis
21 AnthocyaninMonoglycosides and Flavonols in Berry SkinsGrapes were peeled with the help of a scalpel Samples (2 g)were immersed in methanol (40mL) in hermetically closedtubes and placed on a stirring plate at 150 rpm and 25∘CAfter 2 hours the methanolic extracts were acidified with 5formic acid (1 2 vv) filtered through 02120583m PTFE filtersand analysed by HPLC
22 Identification and Quantification of Anthocyanins TheHPLC analyses were performed on a Waters 2695 liquidchromatograph (Waters PA USA) equipped with a Waters2996 diode array detector and a Primesep B2 column (SielcIllinois USA) 25 times 04 cm 5120583m particle size using assolvents water plus 5 formic acid (solvent A) and HPLCgrade acetonitrile (solvent B) at a flow rate of 08mLminminus1Elution was performed with a gradient starting with 5 Bto reach 9 B at 28min 13 B at 30min 21 B at 52min24 B at 65min and 70 B at 75min maintaining thisgradient for 5 minutes Chromatograms were recorded at520 nm (anthocyanins) and 360 nm (flavonols)
Identification of the compounds was carried out bycomparing their UV spectra recorded with the diode arraydetector and those reported in the literature Also an HPLC-MS analysis was made to confirm the identity of each peakAn LC-MSD-Trap VL-01036 liquid chromatograph-ion trapmass detector (Agilent Technologies Waldbronn Germany)equipped with an electrospray ionization (ESI) system wasused Elutionwas performed in theHPLC analysis conditionsdescribed previously with a flow rate of 08mL minminus1 Theheated capillary and voltage were maintained at 350∘C and4 kV respectively Mass scans (MS) were measured frommz100 up tomz 800
Anthocyanins were quantified at 520 nm as malvidin-3-glucoside using malvidin-3-glucoside chloride as externalstandard (Extrasynthese Genay France) Flavonols werequantified at 360 nm as quercetin-3-glucoside using thiscompound as external standard (Sigma Missouri USA)
23 Statistical Data Treatment All the analyses were per-formed with the statistical package Statgraphics 51
3 Results and Discussion
For this study 27 hybrids bearing red grapes and 15 hybridsbearing white grapes from Monastrell times Syrah 32 red and6 white from Monastrell times Cabernet Sauvignon and 13red from Monastrell times Barbera were studied The presenceof white hybrids in Monastrell times Cabernet Sauvignon andMonastrell times Syrah indicates the heterozygous nature ofthe parentals in regard to genes controlling anthocyaninsynthesis whereas Barbera being homozygous [13] does notproduce hybrids bearing white grapes Boss et al [14] and
Kobayashi et al [15] showed that expression of the UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) gene iscritical for anthocyanin biosynthesis in grape Experimentswith the berry skins of white and red cultivars revealed thatthe UFGT gene was expressed in all the red cultivars butnot in the white ones whereas the other genes involved inanthocyanin biosynthesis (Figure 1) are expressed in bothwhite and red cultivars The presence or absence of theenzymeUFGT is controlled byMyb-related regulatory genesand the insertion of the retroelement Gret1 in the promoterregion of VvmybA1 gene appears to be associated withwhite-fruited cultivars when present in a homozygous statePigmented cultivars possess at least one allele at theVvmybA1locus not containing this large retroelement [13 16] as is thecase of parentals of this study
Table 1 shows the results of the anthocyanin analysisfor the studied grapes and Figure 2 the range of concen-trations among the hybrids Syrah grapes contained higherconcentration of anthocyanins than Monastrell grapes Themean concentration in their hybrid grapes (1631mgg freshskin) was slightly higher than in Syrah (1506mgg freshskin) and the maximum value reached by the grapes ofone seedling (3949mgg fresh skin) was twice the valuefound in Syrah Cabernet Sauvignon and Barbera grapesalso showed higher concentration of anthocyanins thanMonastrell and were similar to that of Syrah grapes Themean values of anthocyanin content in the grapes of theirseedlings were slightly lower than in Cabernet Sauvignonand Barbera (1442 and 1208mgg fresh skin respectively)and higher than that shown by Monastrell grapes (740mggfresh skin) and again the maximum value found in theirseedlings was double that of Cabernet Sauvignon and Bar-bera grapes Many hybrids presented grapes with muchhigher concentration than their parentals as can be seenin Figure 2 The appearance of a large number of hybridsin which the anthocyanin concentration is not within therange of concentration of their parental phenotypes is calledtransgressive segregation frequent in intraspecific crossesand in domesticated populations The occurrence of thesegregation of a given traitmanifestedmainly in one direction(as happens in our case most of the hybrids showing highervalues of anthocyanin concentration than the parentals) mayimply that the trait has undergone fairly constant directionalselection or a certain overdominance of the genes controllingphenolic synthesis [17 18] Similar results were found byLiang et al [10] in grapes but our findings differ from thoseof Ju et al [19] in apples that found that crossing between twored-fruited apple cultivars produced less colored progenyA previous work exploring the proanthocyanidin content ofMonastrell times Syrah hybrid grapes also reported this type ofsegregation for this character [20]
As stated previously the presence of the enzyme UFGTis necessary for anthocyanin biosynthesis However thebiosynthesis of the different anthocyanin precursors is drivenupstream of the enzyme UFGT by the activity of F31015840H andF3101584051015840H enzymes which add either a single hydroxyl groupor two to dihydrokaempferol (Figure 1) Once convertedto dihydroquercetin or dihydromyricetin these intermedi-ates flow through common downstream enzymes to form
4 Journal of Analytical Methods in Chemistry
Table 1 Mean values (119899 = 3) of the anthocyanin profile and total anthocyanin content (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids
del cyan pet peon malv nonacylated acylated dihydrox trihydrox Total (mgg)Monastrell
Mean 125 137 100 200 397 552 448 377 623 740Syrah
Mean 72 36 81 109 681 496 503 167 833 1506CS
Mean 96 52 101 74 677 459 541 126 874 1507Barbera
Mean 98 31 139 47 686 656 343 77 923 1402Mon times Sy (27)a
Mean 100 71 86 154 548 432 568 265 734 1631Minimum 57 32 64 65 295 462 297 132 462 386Maximum 243 154 139 375 727 702 713 537 868 3949
Mon times CS (32)b
Mean 124 60 133 105 578 525 472 165 835 1444Minimum 75 35 94 56 444 352 313 95 689 487Maximum 234 121 203 215 685 686 647 311 905 2867
Mon times Bar (13)c
Mean 109 77 133 162 519 720 280 239 761 1208Minimum 41 11 90 44 317 415 101 67 552 161Maximum 170 146 197 364 779 898 585 447 933 2788
abcThe number in parenthesis represents the number of hybrids for each crossing del percentage of delphinidin derivatives cyan percentage of cyanidin derivatives pet percentage of petunidin derivatives peon percentage ofpeonidin derivatives malv percentage of malvidin derivatives nonacylated percentage of nonacylated anthocyanins acylated percentage of acylatedanthocyanins dihydrox percentage of dihydroxylated anthocyanins trihydrox percentage of trihydroxylated anthocyanins and Total (mgg) totalanthocyanin content (mg per g of skin)
disubstituted and trisubstituted anthocyanins when UFGTis expressed and to form other polyphenols (flavanolsflavonols) at different developmental stages All the studiedhybrids bearing red grapes synthesised all five anthocyanins(the dihydroxylated cyaniding peonidin-3-glucosides thetrihydroxylated delphinidin petunidin and malvidin-3-glucosides) together with their acylated derivatives Thismeans that all the parentals and the hybrids expressed func-tional F31015840HandF3101584051015840Hgenes for the synthesis of 3101584041015840-OHand310158404101584051015840-OH anthocyanins as well as methyltransferases (MT)for the methylation of primary anthocyanins As regardsthe percentage of the different anthocyanins in the differentparentals Monastrell grapes were characterized by a highpercentage of cyanidin suggesting a lower F3101584051015840H activitythan in the other varieties A low expression of F3101584051015840H hasbeen associated with cyanidin-based anthocyanins in grapeleafs [21] The percentages of malvidin-based anthocyaninsin Monastrell did not exceed 40 so the total percentageof trihydroxylated anthocyanins was low The percentage oftrihydroxylated anthocyanins reached 83 in Syrah grapes874 in Cabernet Sauvignon grapes and 923 in Barberagrapes The percentage of trihydroxylated anthocyanins waseven higher in some Monastrell times Cabernet Sauvignonand Monastrell times Barbera hybrids reaching values as highas 905 and 93 respectively The mean value of thepercentage of trihydroxylated anthocyanins in the hybridgrapes is close to the mean value between both parentals
The segregation can be fitted to a normal distribution and avery low number of hybrids presented higher or lower valuesthan in the parentals as can be seen in Figure 3These resultswere similar to those obtained during the first screening ofthe anthocyanin profile in Monastrell times Cabernet Sauvignongrapes [22] In spite of the presence of all anthocyaninbiosynthetic enzymes in all the investigated hybrids (sinceall the possible structures were found) a genotype-specificregulation of the structural genes along the core pathway andat the main branching points is presumed to underlie theobservedmethoxylation and hydroxylation variations amongthe grapes from the parentals and those of their hybrid plants
With regard to the percentage of anthocyanin acylationBarbera grapes showed the lowest percentage of acylation(343) followed by Monastrell (448) and CabernetSauvignon showed the highest percentages (54) The meanvalues of the percentage of acylated anthocyanins for Monas-trell times Cabernet Sauvignon hybrids were 47 568 forMonastrell times Syrah (higher than the percentage found inSyrah) while the hybrid grapes from Monastrell times Barberashowed the lowest percentages of acylation (28) As inthe case of the anthocyanin concentration data there was atendency towards higher values of acylation in the hybridsand no hybrid contained only nonacylated anthocyanins
As regards flavonols (Table 2) these flavonoids werepresent in both white and red grapes We could iden-tify mono- (kaempferol) di- (quercetin and isorhamnetin)
Journal of Analytical Methods in Chemistry 5
0
10000
20000
30000
40000
50000
(120583g
g)
Mtimes
S 20
Mtimes
S 76
Mtimes
S 4
Mtimes
S 28
Mon
astre
llM
timesS
66M
timesS
21M
timesS
31M
timesS
0M
timesS
27M
timesS
26M
timesS
62M
timesS
97Sy
rah
Mtimes
S 42
Mtimes
S 56
Mtimes
S 3
Mtimes
S 11
7M
timesS
46M
timesS
38M
timesS
34M
timesS
57M
timesS
75M
timesS
8M
timesS
114
Mtimes
S 11
Mtimes
S 37
Mtimes
S 47
Mtimes
S 71
(a)
0
5000
10000
15000
20000
25000
30000
Mtimes
CS 2
7M
timesCS
163
Mtimes
CS 1
58M
timesCS
99
Mtimes
CS 1
84M
timesCS
37
Mtimes
CS 1
07M
timesCS
7M
onas
trell
Mtimes
CS 7
2M
timesCS
DM
timesCS
EM
timesCS
26
Mtimes
CS 4
Mtimes
CS 9
1M
timesCS
100
Mtimes
CS 1
99M
timesCS
56
Mtimes
CS 1
35M
timesCS
90
Mtimes
CS A
Cabe
rnet
SM
timesCS
136
Mtimes
CS B
Mtimes
CS 8
4M
timesCS
96
Mtimes
CS C
Mtimes
CS 3
8M
timesCS
19
Mtimes
CS 5
9M
timesCS
55
Mtimes
CS 8
0M
timesCS
49
Mtimes
CS 2
09M
timesCS
16
(120583g
g)
(b)
0
5000
10000
15000
20000
25000
30000
Mtimes
B 12
2
Mtimes
B 10
1
Mtimes
B 66
Mtimes
B 55
Mon
astre
ll
Mtimes
B 94
Mtimes
B 11
3
Mtimes
B 75
Mtimes
B 84
Barb
era
Mtimes
B 70
Mtimes
B 10
9
Mtimes
B 12
0
Mtimes
B 12
1
Mtimes
B 69
(120583g
g)
(c)
Figure 2 Concentration of anthocyanins in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c)hybrid grapes (each bar represents the mean value of three samples)
6 Journal of Analytical Methods in Chemistry
0
1
2
3
4
5
6
7
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mtimes
S 62
Mtimes
S 3
Mon
astre
llM
timesS
4M
timesS
76M
timesS
20M
timesS
97M
timesS
66M
timesS
31M
timesS
21M
timesS
56M
timesS
27M
timesS
117
Mtimes
S 57
Mtimes
S 11
Mtimes
S 28
Mtimes
S 75
Mtimes
S 34
Mtimes
S 38
Mtimes
S 0
Mtimes
S 47
Mtimes
S 26
Mtimes
S 8
Mtimes
S 37
Mtimes
S 11
4M
timesS
71M
timesS
42Sy
rah
Mtimes
S 46
(a)
0
2
4
6
8
10
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mon
astre
llM
timesCS
209
Mtimes
CS 1
58M
timesCS
27
Mtimes
CS 1
63M
timesCS
49
Mtimes
CS 1
84M
timesCS
37
Mtimes
CS 4
Mtimes
CS 9
9M
timesCS
96
Mtimes
CS 5
9M
timesCS
7M
timesCS
135
Mtimes
CS 9
1M
timesCS
26
Mtimes
CS 1
00M
timesCS
107
Mtimes
CS 5
6M
timesCS
19
Mtimes
CS 1
6M
timesCS
84
Mtimes
CS 1
36M
timesCS
DM
timesCS
55
Mtimes
CS 7
2Ca
bern
etS
Mtimes
CS 1
99M
timesCS
90
Mtimes
CS C
Mtimes
CS E
Mtimes
CS B
Mtimes
CS 3
8M
timesCS
80
Mtimes
CS A
(b)
0
2
4
6
8
10
12
14
16
Mtimes
B 66
Mon
astre
ll
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 12
1
Mtimes
B 12
2
Mtimes
B 55
Mtimes
B 84
Mtimes
B 69
Mtimes
B 75
Mtimes
B 10
9
Mtimes
B 70
Mtimes
B 94
Barb
era
Mtimes
B 12
0Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
(c)
Figure 3 Trihydroxylateddihydroxylated anthocyanins ratios in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) andMonastrell times Barbera (c) hybrid grapes
Journal of Analytical Methods in Chemistry 7
Table 2 Mean values of the flavonol profile and total flavonolcontent (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids (119899 = 3)
monohydr dihydr trihydrox TotalMonastrell
Mean 109 707 184 056Syrah
Mean 134 442 424 073CS
Mean 147 446 407 019Barbera
Mean 43 533 424 049Red hybrids
Mon times SyMean 104 524 372 073Minimum 53 363 165 018Maximum 160 726 550 183
Mon times CSMean 113 511 376 037Minimum 50 352 203 011Maximum 209 650 583 078
Mon times BarMean 94 619 287 042Minimum 47 429 87 012Maximum 136 839 434 139
White hybridsMon times Sy
Mean 134 841 24 035Minimum 41 746 06 008Maximum 237 947 72 128
Mon times CSMean 200 748 51 008Minimum 168 689 09 002Maximum 259 784 127 018
monohydrox percentage of monohydroxylated flavonols dihydroxpercentage of dihydroxylated flavonols trihydrox percentage of trihy-droxylated flavonols Total total flavonol content (mg per g of skin)
and trihydroxylated (myricetin laricitrin and syringetin)flavonol glycosides (glucosides glucoronides and smallquantities of galactosides) In red grapes the monohydrox-ylated flavonols represented the lowest percentage especiallyin Barbera grapes (43) Monastrell grapes presented a veryhigh percentage of dihydroxylated flavonols (707) muchhigher than in the other varieties and therefore a lowerpercentage of trihydroxylated flavonols whereas BarberaSyrah and Cabernet Sauvignon grapes reached a percentageof trihydroxylated flavonols of around 45ndash50 This factindicates lower F3101584051015840H activity in Monastrell grapes as alsoobserved for the anthocyanins
As regards the flavonol content Cabernet Sauvignongrapes showed low concentrations of flavonols and Monas-trell and Syrah much higher concentrations while the highestvalue was found in red grapes from a hybrid plant fromMonastrell times Syrah (Table 2 Figure 4) reaching values as
high as 183mgg of skin In the hybrids bearing white grapesa lower concentration of flavonols was measured comparedwith that of the hybrids bearing red grapes Azuma et al[23 24] stated that Myb genes besides the regulation of theUFGT expression appear to enhance the expression of allthe genes involved in the anthocyanin biosynthesis pathwaybecause the transcription of all anthocyanin biosynthesisgenes appears to be slightly activated which would explainthe higher concentration of flavonols in red grapes Also inthese white skinned grapes trihydroxylated flavonols werebarely presentMattivi et al [25] studying the flavonol profileof several grape varieties did not detect trihydroxylatedflavonols in white grapes Bogs et al [26] did not findsignificant expression of F3101584051015840H and UFGT in white grapevarieties which suggests a related regulation of F3101584051015840H andUFGT during berry ripening and justifies the almost nullpresence of trihydroxylated flavonols in white grapes F3101584051015840Hwas detected in white grapes var Chardonnay but prior toveraison [26] since it is needed for flavanol biosynthesiswhich seems to be controlled differently indeed no differ-ences in the percentage of trihydroxylated flavanols wereobserved between the red and white grapes arising fromthe cross of Monastrell times Syrah [20] However other studiesstated that flavonol synthase (FLS) was not upregulated whenUFGT was expressed and that the increase in flavonols inred grapes was a consequence of an increase flux through theflavonoid pathway [27]
4 Conclusions
The study of the anthocyanin and flavonol profiles of thegrapes from the hybrid plants can be useful for a targetedinformative metabolomic analysis [28] a tool for selectingpromising grapes according to their profile andor content
Seedlings with grapes presenting very high concentra-tions of anthocyanins and flavonols can be expected fromintraspecific crosses and these resulting grapes could leadto highly colored wines with increased health-related prop-erties In this way three plants arising from Monastrell timesSyrah (hybrids 8 37 and 71) presented anthocyanin andflavonol concentrations higher than 20 and 1mgg fresh skinrespectively (Figures 2 and 4) Also four plants arising fromMonastrell timesCabernet Sauvignon (hybrids 38 59 55 and 80)and two from Monastrell times Barbera (hybrid plants 120 121)showed anthocyanin values higher than 20mgg fresh skinaccompanied with high concentration of flavonols (Figures2 and 4) The hydroxylation pattern which also influenceswine color and its stability will be strongly influenced by theparentals pattern since values higher than that shown by thebest parental in this respect will be difficult to obtain Alsoin the case of the crosses between heterozygous parentals(Monastrell Cabernet Sauvignon and Syrah) hybrids bearingwhite grapes can be obtained some of them with a highconcentration of flavonols that could be of importance in thehealth properties of this fruit and its derived products suchas the wineThe information obtained in this study should behelpful for selecting parentals for breeding programs
8 Journal of Analytical Methods in Chemistry
0
500
1000
1500
2000
(120583g
g)
B-M
timesS
30B-
Mtimes
S 1
B-M
timesS
19M
timesS
28M
timesS
31B-
Mtimes
S 10
0M
timesS
20B-
Mtimes
S 59
B-M
timesS
118
B-M
timesS
9B-
Mtimes
S 14
B-M
timesS
94B-
Mtimes
S 82
B-M
timesS
15M
timesS
26M
timesS
0M
timesS
75B-
Mtimes
S 40
Mtimes
S 38
Mtimes
S 27
Mtimes
S 66
B-M
timesS
73B-
Mtimes
S 65
Mtimes
S 21
Mtimes
S 76
Mon
astre
llM
timesS
11M
timesS
3M
timesS
114
Mtimes
S 62
Mtimes
S 34
Syra
hM
timesS
56M
timesS
117
Mtimes
S 47
Mtimes
S 97
Mtimes
S 4
Mtimes
S 42
Mtimes
S 46
Mtimes
S 71
Mtimes
S 37
B-M
timesS
91M
timesS
8M
timesS
57
(a)
0
200
400
600
800
1000
(120583g
g)
B-M
timesCS
17
B-M
timesCS
13
B-M
timesCS
93
B-M
timesCS
171
B-M
timesCS
146
Mtimes
CS 9
9M
timesCS
27
Mtimes
CS D
Mtimes
CS 1
84M
timesCS
135
Mtimes
CS 4
B-M
timesCS
98
Mtimes
CS 1
07Ca
bern
etS
Mtimes
CS 9
1M
timesCS
7M
timesCS
26
Mtimes
CS 1
36M
timesCS
163
Mtimes
CS 9
6M
timesCS
84
Mtimes
CS 1
58M
timesCS
100
Mtimes
CS 1
99M
timesCS
72
Mtimes
CS E
Mtimes
CS 4
9M
timesCS
BM
timesCS
37
Mtimes
CS 1
9M
timesCS
16
Mtimes
CS 8
0M
timesCS
55
Mon
astre
llM
timesCS
38
Mtimes
CS 9
0M
timesCS
CM
timesCS
59
Mtimes
CS 5
6M
timesCS
209
Mtimes
CS A
(b)
0
200
400
600
800
1000
1200
1400
1600
Mtimes
B 55
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 66
Mtimes
B 75
Mtimes
B 10
9
Barb
era
Mtimes
B 69
Mtimes
B 12
1
Mtimes
B 12
0
Mon
astre
ll
Mtimes
B 70
Mtimes
B 84
(120583g
g)
Mtimes
B 94
Mtimes
B 12
2
(c)
Figure 4 Concentration of flavonols in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c) hybridgrapes (yellow bars indicate white grape bearing hybrids)
Journal of Analytical Methods in Chemistry 9
Acknowledgment
This work was made possible by financial assistance of theMinisterio de Ciencia e Innovacion Project AGL2006-11019
References
[1] F Alen-Ruiz M S Garcıa-Falcon M C Perez-Lamela EMartınez-Carballo and J Simal-Gandara ldquoInfluence of majorpolyphenols on antioxidant activity in Mencia and Brancellaored grapesrdquo Food Chem vol 113 pp 53ndash60 2008
[2] S C Forester and A L Waterhouse ldquoMetabolites are key tounderstanding health effects of wine polyphenolicsrdquo Journal ofNutrition vol 139 no 9 pp 1324Sndash1831S 2009
[3] A Ortega-Regules I Romero-Cascales J M Lopez-Roca J MRos-Garcıa and E Gomez-Plaza ldquoAnthocyanin fingerprint ofgrapes environmental and genetic variationsrdquo Journal of theScience of Food and Agriculture vol 86 no 10 pp 1460ndash14672006
[4] V Cheynier and J Rigaud ldquoHPLC separation and characteri-zation of flavonols in the skins of Vitis vinifera var CinsaultrdquoAmerican Journal of Enology and Viticulture vol 37 pp 248ndash252 1986
[5] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero andI Hermosın-Gutierrez ldquoFlavonol profiles of Vitis vinifera redgrapes and their single-cultivar winesrdquo Journal of Agriculturaland Food Chemistry vol 55 no 3 pp 992ndash1002 2007
[6] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero M VGomez A H Velders and I Hermosın-Gutierrez ldquoFlavonol 3-O-glycosides series of Vitis vinifera Cv Petit Verdot red winegrapesrdquo Journal of Agricultural and Food Chemistry vol 57 no1 pp 209ndash219 2009
[7] P Sarni H Fulcrand V Souillol JM Souquet andV CheynierldquoMechanisms of anthocyanin degradation in grape must likemodel solutionsrdquo Journal of the Science of Food and Agriculturevol 69 no 3 pp 385ndash391 1995
[8] C Malien-Aubert O Dangles andM J Amiot ldquoColor stabilityof commercial anthocyanin-based extracts in relation to thephenolic composition Protective effects by intra- and inter-molecular copigmentationrdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 170ndash176 2001
[9] H Chang H C Eun and S C Hyang ldquoQuantitative Structure-Activity Relationship (QSAR) of antioxidative anthocyanidinsand their glycosidesrdquo Food Science and Biotechnology vol 17 no3 pp 501ndash507 2008
[10] Z Liang C Yang J Yang et al ldquoInheritance of anthocyaninsin berries of Vitis vinifera grapesrdquo Euphytica vol 167 no 1 pp113ndash125 2009
[11] A Bayo-Canha J I Fernandez-Fernandez A Martınez-Cutillas and L Ruiz-Garcıa ldquoPhenotypic segregation andrelationships of agronomic traits in Monastrell times Syrah winegrape progenyrdquo Euphytica vol 186 pp 393ndash407 2012
[12] A F Adam-Blondon C Roux D Claux G Butterlin DMerdinoglu and P This ldquoMapping 245 SSR markers on theVitis vinifera genome a tool for grape geneticsrdquoTheoretical andApplied Genetics vol 109 no 5 pp 1017ndash1027 2004
[13] A R Walker E Lee J Bogs D A J McDavid M R Thomasand S P Robinson ldquoWhite grapes arose through the mutationof two similar and adjacent regulatory genesrdquo Plant Journal vol49 no 5 pp 772ndash785 2007
[14] P K Boss C Davies and S P Robinson ldquoAnalysis of theexpression of anthocyanin pathway genes in developing Vitis
vinifera L cv Shiraz grape berries and the implications forpathway regulationrdquo Plant Physiology vol 111 no 4 pp 1059ndash1066 1996
[15] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002
[16] D Lijavetzky L Ruiz-Garcıa J A Cabezas et al ldquoMoleculargenetics of berry colour variation in table graperdquo MolecularGenetics and Genomics vol 276 no 5 pp 427ndash435 2006
[17] M C de Vicente and S D Tanksley ldquoQTL analysis of transgres-sive segregation in an interspecific tomato crossrdquo Genetics vol134 no 2 pp 585ndash596 1993
[18] L H Rieseberg M A Archer and R K Wayne ldquoTransgressivesegregation adaptation and speciationrdquoHeredity vol 83 no 4pp 363ndash372 1999
[19] Z Ju C Liu Y Yuan YWang andG Liu ldquoColoration potentialanthocyanin accumulation and enzyme activity in fruit ofcommercial apple cultivars and their F1 progenyrdquo ScientiaHorticulturae vol 79 no 1-2 pp 39ndash50 1999
[20] A Hernandez-Jimenez E Gomez-Plaza A Martınez-Cutillasand J A Kennedy ldquoGrape skin and seed proanthocyanidinsfrom Monastrell times Syrah grapesrdquo Journal of Agricultural andFood Chemistry vol 57 no 22 pp 10798ndash10803 2009
[21] H Kobayashi S Suzuki F Tanzawa andT Takayanagispi ldquoLowexpression of flavonoid 3rsquo5rsquo-hydroxylase (F3rsquo5rsquoH) associatedwith cyanidin-based anthocyanins in grape leafrdquo AmericanJournal of Enology and Viticulture vol 60 no 3 pp 362ndash3672009
[22] E Gomez-Plaza R Gil-Munoz A Hernandez-Jimenez JM Lopez-Roca A Ortega-Regules and A Martınez-CutillasldquoStudies on the anthocyanin profile ofVitis vinifera intraspecifichybrids (Monastrell times Cabernet Sauvignon)rdquo European FoodResearch and Technology vol 227 no 2 pp 479ndash484 2008
[23] A Azuma S Kobayashi N Mitani et al ldquoGenomic and geneticanalysis ofMyb-related genes that regulate anthocyanin biosyn-thesis in grape berry skinrdquoTheoretical and Applied Genetics vol117 no 6 pp 1009ndash1019 2008
[24] A Azuma S Kobayashi H Yakushiji M Yamada N Mitaniand A Sato ldquoVvmybA1 genotype determines grape skin colorrdquoVitis vol 46 no 3 pp 154ndash155 2007
[25] FMattivi R Guzzon U VrhovsekM Stefanini and R VelascoldquoMetabolite profiling of grape flavonols and anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7692ndash7702 2006
[26] J Bogs A Ebadi D McDavid and S P Robinson ldquoIdentifi-cation of the flavonoid hydroxylases from grapevine and theirregulation dining fruit developmentrdquo Plant Physiology vol 140no 1 pp 279ndash291 2006
[27] F Quattrocchio A Baudry L Lepiniec and E GrotewoldldquoThe regulation of flavonoid biosynthesisrdquo in The Science ofFlavonoids E Grotewold Ed pp 97ndash122 Springer New YorkNY USA 2008
[28] J M Cevallos-Cevallos J I Reyes-De-Corcuera E EtxeberriaM D Danyluk and G E Rodrick ldquoMetabolomic analysis infood science a reviewrdquo Trends in Food Science and Technologyvol 20 no 11-12 pp 557ndash566 2009
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
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International Journal ofPhotoenergy
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Carbohydrate Chemistry
International Journal of
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Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Analytical Methods in Chemistry
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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Quantum Chemistry
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CatalystsJournal of
Journal of Analytical Methods in Chemistry 3
several clusters of each hybrid vine The size of the samplewas around 300 berries which were bulked and separated in3 subsamples of approximately 100 berries to run triplicateanalyses Grape samples were kept frozen (minus20∘C) untilextraction and analysis
21 AnthocyaninMonoglycosides and Flavonols in Berry SkinsGrapes were peeled with the help of a scalpel Samples (2 g)were immersed in methanol (40mL) in hermetically closedtubes and placed on a stirring plate at 150 rpm and 25∘CAfter 2 hours the methanolic extracts were acidified with 5formic acid (1 2 vv) filtered through 02120583m PTFE filtersand analysed by HPLC
22 Identification and Quantification of Anthocyanins TheHPLC analyses were performed on a Waters 2695 liquidchromatograph (Waters PA USA) equipped with a Waters2996 diode array detector and a Primesep B2 column (SielcIllinois USA) 25 times 04 cm 5120583m particle size using assolvents water plus 5 formic acid (solvent A) and HPLCgrade acetonitrile (solvent B) at a flow rate of 08mLminminus1Elution was performed with a gradient starting with 5 Bto reach 9 B at 28min 13 B at 30min 21 B at 52min24 B at 65min and 70 B at 75min maintaining thisgradient for 5 minutes Chromatograms were recorded at520 nm (anthocyanins) and 360 nm (flavonols)
Identification of the compounds was carried out bycomparing their UV spectra recorded with the diode arraydetector and those reported in the literature Also an HPLC-MS analysis was made to confirm the identity of each peakAn LC-MSD-Trap VL-01036 liquid chromatograph-ion trapmass detector (Agilent Technologies Waldbronn Germany)equipped with an electrospray ionization (ESI) system wasused Elutionwas performed in theHPLC analysis conditionsdescribed previously with a flow rate of 08mL minminus1 Theheated capillary and voltage were maintained at 350∘C and4 kV respectively Mass scans (MS) were measured frommz100 up tomz 800
Anthocyanins were quantified at 520 nm as malvidin-3-glucoside using malvidin-3-glucoside chloride as externalstandard (Extrasynthese Genay France) Flavonols werequantified at 360 nm as quercetin-3-glucoside using thiscompound as external standard (Sigma Missouri USA)
23 Statistical Data Treatment All the analyses were per-formed with the statistical package Statgraphics 51
3 Results and Discussion
For this study 27 hybrids bearing red grapes and 15 hybridsbearing white grapes from Monastrell times Syrah 32 red and6 white from Monastrell times Cabernet Sauvignon and 13red from Monastrell times Barbera were studied The presenceof white hybrids in Monastrell times Cabernet Sauvignon andMonastrell times Syrah indicates the heterozygous nature ofthe parentals in regard to genes controlling anthocyaninsynthesis whereas Barbera being homozygous [13] does notproduce hybrids bearing white grapes Boss et al [14] and
Kobayashi et al [15] showed that expression of the UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) gene iscritical for anthocyanin biosynthesis in grape Experimentswith the berry skins of white and red cultivars revealed thatthe UFGT gene was expressed in all the red cultivars butnot in the white ones whereas the other genes involved inanthocyanin biosynthesis (Figure 1) are expressed in bothwhite and red cultivars The presence or absence of theenzymeUFGT is controlled byMyb-related regulatory genesand the insertion of the retroelement Gret1 in the promoterregion of VvmybA1 gene appears to be associated withwhite-fruited cultivars when present in a homozygous statePigmented cultivars possess at least one allele at theVvmybA1locus not containing this large retroelement [13 16] as is thecase of parentals of this study
Table 1 shows the results of the anthocyanin analysisfor the studied grapes and Figure 2 the range of concen-trations among the hybrids Syrah grapes contained higherconcentration of anthocyanins than Monastrell grapes Themean concentration in their hybrid grapes (1631mgg freshskin) was slightly higher than in Syrah (1506mgg freshskin) and the maximum value reached by the grapes ofone seedling (3949mgg fresh skin) was twice the valuefound in Syrah Cabernet Sauvignon and Barbera grapesalso showed higher concentration of anthocyanins thanMonastrell and were similar to that of Syrah grapes Themean values of anthocyanin content in the grapes of theirseedlings were slightly lower than in Cabernet Sauvignonand Barbera (1442 and 1208mgg fresh skin respectively)and higher than that shown by Monastrell grapes (740mggfresh skin) and again the maximum value found in theirseedlings was double that of Cabernet Sauvignon and Bar-bera grapes Many hybrids presented grapes with muchhigher concentration than their parentals as can be seenin Figure 2 The appearance of a large number of hybridsin which the anthocyanin concentration is not within therange of concentration of their parental phenotypes is calledtransgressive segregation frequent in intraspecific crossesand in domesticated populations The occurrence of thesegregation of a given traitmanifestedmainly in one direction(as happens in our case most of the hybrids showing highervalues of anthocyanin concentration than the parentals) mayimply that the trait has undergone fairly constant directionalselection or a certain overdominance of the genes controllingphenolic synthesis [17 18] Similar results were found byLiang et al [10] in grapes but our findings differ from thoseof Ju et al [19] in apples that found that crossing between twored-fruited apple cultivars produced less colored progenyA previous work exploring the proanthocyanidin content ofMonastrell times Syrah hybrid grapes also reported this type ofsegregation for this character [20]
As stated previously the presence of the enzyme UFGTis necessary for anthocyanin biosynthesis However thebiosynthesis of the different anthocyanin precursors is drivenupstream of the enzyme UFGT by the activity of F31015840H andF3101584051015840H enzymes which add either a single hydroxyl groupor two to dihydrokaempferol (Figure 1) Once convertedto dihydroquercetin or dihydromyricetin these intermedi-ates flow through common downstream enzymes to form
4 Journal of Analytical Methods in Chemistry
Table 1 Mean values (119899 = 3) of the anthocyanin profile and total anthocyanin content (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids
del cyan pet peon malv nonacylated acylated dihydrox trihydrox Total (mgg)Monastrell
Mean 125 137 100 200 397 552 448 377 623 740Syrah
Mean 72 36 81 109 681 496 503 167 833 1506CS
Mean 96 52 101 74 677 459 541 126 874 1507Barbera
Mean 98 31 139 47 686 656 343 77 923 1402Mon times Sy (27)a
Mean 100 71 86 154 548 432 568 265 734 1631Minimum 57 32 64 65 295 462 297 132 462 386Maximum 243 154 139 375 727 702 713 537 868 3949
Mon times CS (32)b
Mean 124 60 133 105 578 525 472 165 835 1444Minimum 75 35 94 56 444 352 313 95 689 487Maximum 234 121 203 215 685 686 647 311 905 2867
Mon times Bar (13)c
Mean 109 77 133 162 519 720 280 239 761 1208Minimum 41 11 90 44 317 415 101 67 552 161Maximum 170 146 197 364 779 898 585 447 933 2788
abcThe number in parenthesis represents the number of hybrids for each crossing del percentage of delphinidin derivatives cyan percentage of cyanidin derivatives pet percentage of petunidin derivatives peon percentage ofpeonidin derivatives malv percentage of malvidin derivatives nonacylated percentage of nonacylated anthocyanins acylated percentage of acylatedanthocyanins dihydrox percentage of dihydroxylated anthocyanins trihydrox percentage of trihydroxylated anthocyanins and Total (mgg) totalanthocyanin content (mg per g of skin)
disubstituted and trisubstituted anthocyanins when UFGTis expressed and to form other polyphenols (flavanolsflavonols) at different developmental stages All the studiedhybrids bearing red grapes synthesised all five anthocyanins(the dihydroxylated cyaniding peonidin-3-glucosides thetrihydroxylated delphinidin petunidin and malvidin-3-glucosides) together with their acylated derivatives Thismeans that all the parentals and the hybrids expressed func-tional F31015840HandF3101584051015840Hgenes for the synthesis of 3101584041015840-OHand310158404101584051015840-OH anthocyanins as well as methyltransferases (MT)for the methylation of primary anthocyanins As regardsthe percentage of the different anthocyanins in the differentparentals Monastrell grapes were characterized by a highpercentage of cyanidin suggesting a lower F3101584051015840H activitythan in the other varieties A low expression of F3101584051015840H hasbeen associated with cyanidin-based anthocyanins in grapeleafs [21] The percentages of malvidin-based anthocyaninsin Monastrell did not exceed 40 so the total percentageof trihydroxylated anthocyanins was low The percentage oftrihydroxylated anthocyanins reached 83 in Syrah grapes874 in Cabernet Sauvignon grapes and 923 in Barberagrapes The percentage of trihydroxylated anthocyanins waseven higher in some Monastrell times Cabernet Sauvignonand Monastrell times Barbera hybrids reaching values as highas 905 and 93 respectively The mean value of thepercentage of trihydroxylated anthocyanins in the hybridgrapes is close to the mean value between both parentals
The segregation can be fitted to a normal distribution and avery low number of hybrids presented higher or lower valuesthan in the parentals as can be seen in Figure 3These resultswere similar to those obtained during the first screening ofthe anthocyanin profile in Monastrell times Cabernet Sauvignongrapes [22] In spite of the presence of all anthocyaninbiosynthetic enzymes in all the investigated hybrids (sinceall the possible structures were found) a genotype-specificregulation of the structural genes along the core pathway andat the main branching points is presumed to underlie theobservedmethoxylation and hydroxylation variations amongthe grapes from the parentals and those of their hybrid plants
With regard to the percentage of anthocyanin acylationBarbera grapes showed the lowest percentage of acylation(343) followed by Monastrell (448) and CabernetSauvignon showed the highest percentages (54) The meanvalues of the percentage of acylated anthocyanins for Monas-trell times Cabernet Sauvignon hybrids were 47 568 forMonastrell times Syrah (higher than the percentage found inSyrah) while the hybrid grapes from Monastrell times Barberashowed the lowest percentages of acylation (28) As inthe case of the anthocyanin concentration data there was atendency towards higher values of acylation in the hybridsand no hybrid contained only nonacylated anthocyanins
As regards flavonols (Table 2) these flavonoids werepresent in both white and red grapes We could iden-tify mono- (kaempferol) di- (quercetin and isorhamnetin)
Journal of Analytical Methods in Chemistry 5
0
10000
20000
30000
40000
50000
(120583g
g)
Mtimes
S 20
Mtimes
S 76
Mtimes
S 4
Mtimes
S 28
Mon
astre
llM
timesS
66M
timesS
21M
timesS
31M
timesS
0M
timesS
27M
timesS
26M
timesS
62M
timesS
97Sy
rah
Mtimes
S 42
Mtimes
S 56
Mtimes
S 3
Mtimes
S 11
7M
timesS
46M
timesS
38M
timesS
34M
timesS
57M
timesS
75M
timesS
8M
timesS
114
Mtimes
S 11
Mtimes
S 37
Mtimes
S 47
Mtimes
S 71
(a)
0
5000
10000
15000
20000
25000
30000
Mtimes
CS 2
7M
timesCS
163
Mtimes
CS 1
58M
timesCS
99
Mtimes
CS 1
84M
timesCS
37
Mtimes
CS 1
07M
timesCS
7M
onas
trell
Mtimes
CS 7
2M
timesCS
DM
timesCS
EM
timesCS
26
Mtimes
CS 4
Mtimes
CS 9
1M
timesCS
100
Mtimes
CS 1
99M
timesCS
56
Mtimes
CS 1
35M
timesCS
90
Mtimes
CS A
Cabe
rnet
SM
timesCS
136
Mtimes
CS B
Mtimes
CS 8
4M
timesCS
96
Mtimes
CS C
Mtimes
CS 3
8M
timesCS
19
Mtimes
CS 5
9M
timesCS
55
Mtimes
CS 8
0M
timesCS
49
Mtimes
CS 2
09M
timesCS
16
(120583g
g)
(b)
0
5000
10000
15000
20000
25000
30000
Mtimes
B 12
2
Mtimes
B 10
1
Mtimes
B 66
Mtimes
B 55
Mon
astre
ll
Mtimes
B 94
Mtimes
B 11
3
Mtimes
B 75
Mtimes
B 84
Barb
era
Mtimes
B 70
Mtimes
B 10
9
Mtimes
B 12
0
Mtimes
B 12
1
Mtimes
B 69
(120583g
g)
(c)
Figure 2 Concentration of anthocyanins in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c)hybrid grapes (each bar represents the mean value of three samples)
6 Journal of Analytical Methods in Chemistry
0
1
2
3
4
5
6
7
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mtimes
S 62
Mtimes
S 3
Mon
astre
llM
timesS
4M
timesS
76M
timesS
20M
timesS
97M
timesS
66M
timesS
31M
timesS
21M
timesS
56M
timesS
27M
timesS
117
Mtimes
S 57
Mtimes
S 11
Mtimes
S 28
Mtimes
S 75
Mtimes
S 34
Mtimes
S 38
Mtimes
S 0
Mtimes
S 47
Mtimes
S 26
Mtimes
S 8
Mtimes
S 37
Mtimes
S 11
4M
timesS
71M
timesS
42Sy
rah
Mtimes
S 46
(a)
0
2
4
6
8
10
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mon
astre
llM
timesCS
209
Mtimes
CS 1
58M
timesCS
27
Mtimes
CS 1
63M
timesCS
49
Mtimes
CS 1
84M
timesCS
37
Mtimes
CS 4
Mtimes
CS 9
9M
timesCS
96
Mtimes
CS 5
9M
timesCS
7M
timesCS
135
Mtimes
CS 9
1M
timesCS
26
Mtimes
CS 1
00M
timesCS
107
Mtimes
CS 5
6M
timesCS
19
Mtimes
CS 1
6M
timesCS
84
Mtimes
CS 1
36M
timesCS
DM
timesCS
55
Mtimes
CS 7
2Ca
bern
etS
Mtimes
CS 1
99M
timesCS
90
Mtimes
CS C
Mtimes
CS E
Mtimes
CS B
Mtimes
CS 3
8M
timesCS
80
Mtimes
CS A
(b)
0
2
4
6
8
10
12
14
16
Mtimes
B 66
Mon
astre
ll
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 12
1
Mtimes
B 12
2
Mtimes
B 55
Mtimes
B 84
Mtimes
B 69
Mtimes
B 75
Mtimes
B 10
9
Mtimes
B 70
Mtimes
B 94
Barb
era
Mtimes
B 12
0Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
(c)
Figure 3 Trihydroxylateddihydroxylated anthocyanins ratios in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) andMonastrell times Barbera (c) hybrid grapes
Journal of Analytical Methods in Chemistry 7
Table 2 Mean values of the flavonol profile and total flavonolcontent (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids (119899 = 3)
monohydr dihydr trihydrox TotalMonastrell
Mean 109 707 184 056Syrah
Mean 134 442 424 073CS
Mean 147 446 407 019Barbera
Mean 43 533 424 049Red hybrids
Mon times SyMean 104 524 372 073Minimum 53 363 165 018Maximum 160 726 550 183
Mon times CSMean 113 511 376 037Minimum 50 352 203 011Maximum 209 650 583 078
Mon times BarMean 94 619 287 042Minimum 47 429 87 012Maximum 136 839 434 139
White hybridsMon times Sy
Mean 134 841 24 035Minimum 41 746 06 008Maximum 237 947 72 128
Mon times CSMean 200 748 51 008Minimum 168 689 09 002Maximum 259 784 127 018
monohydrox percentage of monohydroxylated flavonols dihydroxpercentage of dihydroxylated flavonols trihydrox percentage of trihy-droxylated flavonols Total total flavonol content (mg per g of skin)
and trihydroxylated (myricetin laricitrin and syringetin)flavonol glycosides (glucosides glucoronides and smallquantities of galactosides) In red grapes the monohydrox-ylated flavonols represented the lowest percentage especiallyin Barbera grapes (43) Monastrell grapes presented a veryhigh percentage of dihydroxylated flavonols (707) muchhigher than in the other varieties and therefore a lowerpercentage of trihydroxylated flavonols whereas BarberaSyrah and Cabernet Sauvignon grapes reached a percentageof trihydroxylated flavonols of around 45ndash50 This factindicates lower F3101584051015840H activity in Monastrell grapes as alsoobserved for the anthocyanins
As regards the flavonol content Cabernet Sauvignongrapes showed low concentrations of flavonols and Monas-trell and Syrah much higher concentrations while the highestvalue was found in red grapes from a hybrid plant fromMonastrell times Syrah (Table 2 Figure 4) reaching values as
high as 183mgg of skin In the hybrids bearing white grapesa lower concentration of flavonols was measured comparedwith that of the hybrids bearing red grapes Azuma et al[23 24] stated that Myb genes besides the regulation of theUFGT expression appear to enhance the expression of allthe genes involved in the anthocyanin biosynthesis pathwaybecause the transcription of all anthocyanin biosynthesisgenes appears to be slightly activated which would explainthe higher concentration of flavonols in red grapes Also inthese white skinned grapes trihydroxylated flavonols werebarely presentMattivi et al [25] studying the flavonol profileof several grape varieties did not detect trihydroxylatedflavonols in white grapes Bogs et al [26] did not findsignificant expression of F3101584051015840H and UFGT in white grapevarieties which suggests a related regulation of F3101584051015840H andUFGT during berry ripening and justifies the almost nullpresence of trihydroxylated flavonols in white grapes F3101584051015840Hwas detected in white grapes var Chardonnay but prior toveraison [26] since it is needed for flavanol biosynthesiswhich seems to be controlled differently indeed no differ-ences in the percentage of trihydroxylated flavanols wereobserved between the red and white grapes arising fromthe cross of Monastrell times Syrah [20] However other studiesstated that flavonol synthase (FLS) was not upregulated whenUFGT was expressed and that the increase in flavonols inred grapes was a consequence of an increase flux through theflavonoid pathway [27]
4 Conclusions
The study of the anthocyanin and flavonol profiles of thegrapes from the hybrid plants can be useful for a targetedinformative metabolomic analysis [28] a tool for selectingpromising grapes according to their profile andor content
Seedlings with grapes presenting very high concentra-tions of anthocyanins and flavonols can be expected fromintraspecific crosses and these resulting grapes could leadto highly colored wines with increased health-related prop-erties In this way three plants arising from Monastrell timesSyrah (hybrids 8 37 and 71) presented anthocyanin andflavonol concentrations higher than 20 and 1mgg fresh skinrespectively (Figures 2 and 4) Also four plants arising fromMonastrell timesCabernet Sauvignon (hybrids 38 59 55 and 80)and two from Monastrell times Barbera (hybrid plants 120 121)showed anthocyanin values higher than 20mgg fresh skinaccompanied with high concentration of flavonols (Figures2 and 4) The hydroxylation pattern which also influenceswine color and its stability will be strongly influenced by theparentals pattern since values higher than that shown by thebest parental in this respect will be difficult to obtain Alsoin the case of the crosses between heterozygous parentals(Monastrell Cabernet Sauvignon and Syrah) hybrids bearingwhite grapes can be obtained some of them with a highconcentration of flavonols that could be of importance in thehealth properties of this fruit and its derived products suchas the wineThe information obtained in this study should behelpful for selecting parentals for breeding programs
8 Journal of Analytical Methods in Chemistry
0
500
1000
1500
2000
(120583g
g)
B-M
timesS
30B-
Mtimes
S 1
B-M
timesS
19M
timesS
28M
timesS
31B-
Mtimes
S 10
0M
timesS
20B-
Mtimes
S 59
B-M
timesS
118
B-M
timesS
9B-
Mtimes
S 14
B-M
timesS
94B-
Mtimes
S 82
B-M
timesS
15M
timesS
26M
timesS
0M
timesS
75B-
Mtimes
S 40
Mtimes
S 38
Mtimes
S 27
Mtimes
S 66
B-M
timesS
73B-
Mtimes
S 65
Mtimes
S 21
Mtimes
S 76
Mon
astre
llM
timesS
11M
timesS
3M
timesS
114
Mtimes
S 62
Mtimes
S 34
Syra
hM
timesS
56M
timesS
117
Mtimes
S 47
Mtimes
S 97
Mtimes
S 4
Mtimes
S 42
Mtimes
S 46
Mtimes
S 71
Mtimes
S 37
B-M
timesS
91M
timesS
8M
timesS
57
(a)
0
200
400
600
800
1000
(120583g
g)
B-M
timesCS
17
B-M
timesCS
13
B-M
timesCS
93
B-M
timesCS
171
B-M
timesCS
146
Mtimes
CS 9
9M
timesCS
27
Mtimes
CS D
Mtimes
CS 1
84M
timesCS
135
Mtimes
CS 4
B-M
timesCS
98
Mtimes
CS 1
07Ca
bern
etS
Mtimes
CS 9
1M
timesCS
7M
timesCS
26
Mtimes
CS 1
36M
timesCS
163
Mtimes
CS 9
6M
timesCS
84
Mtimes
CS 1
58M
timesCS
100
Mtimes
CS 1
99M
timesCS
72
Mtimes
CS E
Mtimes
CS 4
9M
timesCS
BM
timesCS
37
Mtimes
CS 1
9M
timesCS
16
Mtimes
CS 8
0M
timesCS
55
Mon
astre
llM
timesCS
38
Mtimes
CS 9
0M
timesCS
CM
timesCS
59
Mtimes
CS 5
6M
timesCS
209
Mtimes
CS A
(b)
0
200
400
600
800
1000
1200
1400
1600
Mtimes
B 55
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 66
Mtimes
B 75
Mtimes
B 10
9
Barb
era
Mtimes
B 69
Mtimes
B 12
1
Mtimes
B 12
0
Mon
astre
ll
Mtimes
B 70
Mtimes
B 84
(120583g
g)
Mtimes
B 94
Mtimes
B 12
2
(c)
Figure 4 Concentration of flavonols in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c) hybridgrapes (yellow bars indicate white grape bearing hybrids)
Journal of Analytical Methods in Chemistry 9
Acknowledgment
This work was made possible by financial assistance of theMinisterio de Ciencia e Innovacion Project AGL2006-11019
References
[1] F Alen-Ruiz M S Garcıa-Falcon M C Perez-Lamela EMartınez-Carballo and J Simal-Gandara ldquoInfluence of majorpolyphenols on antioxidant activity in Mencia and Brancellaored grapesrdquo Food Chem vol 113 pp 53ndash60 2008
[2] S C Forester and A L Waterhouse ldquoMetabolites are key tounderstanding health effects of wine polyphenolicsrdquo Journal ofNutrition vol 139 no 9 pp 1324Sndash1831S 2009
[3] A Ortega-Regules I Romero-Cascales J M Lopez-Roca J MRos-Garcıa and E Gomez-Plaza ldquoAnthocyanin fingerprint ofgrapes environmental and genetic variationsrdquo Journal of theScience of Food and Agriculture vol 86 no 10 pp 1460ndash14672006
[4] V Cheynier and J Rigaud ldquoHPLC separation and characteri-zation of flavonols in the skins of Vitis vinifera var CinsaultrdquoAmerican Journal of Enology and Viticulture vol 37 pp 248ndash252 1986
[5] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero andI Hermosın-Gutierrez ldquoFlavonol profiles of Vitis vinifera redgrapes and their single-cultivar winesrdquo Journal of Agriculturaland Food Chemistry vol 55 no 3 pp 992ndash1002 2007
[6] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero M VGomez A H Velders and I Hermosın-Gutierrez ldquoFlavonol 3-O-glycosides series of Vitis vinifera Cv Petit Verdot red winegrapesrdquo Journal of Agricultural and Food Chemistry vol 57 no1 pp 209ndash219 2009
[7] P Sarni H Fulcrand V Souillol JM Souquet andV CheynierldquoMechanisms of anthocyanin degradation in grape must likemodel solutionsrdquo Journal of the Science of Food and Agriculturevol 69 no 3 pp 385ndash391 1995
[8] C Malien-Aubert O Dangles andM J Amiot ldquoColor stabilityof commercial anthocyanin-based extracts in relation to thephenolic composition Protective effects by intra- and inter-molecular copigmentationrdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 170ndash176 2001
[9] H Chang H C Eun and S C Hyang ldquoQuantitative Structure-Activity Relationship (QSAR) of antioxidative anthocyanidinsand their glycosidesrdquo Food Science and Biotechnology vol 17 no3 pp 501ndash507 2008
[10] Z Liang C Yang J Yang et al ldquoInheritance of anthocyaninsin berries of Vitis vinifera grapesrdquo Euphytica vol 167 no 1 pp113ndash125 2009
[11] A Bayo-Canha J I Fernandez-Fernandez A Martınez-Cutillas and L Ruiz-Garcıa ldquoPhenotypic segregation andrelationships of agronomic traits in Monastrell times Syrah winegrape progenyrdquo Euphytica vol 186 pp 393ndash407 2012
[12] A F Adam-Blondon C Roux D Claux G Butterlin DMerdinoglu and P This ldquoMapping 245 SSR markers on theVitis vinifera genome a tool for grape geneticsrdquoTheoretical andApplied Genetics vol 109 no 5 pp 1017ndash1027 2004
[13] A R Walker E Lee J Bogs D A J McDavid M R Thomasand S P Robinson ldquoWhite grapes arose through the mutationof two similar and adjacent regulatory genesrdquo Plant Journal vol49 no 5 pp 772ndash785 2007
[14] P K Boss C Davies and S P Robinson ldquoAnalysis of theexpression of anthocyanin pathway genes in developing Vitis
vinifera L cv Shiraz grape berries and the implications forpathway regulationrdquo Plant Physiology vol 111 no 4 pp 1059ndash1066 1996
[15] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002
[16] D Lijavetzky L Ruiz-Garcıa J A Cabezas et al ldquoMoleculargenetics of berry colour variation in table graperdquo MolecularGenetics and Genomics vol 276 no 5 pp 427ndash435 2006
[17] M C de Vicente and S D Tanksley ldquoQTL analysis of transgres-sive segregation in an interspecific tomato crossrdquo Genetics vol134 no 2 pp 585ndash596 1993
[18] L H Rieseberg M A Archer and R K Wayne ldquoTransgressivesegregation adaptation and speciationrdquoHeredity vol 83 no 4pp 363ndash372 1999
[19] Z Ju C Liu Y Yuan YWang andG Liu ldquoColoration potentialanthocyanin accumulation and enzyme activity in fruit ofcommercial apple cultivars and their F1 progenyrdquo ScientiaHorticulturae vol 79 no 1-2 pp 39ndash50 1999
[20] A Hernandez-Jimenez E Gomez-Plaza A Martınez-Cutillasand J A Kennedy ldquoGrape skin and seed proanthocyanidinsfrom Monastrell times Syrah grapesrdquo Journal of Agricultural andFood Chemistry vol 57 no 22 pp 10798ndash10803 2009
[21] H Kobayashi S Suzuki F Tanzawa andT Takayanagispi ldquoLowexpression of flavonoid 3rsquo5rsquo-hydroxylase (F3rsquo5rsquoH) associatedwith cyanidin-based anthocyanins in grape leafrdquo AmericanJournal of Enology and Viticulture vol 60 no 3 pp 362ndash3672009
[22] E Gomez-Plaza R Gil-Munoz A Hernandez-Jimenez JM Lopez-Roca A Ortega-Regules and A Martınez-CutillasldquoStudies on the anthocyanin profile ofVitis vinifera intraspecifichybrids (Monastrell times Cabernet Sauvignon)rdquo European FoodResearch and Technology vol 227 no 2 pp 479ndash484 2008
[23] A Azuma S Kobayashi N Mitani et al ldquoGenomic and geneticanalysis ofMyb-related genes that regulate anthocyanin biosyn-thesis in grape berry skinrdquoTheoretical and Applied Genetics vol117 no 6 pp 1009ndash1019 2008
[24] A Azuma S Kobayashi H Yakushiji M Yamada N Mitaniand A Sato ldquoVvmybA1 genotype determines grape skin colorrdquoVitis vol 46 no 3 pp 154ndash155 2007
[25] FMattivi R Guzzon U VrhovsekM Stefanini and R VelascoldquoMetabolite profiling of grape flavonols and anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7692ndash7702 2006
[26] J Bogs A Ebadi D McDavid and S P Robinson ldquoIdentifi-cation of the flavonoid hydroxylases from grapevine and theirregulation dining fruit developmentrdquo Plant Physiology vol 140no 1 pp 279ndash291 2006
[27] F Quattrocchio A Baudry L Lepiniec and E GrotewoldldquoThe regulation of flavonoid biosynthesisrdquo in The Science ofFlavonoids E Grotewold Ed pp 97ndash122 Springer New YorkNY USA 2008
[28] J M Cevallos-Cevallos J I Reyes-De-Corcuera E EtxeberriaM D Danyluk and G E Rodrick ldquoMetabolomic analysis infood science a reviewrdquo Trends in Food Science and Technologyvol 20 no 11-12 pp 557ndash566 2009
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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ElectrochemistryInternational Journal of
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CatalystsJournal of
4 Journal of Analytical Methods in Chemistry
Table 1 Mean values (119899 = 3) of the anthocyanin profile and total anthocyanin content (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids
del cyan pet peon malv nonacylated acylated dihydrox trihydrox Total (mgg)Monastrell
Mean 125 137 100 200 397 552 448 377 623 740Syrah
Mean 72 36 81 109 681 496 503 167 833 1506CS
Mean 96 52 101 74 677 459 541 126 874 1507Barbera
Mean 98 31 139 47 686 656 343 77 923 1402Mon times Sy (27)a
Mean 100 71 86 154 548 432 568 265 734 1631Minimum 57 32 64 65 295 462 297 132 462 386Maximum 243 154 139 375 727 702 713 537 868 3949
Mon times CS (32)b
Mean 124 60 133 105 578 525 472 165 835 1444Minimum 75 35 94 56 444 352 313 95 689 487Maximum 234 121 203 215 685 686 647 311 905 2867
Mon times Bar (13)c
Mean 109 77 133 162 519 720 280 239 761 1208Minimum 41 11 90 44 317 415 101 67 552 161Maximum 170 146 197 364 779 898 585 447 933 2788
abcThe number in parenthesis represents the number of hybrids for each crossing del percentage of delphinidin derivatives cyan percentage of cyanidin derivatives pet percentage of petunidin derivatives peon percentage ofpeonidin derivatives malv percentage of malvidin derivatives nonacylated percentage of nonacylated anthocyanins acylated percentage of acylatedanthocyanins dihydrox percentage of dihydroxylated anthocyanins trihydrox percentage of trihydroxylated anthocyanins and Total (mgg) totalanthocyanin content (mg per g of skin)
disubstituted and trisubstituted anthocyanins when UFGTis expressed and to form other polyphenols (flavanolsflavonols) at different developmental stages All the studiedhybrids bearing red grapes synthesised all five anthocyanins(the dihydroxylated cyaniding peonidin-3-glucosides thetrihydroxylated delphinidin petunidin and malvidin-3-glucosides) together with their acylated derivatives Thismeans that all the parentals and the hybrids expressed func-tional F31015840HandF3101584051015840Hgenes for the synthesis of 3101584041015840-OHand310158404101584051015840-OH anthocyanins as well as methyltransferases (MT)for the methylation of primary anthocyanins As regardsthe percentage of the different anthocyanins in the differentparentals Monastrell grapes were characterized by a highpercentage of cyanidin suggesting a lower F3101584051015840H activitythan in the other varieties A low expression of F3101584051015840H hasbeen associated with cyanidin-based anthocyanins in grapeleafs [21] The percentages of malvidin-based anthocyaninsin Monastrell did not exceed 40 so the total percentageof trihydroxylated anthocyanins was low The percentage oftrihydroxylated anthocyanins reached 83 in Syrah grapes874 in Cabernet Sauvignon grapes and 923 in Barberagrapes The percentage of trihydroxylated anthocyanins waseven higher in some Monastrell times Cabernet Sauvignonand Monastrell times Barbera hybrids reaching values as highas 905 and 93 respectively The mean value of thepercentage of trihydroxylated anthocyanins in the hybridgrapes is close to the mean value between both parentals
The segregation can be fitted to a normal distribution and avery low number of hybrids presented higher or lower valuesthan in the parentals as can be seen in Figure 3These resultswere similar to those obtained during the first screening ofthe anthocyanin profile in Monastrell times Cabernet Sauvignongrapes [22] In spite of the presence of all anthocyaninbiosynthetic enzymes in all the investigated hybrids (sinceall the possible structures were found) a genotype-specificregulation of the structural genes along the core pathway andat the main branching points is presumed to underlie theobservedmethoxylation and hydroxylation variations amongthe grapes from the parentals and those of their hybrid plants
With regard to the percentage of anthocyanin acylationBarbera grapes showed the lowest percentage of acylation(343) followed by Monastrell (448) and CabernetSauvignon showed the highest percentages (54) The meanvalues of the percentage of acylated anthocyanins for Monas-trell times Cabernet Sauvignon hybrids were 47 568 forMonastrell times Syrah (higher than the percentage found inSyrah) while the hybrid grapes from Monastrell times Barberashowed the lowest percentages of acylation (28) As inthe case of the anthocyanin concentration data there was atendency towards higher values of acylation in the hybridsand no hybrid contained only nonacylated anthocyanins
As regards flavonols (Table 2) these flavonoids werepresent in both white and red grapes We could iden-tify mono- (kaempferol) di- (quercetin and isorhamnetin)
Journal of Analytical Methods in Chemistry 5
0
10000
20000
30000
40000
50000
(120583g
g)
Mtimes
S 20
Mtimes
S 76
Mtimes
S 4
Mtimes
S 28
Mon
astre
llM
timesS
66M
timesS
21M
timesS
31M
timesS
0M
timesS
27M
timesS
26M
timesS
62M
timesS
97Sy
rah
Mtimes
S 42
Mtimes
S 56
Mtimes
S 3
Mtimes
S 11
7M
timesS
46M
timesS
38M
timesS
34M
timesS
57M
timesS
75M
timesS
8M
timesS
114
Mtimes
S 11
Mtimes
S 37
Mtimes
S 47
Mtimes
S 71
(a)
0
5000
10000
15000
20000
25000
30000
Mtimes
CS 2
7M
timesCS
163
Mtimes
CS 1
58M
timesCS
99
Mtimes
CS 1
84M
timesCS
37
Mtimes
CS 1
07M
timesCS
7M
onas
trell
Mtimes
CS 7
2M
timesCS
DM
timesCS
EM
timesCS
26
Mtimes
CS 4
Mtimes
CS 9
1M
timesCS
100
Mtimes
CS 1
99M
timesCS
56
Mtimes
CS 1
35M
timesCS
90
Mtimes
CS A
Cabe
rnet
SM
timesCS
136
Mtimes
CS B
Mtimes
CS 8
4M
timesCS
96
Mtimes
CS C
Mtimes
CS 3
8M
timesCS
19
Mtimes
CS 5
9M
timesCS
55
Mtimes
CS 8
0M
timesCS
49
Mtimes
CS 2
09M
timesCS
16
(120583g
g)
(b)
0
5000
10000
15000
20000
25000
30000
Mtimes
B 12
2
Mtimes
B 10
1
Mtimes
B 66
Mtimes
B 55
Mon
astre
ll
Mtimes
B 94
Mtimes
B 11
3
Mtimes
B 75
Mtimes
B 84
Barb
era
Mtimes
B 70
Mtimes
B 10
9
Mtimes
B 12
0
Mtimes
B 12
1
Mtimes
B 69
(120583g
g)
(c)
Figure 2 Concentration of anthocyanins in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c)hybrid grapes (each bar represents the mean value of three samples)
6 Journal of Analytical Methods in Chemistry
0
1
2
3
4
5
6
7
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mtimes
S 62
Mtimes
S 3
Mon
astre
llM
timesS
4M
timesS
76M
timesS
20M
timesS
97M
timesS
66M
timesS
31M
timesS
21M
timesS
56M
timesS
27M
timesS
117
Mtimes
S 57
Mtimes
S 11
Mtimes
S 28
Mtimes
S 75
Mtimes
S 34
Mtimes
S 38
Mtimes
S 0
Mtimes
S 47
Mtimes
S 26
Mtimes
S 8
Mtimes
S 37
Mtimes
S 11
4M
timesS
71M
timesS
42Sy
rah
Mtimes
S 46
(a)
0
2
4
6
8
10
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mon
astre
llM
timesCS
209
Mtimes
CS 1
58M
timesCS
27
Mtimes
CS 1
63M
timesCS
49
Mtimes
CS 1
84M
timesCS
37
Mtimes
CS 4
Mtimes
CS 9
9M
timesCS
96
Mtimes
CS 5
9M
timesCS
7M
timesCS
135
Mtimes
CS 9
1M
timesCS
26
Mtimes
CS 1
00M
timesCS
107
Mtimes
CS 5
6M
timesCS
19
Mtimes
CS 1
6M
timesCS
84
Mtimes
CS 1
36M
timesCS
DM
timesCS
55
Mtimes
CS 7
2Ca
bern
etS
Mtimes
CS 1
99M
timesCS
90
Mtimes
CS C
Mtimes
CS E
Mtimes
CS B
Mtimes
CS 3
8M
timesCS
80
Mtimes
CS A
(b)
0
2
4
6
8
10
12
14
16
Mtimes
B 66
Mon
astre
ll
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 12
1
Mtimes
B 12
2
Mtimes
B 55
Mtimes
B 84
Mtimes
B 69
Mtimes
B 75
Mtimes
B 10
9
Mtimes
B 70
Mtimes
B 94
Barb
era
Mtimes
B 12
0Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
(c)
Figure 3 Trihydroxylateddihydroxylated anthocyanins ratios in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) andMonastrell times Barbera (c) hybrid grapes
Journal of Analytical Methods in Chemistry 7
Table 2 Mean values of the flavonol profile and total flavonolcontent (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids (119899 = 3)
monohydr dihydr trihydrox TotalMonastrell
Mean 109 707 184 056Syrah
Mean 134 442 424 073CS
Mean 147 446 407 019Barbera
Mean 43 533 424 049Red hybrids
Mon times SyMean 104 524 372 073Minimum 53 363 165 018Maximum 160 726 550 183
Mon times CSMean 113 511 376 037Minimum 50 352 203 011Maximum 209 650 583 078
Mon times BarMean 94 619 287 042Minimum 47 429 87 012Maximum 136 839 434 139
White hybridsMon times Sy
Mean 134 841 24 035Minimum 41 746 06 008Maximum 237 947 72 128
Mon times CSMean 200 748 51 008Minimum 168 689 09 002Maximum 259 784 127 018
monohydrox percentage of monohydroxylated flavonols dihydroxpercentage of dihydroxylated flavonols trihydrox percentage of trihy-droxylated flavonols Total total flavonol content (mg per g of skin)
and trihydroxylated (myricetin laricitrin and syringetin)flavonol glycosides (glucosides glucoronides and smallquantities of galactosides) In red grapes the monohydrox-ylated flavonols represented the lowest percentage especiallyin Barbera grapes (43) Monastrell grapes presented a veryhigh percentage of dihydroxylated flavonols (707) muchhigher than in the other varieties and therefore a lowerpercentage of trihydroxylated flavonols whereas BarberaSyrah and Cabernet Sauvignon grapes reached a percentageof trihydroxylated flavonols of around 45ndash50 This factindicates lower F3101584051015840H activity in Monastrell grapes as alsoobserved for the anthocyanins
As regards the flavonol content Cabernet Sauvignongrapes showed low concentrations of flavonols and Monas-trell and Syrah much higher concentrations while the highestvalue was found in red grapes from a hybrid plant fromMonastrell times Syrah (Table 2 Figure 4) reaching values as
high as 183mgg of skin In the hybrids bearing white grapesa lower concentration of flavonols was measured comparedwith that of the hybrids bearing red grapes Azuma et al[23 24] stated that Myb genes besides the regulation of theUFGT expression appear to enhance the expression of allthe genes involved in the anthocyanin biosynthesis pathwaybecause the transcription of all anthocyanin biosynthesisgenes appears to be slightly activated which would explainthe higher concentration of flavonols in red grapes Also inthese white skinned grapes trihydroxylated flavonols werebarely presentMattivi et al [25] studying the flavonol profileof several grape varieties did not detect trihydroxylatedflavonols in white grapes Bogs et al [26] did not findsignificant expression of F3101584051015840H and UFGT in white grapevarieties which suggests a related regulation of F3101584051015840H andUFGT during berry ripening and justifies the almost nullpresence of trihydroxylated flavonols in white grapes F3101584051015840Hwas detected in white grapes var Chardonnay but prior toveraison [26] since it is needed for flavanol biosynthesiswhich seems to be controlled differently indeed no differ-ences in the percentage of trihydroxylated flavanols wereobserved between the red and white grapes arising fromthe cross of Monastrell times Syrah [20] However other studiesstated that flavonol synthase (FLS) was not upregulated whenUFGT was expressed and that the increase in flavonols inred grapes was a consequence of an increase flux through theflavonoid pathway [27]
4 Conclusions
The study of the anthocyanin and flavonol profiles of thegrapes from the hybrid plants can be useful for a targetedinformative metabolomic analysis [28] a tool for selectingpromising grapes according to their profile andor content
Seedlings with grapes presenting very high concentra-tions of anthocyanins and flavonols can be expected fromintraspecific crosses and these resulting grapes could leadto highly colored wines with increased health-related prop-erties In this way three plants arising from Monastrell timesSyrah (hybrids 8 37 and 71) presented anthocyanin andflavonol concentrations higher than 20 and 1mgg fresh skinrespectively (Figures 2 and 4) Also four plants arising fromMonastrell timesCabernet Sauvignon (hybrids 38 59 55 and 80)and two from Monastrell times Barbera (hybrid plants 120 121)showed anthocyanin values higher than 20mgg fresh skinaccompanied with high concentration of flavonols (Figures2 and 4) The hydroxylation pattern which also influenceswine color and its stability will be strongly influenced by theparentals pattern since values higher than that shown by thebest parental in this respect will be difficult to obtain Alsoin the case of the crosses between heterozygous parentals(Monastrell Cabernet Sauvignon and Syrah) hybrids bearingwhite grapes can be obtained some of them with a highconcentration of flavonols that could be of importance in thehealth properties of this fruit and its derived products suchas the wineThe information obtained in this study should behelpful for selecting parentals for breeding programs
8 Journal of Analytical Methods in Chemistry
0
500
1000
1500
2000
(120583g
g)
B-M
timesS
30B-
Mtimes
S 1
B-M
timesS
19M
timesS
28M
timesS
31B-
Mtimes
S 10
0M
timesS
20B-
Mtimes
S 59
B-M
timesS
118
B-M
timesS
9B-
Mtimes
S 14
B-M
timesS
94B-
Mtimes
S 82
B-M
timesS
15M
timesS
26M
timesS
0M
timesS
75B-
Mtimes
S 40
Mtimes
S 38
Mtimes
S 27
Mtimes
S 66
B-M
timesS
73B-
Mtimes
S 65
Mtimes
S 21
Mtimes
S 76
Mon
astre
llM
timesS
11M
timesS
3M
timesS
114
Mtimes
S 62
Mtimes
S 34
Syra
hM
timesS
56M
timesS
117
Mtimes
S 47
Mtimes
S 97
Mtimes
S 4
Mtimes
S 42
Mtimes
S 46
Mtimes
S 71
Mtimes
S 37
B-M
timesS
91M
timesS
8M
timesS
57
(a)
0
200
400
600
800
1000
(120583g
g)
B-M
timesCS
17
B-M
timesCS
13
B-M
timesCS
93
B-M
timesCS
171
B-M
timesCS
146
Mtimes
CS 9
9M
timesCS
27
Mtimes
CS D
Mtimes
CS 1
84M
timesCS
135
Mtimes
CS 4
B-M
timesCS
98
Mtimes
CS 1
07Ca
bern
etS
Mtimes
CS 9
1M
timesCS
7M
timesCS
26
Mtimes
CS 1
36M
timesCS
163
Mtimes
CS 9
6M
timesCS
84
Mtimes
CS 1
58M
timesCS
100
Mtimes
CS 1
99M
timesCS
72
Mtimes
CS E
Mtimes
CS 4
9M
timesCS
BM
timesCS
37
Mtimes
CS 1
9M
timesCS
16
Mtimes
CS 8
0M
timesCS
55
Mon
astre
llM
timesCS
38
Mtimes
CS 9
0M
timesCS
CM
timesCS
59
Mtimes
CS 5
6M
timesCS
209
Mtimes
CS A
(b)
0
200
400
600
800
1000
1200
1400
1600
Mtimes
B 55
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 66
Mtimes
B 75
Mtimes
B 10
9
Barb
era
Mtimes
B 69
Mtimes
B 12
1
Mtimes
B 12
0
Mon
astre
ll
Mtimes
B 70
Mtimes
B 84
(120583g
g)
Mtimes
B 94
Mtimes
B 12
2
(c)
Figure 4 Concentration of flavonols in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c) hybridgrapes (yellow bars indicate white grape bearing hybrids)
Journal of Analytical Methods in Chemistry 9
Acknowledgment
This work was made possible by financial assistance of theMinisterio de Ciencia e Innovacion Project AGL2006-11019
References
[1] F Alen-Ruiz M S Garcıa-Falcon M C Perez-Lamela EMartınez-Carballo and J Simal-Gandara ldquoInfluence of majorpolyphenols on antioxidant activity in Mencia and Brancellaored grapesrdquo Food Chem vol 113 pp 53ndash60 2008
[2] S C Forester and A L Waterhouse ldquoMetabolites are key tounderstanding health effects of wine polyphenolicsrdquo Journal ofNutrition vol 139 no 9 pp 1324Sndash1831S 2009
[3] A Ortega-Regules I Romero-Cascales J M Lopez-Roca J MRos-Garcıa and E Gomez-Plaza ldquoAnthocyanin fingerprint ofgrapes environmental and genetic variationsrdquo Journal of theScience of Food and Agriculture vol 86 no 10 pp 1460ndash14672006
[4] V Cheynier and J Rigaud ldquoHPLC separation and characteri-zation of flavonols in the skins of Vitis vinifera var CinsaultrdquoAmerican Journal of Enology and Viticulture vol 37 pp 248ndash252 1986
[5] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero andI Hermosın-Gutierrez ldquoFlavonol profiles of Vitis vinifera redgrapes and their single-cultivar winesrdquo Journal of Agriculturaland Food Chemistry vol 55 no 3 pp 992ndash1002 2007
[6] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero M VGomez A H Velders and I Hermosın-Gutierrez ldquoFlavonol 3-O-glycosides series of Vitis vinifera Cv Petit Verdot red winegrapesrdquo Journal of Agricultural and Food Chemistry vol 57 no1 pp 209ndash219 2009
[7] P Sarni H Fulcrand V Souillol JM Souquet andV CheynierldquoMechanisms of anthocyanin degradation in grape must likemodel solutionsrdquo Journal of the Science of Food and Agriculturevol 69 no 3 pp 385ndash391 1995
[8] C Malien-Aubert O Dangles andM J Amiot ldquoColor stabilityof commercial anthocyanin-based extracts in relation to thephenolic composition Protective effects by intra- and inter-molecular copigmentationrdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 170ndash176 2001
[9] H Chang H C Eun and S C Hyang ldquoQuantitative Structure-Activity Relationship (QSAR) of antioxidative anthocyanidinsand their glycosidesrdquo Food Science and Biotechnology vol 17 no3 pp 501ndash507 2008
[10] Z Liang C Yang J Yang et al ldquoInheritance of anthocyaninsin berries of Vitis vinifera grapesrdquo Euphytica vol 167 no 1 pp113ndash125 2009
[11] A Bayo-Canha J I Fernandez-Fernandez A Martınez-Cutillas and L Ruiz-Garcıa ldquoPhenotypic segregation andrelationships of agronomic traits in Monastrell times Syrah winegrape progenyrdquo Euphytica vol 186 pp 393ndash407 2012
[12] A F Adam-Blondon C Roux D Claux G Butterlin DMerdinoglu and P This ldquoMapping 245 SSR markers on theVitis vinifera genome a tool for grape geneticsrdquoTheoretical andApplied Genetics vol 109 no 5 pp 1017ndash1027 2004
[13] A R Walker E Lee J Bogs D A J McDavid M R Thomasand S P Robinson ldquoWhite grapes arose through the mutationof two similar and adjacent regulatory genesrdquo Plant Journal vol49 no 5 pp 772ndash785 2007
[14] P K Boss C Davies and S P Robinson ldquoAnalysis of theexpression of anthocyanin pathway genes in developing Vitis
vinifera L cv Shiraz grape berries and the implications forpathway regulationrdquo Plant Physiology vol 111 no 4 pp 1059ndash1066 1996
[15] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002
[16] D Lijavetzky L Ruiz-Garcıa J A Cabezas et al ldquoMoleculargenetics of berry colour variation in table graperdquo MolecularGenetics and Genomics vol 276 no 5 pp 427ndash435 2006
[17] M C de Vicente and S D Tanksley ldquoQTL analysis of transgres-sive segregation in an interspecific tomato crossrdquo Genetics vol134 no 2 pp 585ndash596 1993
[18] L H Rieseberg M A Archer and R K Wayne ldquoTransgressivesegregation adaptation and speciationrdquoHeredity vol 83 no 4pp 363ndash372 1999
[19] Z Ju C Liu Y Yuan YWang andG Liu ldquoColoration potentialanthocyanin accumulation and enzyme activity in fruit ofcommercial apple cultivars and their F1 progenyrdquo ScientiaHorticulturae vol 79 no 1-2 pp 39ndash50 1999
[20] A Hernandez-Jimenez E Gomez-Plaza A Martınez-Cutillasand J A Kennedy ldquoGrape skin and seed proanthocyanidinsfrom Monastrell times Syrah grapesrdquo Journal of Agricultural andFood Chemistry vol 57 no 22 pp 10798ndash10803 2009
[21] H Kobayashi S Suzuki F Tanzawa andT Takayanagispi ldquoLowexpression of flavonoid 3rsquo5rsquo-hydroxylase (F3rsquo5rsquoH) associatedwith cyanidin-based anthocyanins in grape leafrdquo AmericanJournal of Enology and Viticulture vol 60 no 3 pp 362ndash3672009
[22] E Gomez-Plaza R Gil-Munoz A Hernandez-Jimenez JM Lopez-Roca A Ortega-Regules and A Martınez-CutillasldquoStudies on the anthocyanin profile ofVitis vinifera intraspecifichybrids (Monastrell times Cabernet Sauvignon)rdquo European FoodResearch and Technology vol 227 no 2 pp 479ndash484 2008
[23] A Azuma S Kobayashi N Mitani et al ldquoGenomic and geneticanalysis ofMyb-related genes that regulate anthocyanin biosyn-thesis in grape berry skinrdquoTheoretical and Applied Genetics vol117 no 6 pp 1009ndash1019 2008
[24] A Azuma S Kobayashi H Yakushiji M Yamada N Mitaniand A Sato ldquoVvmybA1 genotype determines grape skin colorrdquoVitis vol 46 no 3 pp 154ndash155 2007
[25] FMattivi R Guzzon U VrhovsekM Stefanini and R VelascoldquoMetabolite profiling of grape flavonols and anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7692ndash7702 2006
[26] J Bogs A Ebadi D McDavid and S P Robinson ldquoIdentifi-cation of the flavonoid hydroxylases from grapevine and theirregulation dining fruit developmentrdquo Plant Physiology vol 140no 1 pp 279ndash291 2006
[27] F Quattrocchio A Baudry L Lepiniec and E GrotewoldldquoThe regulation of flavonoid biosynthesisrdquo in The Science ofFlavonoids E Grotewold Ed pp 97ndash122 Springer New YorkNY USA 2008
[28] J M Cevallos-Cevallos J I Reyes-De-Corcuera E EtxeberriaM D Danyluk and G E Rodrick ldquoMetabolomic analysis infood science a reviewrdquo Trends in Food Science and Technologyvol 20 no 11-12 pp 557ndash566 2009
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
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Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Analytical Methods in Chemistry
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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
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Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
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Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Analytical Methods in Chemistry 5
0
10000
20000
30000
40000
50000
(120583g
g)
Mtimes
S 20
Mtimes
S 76
Mtimes
S 4
Mtimes
S 28
Mon
astre
llM
timesS
66M
timesS
21M
timesS
31M
timesS
0M
timesS
27M
timesS
26M
timesS
62M
timesS
97Sy
rah
Mtimes
S 42
Mtimes
S 56
Mtimes
S 3
Mtimes
S 11
7M
timesS
46M
timesS
38M
timesS
34M
timesS
57M
timesS
75M
timesS
8M
timesS
114
Mtimes
S 11
Mtimes
S 37
Mtimes
S 47
Mtimes
S 71
(a)
0
5000
10000
15000
20000
25000
30000
Mtimes
CS 2
7M
timesCS
163
Mtimes
CS 1
58M
timesCS
99
Mtimes
CS 1
84M
timesCS
37
Mtimes
CS 1
07M
timesCS
7M
onas
trell
Mtimes
CS 7
2M
timesCS
DM
timesCS
EM
timesCS
26
Mtimes
CS 4
Mtimes
CS 9
1M
timesCS
100
Mtimes
CS 1
99M
timesCS
56
Mtimes
CS 1
35M
timesCS
90
Mtimes
CS A
Cabe
rnet
SM
timesCS
136
Mtimes
CS B
Mtimes
CS 8
4M
timesCS
96
Mtimes
CS C
Mtimes
CS 3
8M
timesCS
19
Mtimes
CS 5
9M
timesCS
55
Mtimes
CS 8
0M
timesCS
49
Mtimes
CS 2
09M
timesCS
16
(120583g
g)
(b)
0
5000
10000
15000
20000
25000
30000
Mtimes
B 12
2
Mtimes
B 10
1
Mtimes
B 66
Mtimes
B 55
Mon
astre
ll
Mtimes
B 94
Mtimes
B 11
3
Mtimes
B 75
Mtimes
B 84
Barb
era
Mtimes
B 70
Mtimes
B 10
9
Mtimes
B 12
0
Mtimes
B 12
1
Mtimes
B 69
(120583g
g)
(c)
Figure 2 Concentration of anthocyanins in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c)hybrid grapes (each bar represents the mean value of three samples)
6 Journal of Analytical Methods in Chemistry
0
1
2
3
4
5
6
7
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mtimes
S 62
Mtimes
S 3
Mon
astre
llM
timesS
4M
timesS
76M
timesS
20M
timesS
97M
timesS
66M
timesS
31M
timesS
21M
timesS
56M
timesS
27M
timesS
117
Mtimes
S 57
Mtimes
S 11
Mtimes
S 28
Mtimes
S 75
Mtimes
S 34
Mtimes
S 38
Mtimes
S 0
Mtimes
S 47
Mtimes
S 26
Mtimes
S 8
Mtimes
S 37
Mtimes
S 11
4M
timesS
71M
timesS
42Sy
rah
Mtimes
S 46
(a)
0
2
4
6
8
10
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mon
astre
llM
timesCS
209
Mtimes
CS 1
58M
timesCS
27
Mtimes
CS 1
63M
timesCS
49
Mtimes
CS 1
84M
timesCS
37
Mtimes
CS 4
Mtimes
CS 9
9M
timesCS
96
Mtimes
CS 5
9M
timesCS
7M
timesCS
135
Mtimes
CS 9
1M
timesCS
26
Mtimes
CS 1
00M
timesCS
107
Mtimes
CS 5
6M
timesCS
19
Mtimes
CS 1
6M
timesCS
84
Mtimes
CS 1
36M
timesCS
DM
timesCS
55
Mtimes
CS 7
2Ca
bern
etS
Mtimes
CS 1
99M
timesCS
90
Mtimes
CS C
Mtimes
CS E
Mtimes
CS B
Mtimes
CS 3
8M
timesCS
80
Mtimes
CS A
(b)
0
2
4
6
8
10
12
14
16
Mtimes
B 66
Mon
astre
ll
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 12
1
Mtimes
B 12
2
Mtimes
B 55
Mtimes
B 84
Mtimes
B 69
Mtimes
B 75
Mtimes
B 10
9
Mtimes
B 70
Mtimes
B 94
Barb
era
Mtimes
B 12
0Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
(c)
Figure 3 Trihydroxylateddihydroxylated anthocyanins ratios in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) andMonastrell times Barbera (c) hybrid grapes
Journal of Analytical Methods in Chemistry 7
Table 2 Mean values of the flavonol profile and total flavonolcontent (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids (119899 = 3)
monohydr dihydr trihydrox TotalMonastrell
Mean 109 707 184 056Syrah
Mean 134 442 424 073CS
Mean 147 446 407 019Barbera
Mean 43 533 424 049Red hybrids
Mon times SyMean 104 524 372 073Minimum 53 363 165 018Maximum 160 726 550 183
Mon times CSMean 113 511 376 037Minimum 50 352 203 011Maximum 209 650 583 078
Mon times BarMean 94 619 287 042Minimum 47 429 87 012Maximum 136 839 434 139
White hybridsMon times Sy
Mean 134 841 24 035Minimum 41 746 06 008Maximum 237 947 72 128
Mon times CSMean 200 748 51 008Minimum 168 689 09 002Maximum 259 784 127 018
monohydrox percentage of monohydroxylated flavonols dihydroxpercentage of dihydroxylated flavonols trihydrox percentage of trihy-droxylated flavonols Total total flavonol content (mg per g of skin)
and trihydroxylated (myricetin laricitrin and syringetin)flavonol glycosides (glucosides glucoronides and smallquantities of galactosides) In red grapes the monohydrox-ylated flavonols represented the lowest percentage especiallyin Barbera grapes (43) Monastrell grapes presented a veryhigh percentage of dihydroxylated flavonols (707) muchhigher than in the other varieties and therefore a lowerpercentage of trihydroxylated flavonols whereas BarberaSyrah and Cabernet Sauvignon grapes reached a percentageof trihydroxylated flavonols of around 45ndash50 This factindicates lower F3101584051015840H activity in Monastrell grapes as alsoobserved for the anthocyanins
As regards the flavonol content Cabernet Sauvignongrapes showed low concentrations of flavonols and Monas-trell and Syrah much higher concentrations while the highestvalue was found in red grapes from a hybrid plant fromMonastrell times Syrah (Table 2 Figure 4) reaching values as
high as 183mgg of skin In the hybrids bearing white grapesa lower concentration of flavonols was measured comparedwith that of the hybrids bearing red grapes Azuma et al[23 24] stated that Myb genes besides the regulation of theUFGT expression appear to enhance the expression of allthe genes involved in the anthocyanin biosynthesis pathwaybecause the transcription of all anthocyanin biosynthesisgenes appears to be slightly activated which would explainthe higher concentration of flavonols in red grapes Also inthese white skinned grapes trihydroxylated flavonols werebarely presentMattivi et al [25] studying the flavonol profileof several grape varieties did not detect trihydroxylatedflavonols in white grapes Bogs et al [26] did not findsignificant expression of F3101584051015840H and UFGT in white grapevarieties which suggests a related regulation of F3101584051015840H andUFGT during berry ripening and justifies the almost nullpresence of trihydroxylated flavonols in white grapes F3101584051015840Hwas detected in white grapes var Chardonnay but prior toveraison [26] since it is needed for flavanol biosynthesiswhich seems to be controlled differently indeed no differ-ences in the percentage of trihydroxylated flavanols wereobserved between the red and white grapes arising fromthe cross of Monastrell times Syrah [20] However other studiesstated that flavonol synthase (FLS) was not upregulated whenUFGT was expressed and that the increase in flavonols inred grapes was a consequence of an increase flux through theflavonoid pathway [27]
4 Conclusions
The study of the anthocyanin and flavonol profiles of thegrapes from the hybrid plants can be useful for a targetedinformative metabolomic analysis [28] a tool for selectingpromising grapes according to their profile andor content
Seedlings with grapes presenting very high concentra-tions of anthocyanins and flavonols can be expected fromintraspecific crosses and these resulting grapes could leadto highly colored wines with increased health-related prop-erties In this way three plants arising from Monastrell timesSyrah (hybrids 8 37 and 71) presented anthocyanin andflavonol concentrations higher than 20 and 1mgg fresh skinrespectively (Figures 2 and 4) Also four plants arising fromMonastrell timesCabernet Sauvignon (hybrids 38 59 55 and 80)and two from Monastrell times Barbera (hybrid plants 120 121)showed anthocyanin values higher than 20mgg fresh skinaccompanied with high concentration of flavonols (Figures2 and 4) The hydroxylation pattern which also influenceswine color and its stability will be strongly influenced by theparentals pattern since values higher than that shown by thebest parental in this respect will be difficult to obtain Alsoin the case of the crosses between heterozygous parentals(Monastrell Cabernet Sauvignon and Syrah) hybrids bearingwhite grapes can be obtained some of them with a highconcentration of flavonols that could be of importance in thehealth properties of this fruit and its derived products suchas the wineThe information obtained in this study should behelpful for selecting parentals for breeding programs
8 Journal of Analytical Methods in Chemistry
0
500
1000
1500
2000
(120583g
g)
B-M
timesS
30B-
Mtimes
S 1
B-M
timesS
19M
timesS
28M
timesS
31B-
Mtimes
S 10
0M
timesS
20B-
Mtimes
S 59
B-M
timesS
118
B-M
timesS
9B-
Mtimes
S 14
B-M
timesS
94B-
Mtimes
S 82
B-M
timesS
15M
timesS
26M
timesS
0M
timesS
75B-
Mtimes
S 40
Mtimes
S 38
Mtimes
S 27
Mtimes
S 66
B-M
timesS
73B-
Mtimes
S 65
Mtimes
S 21
Mtimes
S 76
Mon
astre
llM
timesS
11M
timesS
3M
timesS
114
Mtimes
S 62
Mtimes
S 34
Syra
hM
timesS
56M
timesS
117
Mtimes
S 47
Mtimes
S 97
Mtimes
S 4
Mtimes
S 42
Mtimes
S 46
Mtimes
S 71
Mtimes
S 37
B-M
timesS
91M
timesS
8M
timesS
57
(a)
0
200
400
600
800
1000
(120583g
g)
B-M
timesCS
17
B-M
timesCS
13
B-M
timesCS
93
B-M
timesCS
171
B-M
timesCS
146
Mtimes
CS 9
9M
timesCS
27
Mtimes
CS D
Mtimes
CS 1
84M
timesCS
135
Mtimes
CS 4
B-M
timesCS
98
Mtimes
CS 1
07Ca
bern
etS
Mtimes
CS 9
1M
timesCS
7M
timesCS
26
Mtimes
CS 1
36M
timesCS
163
Mtimes
CS 9
6M
timesCS
84
Mtimes
CS 1
58M
timesCS
100
Mtimes
CS 1
99M
timesCS
72
Mtimes
CS E
Mtimes
CS 4
9M
timesCS
BM
timesCS
37
Mtimes
CS 1
9M
timesCS
16
Mtimes
CS 8
0M
timesCS
55
Mon
astre
llM
timesCS
38
Mtimes
CS 9
0M
timesCS
CM
timesCS
59
Mtimes
CS 5
6M
timesCS
209
Mtimes
CS A
(b)
0
200
400
600
800
1000
1200
1400
1600
Mtimes
B 55
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 66
Mtimes
B 75
Mtimes
B 10
9
Barb
era
Mtimes
B 69
Mtimes
B 12
1
Mtimes
B 12
0
Mon
astre
ll
Mtimes
B 70
Mtimes
B 84
(120583g
g)
Mtimes
B 94
Mtimes
B 12
2
(c)
Figure 4 Concentration of flavonols in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c) hybridgrapes (yellow bars indicate white grape bearing hybrids)
Journal of Analytical Methods in Chemistry 9
Acknowledgment
This work was made possible by financial assistance of theMinisterio de Ciencia e Innovacion Project AGL2006-11019
References
[1] F Alen-Ruiz M S Garcıa-Falcon M C Perez-Lamela EMartınez-Carballo and J Simal-Gandara ldquoInfluence of majorpolyphenols on antioxidant activity in Mencia and Brancellaored grapesrdquo Food Chem vol 113 pp 53ndash60 2008
[2] S C Forester and A L Waterhouse ldquoMetabolites are key tounderstanding health effects of wine polyphenolicsrdquo Journal ofNutrition vol 139 no 9 pp 1324Sndash1831S 2009
[3] A Ortega-Regules I Romero-Cascales J M Lopez-Roca J MRos-Garcıa and E Gomez-Plaza ldquoAnthocyanin fingerprint ofgrapes environmental and genetic variationsrdquo Journal of theScience of Food and Agriculture vol 86 no 10 pp 1460ndash14672006
[4] V Cheynier and J Rigaud ldquoHPLC separation and characteri-zation of flavonols in the skins of Vitis vinifera var CinsaultrdquoAmerican Journal of Enology and Viticulture vol 37 pp 248ndash252 1986
[5] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero andI Hermosın-Gutierrez ldquoFlavonol profiles of Vitis vinifera redgrapes and their single-cultivar winesrdquo Journal of Agriculturaland Food Chemistry vol 55 no 3 pp 992ndash1002 2007
[6] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero M VGomez A H Velders and I Hermosın-Gutierrez ldquoFlavonol 3-O-glycosides series of Vitis vinifera Cv Petit Verdot red winegrapesrdquo Journal of Agricultural and Food Chemistry vol 57 no1 pp 209ndash219 2009
[7] P Sarni H Fulcrand V Souillol JM Souquet andV CheynierldquoMechanisms of anthocyanin degradation in grape must likemodel solutionsrdquo Journal of the Science of Food and Agriculturevol 69 no 3 pp 385ndash391 1995
[8] C Malien-Aubert O Dangles andM J Amiot ldquoColor stabilityof commercial anthocyanin-based extracts in relation to thephenolic composition Protective effects by intra- and inter-molecular copigmentationrdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 170ndash176 2001
[9] H Chang H C Eun and S C Hyang ldquoQuantitative Structure-Activity Relationship (QSAR) of antioxidative anthocyanidinsand their glycosidesrdquo Food Science and Biotechnology vol 17 no3 pp 501ndash507 2008
[10] Z Liang C Yang J Yang et al ldquoInheritance of anthocyaninsin berries of Vitis vinifera grapesrdquo Euphytica vol 167 no 1 pp113ndash125 2009
[11] A Bayo-Canha J I Fernandez-Fernandez A Martınez-Cutillas and L Ruiz-Garcıa ldquoPhenotypic segregation andrelationships of agronomic traits in Monastrell times Syrah winegrape progenyrdquo Euphytica vol 186 pp 393ndash407 2012
[12] A F Adam-Blondon C Roux D Claux G Butterlin DMerdinoglu and P This ldquoMapping 245 SSR markers on theVitis vinifera genome a tool for grape geneticsrdquoTheoretical andApplied Genetics vol 109 no 5 pp 1017ndash1027 2004
[13] A R Walker E Lee J Bogs D A J McDavid M R Thomasand S P Robinson ldquoWhite grapes arose through the mutationof two similar and adjacent regulatory genesrdquo Plant Journal vol49 no 5 pp 772ndash785 2007
[14] P K Boss C Davies and S P Robinson ldquoAnalysis of theexpression of anthocyanin pathway genes in developing Vitis
vinifera L cv Shiraz grape berries and the implications forpathway regulationrdquo Plant Physiology vol 111 no 4 pp 1059ndash1066 1996
[15] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002
[16] D Lijavetzky L Ruiz-Garcıa J A Cabezas et al ldquoMoleculargenetics of berry colour variation in table graperdquo MolecularGenetics and Genomics vol 276 no 5 pp 427ndash435 2006
[17] M C de Vicente and S D Tanksley ldquoQTL analysis of transgres-sive segregation in an interspecific tomato crossrdquo Genetics vol134 no 2 pp 585ndash596 1993
[18] L H Rieseberg M A Archer and R K Wayne ldquoTransgressivesegregation adaptation and speciationrdquoHeredity vol 83 no 4pp 363ndash372 1999
[19] Z Ju C Liu Y Yuan YWang andG Liu ldquoColoration potentialanthocyanin accumulation and enzyme activity in fruit ofcommercial apple cultivars and their F1 progenyrdquo ScientiaHorticulturae vol 79 no 1-2 pp 39ndash50 1999
[20] A Hernandez-Jimenez E Gomez-Plaza A Martınez-Cutillasand J A Kennedy ldquoGrape skin and seed proanthocyanidinsfrom Monastrell times Syrah grapesrdquo Journal of Agricultural andFood Chemistry vol 57 no 22 pp 10798ndash10803 2009
[21] H Kobayashi S Suzuki F Tanzawa andT Takayanagispi ldquoLowexpression of flavonoid 3rsquo5rsquo-hydroxylase (F3rsquo5rsquoH) associatedwith cyanidin-based anthocyanins in grape leafrdquo AmericanJournal of Enology and Viticulture vol 60 no 3 pp 362ndash3672009
[22] E Gomez-Plaza R Gil-Munoz A Hernandez-Jimenez JM Lopez-Roca A Ortega-Regules and A Martınez-CutillasldquoStudies on the anthocyanin profile ofVitis vinifera intraspecifichybrids (Monastrell times Cabernet Sauvignon)rdquo European FoodResearch and Technology vol 227 no 2 pp 479ndash484 2008
[23] A Azuma S Kobayashi N Mitani et al ldquoGenomic and geneticanalysis ofMyb-related genes that regulate anthocyanin biosyn-thesis in grape berry skinrdquoTheoretical and Applied Genetics vol117 no 6 pp 1009ndash1019 2008
[24] A Azuma S Kobayashi H Yakushiji M Yamada N Mitaniand A Sato ldquoVvmybA1 genotype determines grape skin colorrdquoVitis vol 46 no 3 pp 154ndash155 2007
[25] FMattivi R Guzzon U VrhovsekM Stefanini and R VelascoldquoMetabolite profiling of grape flavonols and anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7692ndash7702 2006
[26] J Bogs A Ebadi D McDavid and S P Robinson ldquoIdentifi-cation of the flavonoid hydroxylases from grapevine and theirregulation dining fruit developmentrdquo Plant Physiology vol 140no 1 pp 279ndash291 2006
[27] F Quattrocchio A Baudry L Lepiniec and E GrotewoldldquoThe regulation of flavonoid biosynthesisrdquo in The Science ofFlavonoids E Grotewold Ed pp 97ndash122 Springer New YorkNY USA 2008
[28] J M Cevallos-Cevallos J I Reyes-De-Corcuera E EtxeberriaM D Danyluk and G E Rodrick ldquoMetabolomic analysis infood science a reviewrdquo Trends in Food Science and Technologyvol 20 no 11-12 pp 557ndash566 2009
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
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Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
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Quantum Chemistry
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Organic Chemistry International
ElectrochemistryInternational Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
6 Journal of Analytical Methods in Chemistry
0
1
2
3
4
5
6
7
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mtimes
S 62
Mtimes
S 3
Mon
astre
llM
timesS
4M
timesS
76M
timesS
20M
timesS
97M
timesS
66M
timesS
31M
timesS
21M
timesS
56M
timesS
27M
timesS
117
Mtimes
S 57
Mtimes
S 11
Mtimes
S 28
Mtimes
S 75
Mtimes
S 34
Mtimes
S 38
Mtimes
S 0
Mtimes
S 47
Mtimes
S 26
Mtimes
S 8
Mtimes
S 37
Mtimes
S 11
4M
timesS
71M
timesS
42Sy
rah
Mtimes
S 46
(a)
0
2
4
6
8
10
Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
Mon
astre
llM
timesCS
209
Mtimes
CS 1
58M
timesCS
27
Mtimes
CS 1
63M
timesCS
49
Mtimes
CS 1
84M
timesCS
37
Mtimes
CS 4
Mtimes
CS 9
9M
timesCS
96
Mtimes
CS 5
9M
timesCS
7M
timesCS
135
Mtimes
CS 9
1M
timesCS
26
Mtimes
CS 1
00M
timesCS
107
Mtimes
CS 5
6M
timesCS
19
Mtimes
CS 1
6M
timesCS
84
Mtimes
CS 1
36M
timesCS
DM
timesCS
55
Mtimes
CS 7
2Ca
bern
etS
Mtimes
CS 1
99M
timesCS
90
Mtimes
CS C
Mtimes
CS E
Mtimes
CS B
Mtimes
CS 3
8M
timesCS
80
Mtimes
CS A
(b)
0
2
4
6
8
10
12
14
16
Mtimes
B 66
Mon
astre
ll
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 12
1
Mtimes
B 12
2
Mtimes
B 55
Mtimes
B 84
Mtimes
B 69
Mtimes
B 75
Mtimes
B 10
9
Mtimes
B 70
Mtimes
B 94
Barb
era
Mtimes
B 12
0Trih
ydro
xylat
edd
ihyd
roxy
late
d an
thoc
yani
ns
(c)
Figure 3 Trihydroxylateddihydroxylated anthocyanins ratios in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) andMonastrell times Barbera (c) hybrid grapes
Journal of Analytical Methods in Chemistry 7
Table 2 Mean values of the flavonol profile and total flavonolcontent (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids (119899 = 3)
monohydr dihydr trihydrox TotalMonastrell
Mean 109 707 184 056Syrah
Mean 134 442 424 073CS
Mean 147 446 407 019Barbera
Mean 43 533 424 049Red hybrids
Mon times SyMean 104 524 372 073Minimum 53 363 165 018Maximum 160 726 550 183
Mon times CSMean 113 511 376 037Minimum 50 352 203 011Maximum 209 650 583 078
Mon times BarMean 94 619 287 042Minimum 47 429 87 012Maximum 136 839 434 139
White hybridsMon times Sy
Mean 134 841 24 035Minimum 41 746 06 008Maximum 237 947 72 128
Mon times CSMean 200 748 51 008Minimum 168 689 09 002Maximum 259 784 127 018
monohydrox percentage of monohydroxylated flavonols dihydroxpercentage of dihydroxylated flavonols trihydrox percentage of trihy-droxylated flavonols Total total flavonol content (mg per g of skin)
and trihydroxylated (myricetin laricitrin and syringetin)flavonol glycosides (glucosides glucoronides and smallquantities of galactosides) In red grapes the monohydrox-ylated flavonols represented the lowest percentage especiallyin Barbera grapes (43) Monastrell grapes presented a veryhigh percentage of dihydroxylated flavonols (707) muchhigher than in the other varieties and therefore a lowerpercentage of trihydroxylated flavonols whereas BarberaSyrah and Cabernet Sauvignon grapes reached a percentageof trihydroxylated flavonols of around 45ndash50 This factindicates lower F3101584051015840H activity in Monastrell grapes as alsoobserved for the anthocyanins
As regards the flavonol content Cabernet Sauvignongrapes showed low concentrations of flavonols and Monas-trell and Syrah much higher concentrations while the highestvalue was found in red grapes from a hybrid plant fromMonastrell times Syrah (Table 2 Figure 4) reaching values as
high as 183mgg of skin In the hybrids bearing white grapesa lower concentration of flavonols was measured comparedwith that of the hybrids bearing red grapes Azuma et al[23 24] stated that Myb genes besides the regulation of theUFGT expression appear to enhance the expression of allthe genes involved in the anthocyanin biosynthesis pathwaybecause the transcription of all anthocyanin biosynthesisgenes appears to be slightly activated which would explainthe higher concentration of flavonols in red grapes Also inthese white skinned grapes trihydroxylated flavonols werebarely presentMattivi et al [25] studying the flavonol profileof several grape varieties did not detect trihydroxylatedflavonols in white grapes Bogs et al [26] did not findsignificant expression of F3101584051015840H and UFGT in white grapevarieties which suggests a related regulation of F3101584051015840H andUFGT during berry ripening and justifies the almost nullpresence of trihydroxylated flavonols in white grapes F3101584051015840Hwas detected in white grapes var Chardonnay but prior toveraison [26] since it is needed for flavanol biosynthesiswhich seems to be controlled differently indeed no differ-ences in the percentage of trihydroxylated flavanols wereobserved between the red and white grapes arising fromthe cross of Monastrell times Syrah [20] However other studiesstated that flavonol synthase (FLS) was not upregulated whenUFGT was expressed and that the increase in flavonols inred grapes was a consequence of an increase flux through theflavonoid pathway [27]
4 Conclusions
The study of the anthocyanin and flavonol profiles of thegrapes from the hybrid plants can be useful for a targetedinformative metabolomic analysis [28] a tool for selectingpromising grapes according to their profile andor content
Seedlings with grapes presenting very high concentra-tions of anthocyanins and flavonols can be expected fromintraspecific crosses and these resulting grapes could leadto highly colored wines with increased health-related prop-erties In this way three plants arising from Monastrell timesSyrah (hybrids 8 37 and 71) presented anthocyanin andflavonol concentrations higher than 20 and 1mgg fresh skinrespectively (Figures 2 and 4) Also four plants arising fromMonastrell timesCabernet Sauvignon (hybrids 38 59 55 and 80)and two from Monastrell times Barbera (hybrid plants 120 121)showed anthocyanin values higher than 20mgg fresh skinaccompanied with high concentration of flavonols (Figures2 and 4) The hydroxylation pattern which also influenceswine color and its stability will be strongly influenced by theparentals pattern since values higher than that shown by thebest parental in this respect will be difficult to obtain Alsoin the case of the crosses between heterozygous parentals(Monastrell Cabernet Sauvignon and Syrah) hybrids bearingwhite grapes can be obtained some of them with a highconcentration of flavonols that could be of importance in thehealth properties of this fruit and its derived products suchas the wineThe information obtained in this study should behelpful for selecting parentals for breeding programs
8 Journal of Analytical Methods in Chemistry
0
500
1000
1500
2000
(120583g
g)
B-M
timesS
30B-
Mtimes
S 1
B-M
timesS
19M
timesS
28M
timesS
31B-
Mtimes
S 10
0M
timesS
20B-
Mtimes
S 59
B-M
timesS
118
B-M
timesS
9B-
Mtimes
S 14
B-M
timesS
94B-
Mtimes
S 82
B-M
timesS
15M
timesS
26M
timesS
0M
timesS
75B-
Mtimes
S 40
Mtimes
S 38
Mtimes
S 27
Mtimes
S 66
B-M
timesS
73B-
Mtimes
S 65
Mtimes
S 21
Mtimes
S 76
Mon
astre
llM
timesS
11M
timesS
3M
timesS
114
Mtimes
S 62
Mtimes
S 34
Syra
hM
timesS
56M
timesS
117
Mtimes
S 47
Mtimes
S 97
Mtimes
S 4
Mtimes
S 42
Mtimes
S 46
Mtimes
S 71
Mtimes
S 37
B-M
timesS
91M
timesS
8M
timesS
57
(a)
0
200
400
600
800
1000
(120583g
g)
B-M
timesCS
17
B-M
timesCS
13
B-M
timesCS
93
B-M
timesCS
171
B-M
timesCS
146
Mtimes
CS 9
9M
timesCS
27
Mtimes
CS D
Mtimes
CS 1
84M
timesCS
135
Mtimes
CS 4
B-M
timesCS
98
Mtimes
CS 1
07Ca
bern
etS
Mtimes
CS 9
1M
timesCS
7M
timesCS
26
Mtimes
CS 1
36M
timesCS
163
Mtimes
CS 9
6M
timesCS
84
Mtimes
CS 1
58M
timesCS
100
Mtimes
CS 1
99M
timesCS
72
Mtimes
CS E
Mtimes
CS 4
9M
timesCS
BM
timesCS
37
Mtimes
CS 1
9M
timesCS
16
Mtimes
CS 8
0M
timesCS
55
Mon
astre
llM
timesCS
38
Mtimes
CS 9
0M
timesCS
CM
timesCS
59
Mtimes
CS 5
6M
timesCS
209
Mtimes
CS A
(b)
0
200
400
600
800
1000
1200
1400
1600
Mtimes
B 55
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 66
Mtimes
B 75
Mtimes
B 10
9
Barb
era
Mtimes
B 69
Mtimes
B 12
1
Mtimes
B 12
0
Mon
astre
ll
Mtimes
B 70
Mtimes
B 84
(120583g
g)
Mtimes
B 94
Mtimes
B 12
2
(c)
Figure 4 Concentration of flavonols in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c) hybridgrapes (yellow bars indicate white grape bearing hybrids)
Journal of Analytical Methods in Chemistry 9
Acknowledgment
This work was made possible by financial assistance of theMinisterio de Ciencia e Innovacion Project AGL2006-11019
References
[1] F Alen-Ruiz M S Garcıa-Falcon M C Perez-Lamela EMartınez-Carballo and J Simal-Gandara ldquoInfluence of majorpolyphenols on antioxidant activity in Mencia and Brancellaored grapesrdquo Food Chem vol 113 pp 53ndash60 2008
[2] S C Forester and A L Waterhouse ldquoMetabolites are key tounderstanding health effects of wine polyphenolicsrdquo Journal ofNutrition vol 139 no 9 pp 1324Sndash1831S 2009
[3] A Ortega-Regules I Romero-Cascales J M Lopez-Roca J MRos-Garcıa and E Gomez-Plaza ldquoAnthocyanin fingerprint ofgrapes environmental and genetic variationsrdquo Journal of theScience of Food and Agriculture vol 86 no 10 pp 1460ndash14672006
[4] V Cheynier and J Rigaud ldquoHPLC separation and characteri-zation of flavonols in the skins of Vitis vinifera var CinsaultrdquoAmerican Journal of Enology and Viticulture vol 37 pp 248ndash252 1986
[5] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero andI Hermosın-Gutierrez ldquoFlavonol profiles of Vitis vinifera redgrapes and their single-cultivar winesrdquo Journal of Agriculturaland Food Chemistry vol 55 no 3 pp 992ndash1002 2007
[6] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero M VGomez A H Velders and I Hermosın-Gutierrez ldquoFlavonol 3-O-glycosides series of Vitis vinifera Cv Petit Verdot red winegrapesrdquo Journal of Agricultural and Food Chemistry vol 57 no1 pp 209ndash219 2009
[7] P Sarni H Fulcrand V Souillol JM Souquet andV CheynierldquoMechanisms of anthocyanin degradation in grape must likemodel solutionsrdquo Journal of the Science of Food and Agriculturevol 69 no 3 pp 385ndash391 1995
[8] C Malien-Aubert O Dangles andM J Amiot ldquoColor stabilityof commercial anthocyanin-based extracts in relation to thephenolic composition Protective effects by intra- and inter-molecular copigmentationrdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 170ndash176 2001
[9] H Chang H C Eun and S C Hyang ldquoQuantitative Structure-Activity Relationship (QSAR) of antioxidative anthocyanidinsand their glycosidesrdquo Food Science and Biotechnology vol 17 no3 pp 501ndash507 2008
[10] Z Liang C Yang J Yang et al ldquoInheritance of anthocyaninsin berries of Vitis vinifera grapesrdquo Euphytica vol 167 no 1 pp113ndash125 2009
[11] A Bayo-Canha J I Fernandez-Fernandez A Martınez-Cutillas and L Ruiz-Garcıa ldquoPhenotypic segregation andrelationships of agronomic traits in Monastrell times Syrah winegrape progenyrdquo Euphytica vol 186 pp 393ndash407 2012
[12] A F Adam-Blondon C Roux D Claux G Butterlin DMerdinoglu and P This ldquoMapping 245 SSR markers on theVitis vinifera genome a tool for grape geneticsrdquoTheoretical andApplied Genetics vol 109 no 5 pp 1017ndash1027 2004
[13] A R Walker E Lee J Bogs D A J McDavid M R Thomasand S P Robinson ldquoWhite grapes arose through the mutationof two similar and adjacent regulatory genesrdquo Plant Journal vol49 no 5 pp 772ndash785 2007
[14] P K Boss C Davies and S P Robinson ldquoAnalysis of theexpression of anthocyanin pathway genes in developing Vitis
vinifera L cv Shiraz grape berries and the implications forpathway regulationrdquo Plant Physiology vol 111 no 4 pp 1059ndash1066 1996
[15] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002
[16] D Lijavetzky L Ruiz-Garcıa J A Cabezas et al ldquoMoleculargenetics of berry colour variation in table graperdquo MolecularGenetics and Genomics vol 276 no 5 pp 427ndash435 2006
[17] M C de Vicente and S D Tanksley ldquoQTL analysis of transgres-sive segregation in an interspecific tomato crossrdquo Genetics vol134 no 2 pp 585ndash596 1993
[18] L H Rieseberg M A Archer and R K Wayne ldquoTransgressivesegregation adaptation and speciationrdquoHeredity vol 83 no 4pp 363ndash372 1999
[19] Z Ju C Liu Y Yuan YWang andG Liu ldquoColoration potentialanthocyanin accumulation and enzyme activity in fruit ofcommercial apple cultivars and their F1 progenyrdquo ScientiaHorticulturae vol 79 no 1-2 pp 39ndash50 1999
[20] A Hernandez-Jimenez E Gomez-Plaza A Martınez-Cutillasand J A Kennedy ldquoGrape skin and seed proanthocyanidinsfrom Monastrell times Syrah grapesrdquo Journal of Agricultural andFood Chemistry vol 57 no 22 pp 10798ndash10803 2009
[21] H Kobayashi S Suzuki F Tanzawa andT Takayanagispi ldquoLowexpression of flavonoid 3rsquo5rsquo-hydroxylase (F3rsquo5rsquoH) associatedwith cyanidin-based anthocyanins in grape leafrdquo AmericanJournal of Enology and Viticulture vol 60 no 3 pp 362ndash3672009
[22] E Gomez-Plaza R Gil-Munoz A Hernandez-Jimenez JM Lopez-Roca A Ortega-Regules and A Martınez-CutillasldquoStudies on the anthocyanin profile ofVitis vinifera intraspecifichybrids (Monastrell times Cabernet Sauvignon)rdquo European FoodResearch and Technology vol 227 no 2 pp 479ndash484 2008
[23] A Azuma S Kobayashi N Mitani et al ldquoGenomic and geneticanalysis ofMyb-related genes that regulate anthocyanin biosyn-thesis in grape berry skinrdquoTheoretical and Applied Genetics vol117 no 6 pp 1009ndash1019 2008
[24] A Azuma S Kobayashi H Yakushiji M Yamada N Mitaniand A Sato ldquoVvmybA1 genotype determines grape skin colorrdquoVitis vol 46 no 3 pp 154ndash155 2007
[25] FMattivi R Guzzon U VrhovsekM Stefanini and R VelascoldquoMetabolite profiling of grape flavonols and anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7692ndash7702 2006
[26] J Bogs A Ebadi D McDavid and S P Robinson ldquoIdentifi-cation of the flavonoid hydroxylases from grapevine and theirregulation dining fruit developmentrdquo Plant Physiology vol 140no 1 pp 279ndash291 2006
[27] F Quattrocchio A Baudry L Lepiniec and E GrotewoldldquoThe regulation of flavonoid biosynthesisrdquo in The Science ofFlavonoids E Grotewold Ed pp 97ndash122 Springer New YorkNY USA 2008
[28] J M Cevallos-Cevallos J I Reyes-De-Corcuera E EtxeberriaM D Danyluk and G E Rodrick ldquoMetabolomic analysis infood science a reviewrdquo Trends in Food Science and Technologyvol 20 no 11-12 pp 557ndash566 2009
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Analytical Methods in Chemistry 7
Table 2 Mean values of the flavonol profile and total flavonolcontent (mgg fresh skin) in Monastrell Syrah and CabernetSauvignon grapes and their hybrids (119899 = 3)
monohydr dihydr trihydrox TotalMonastrell
Mean 109 707 184 056Syrah
Mean 134 442 424 073CS
Mean 147 446 407 019Barbera
Mean 43 533 424 049Red hybrids
Mon times SyMean 104 524 372 073Minimum 53 363 165 018Maximum 160 726 550 183
Mon times CSMean 113 511 376 037Minimum 50 352 203 011Maximum 209 650 583 078
Mon times BarMean 94 619 287 042Minimum 47 429 87 012Maximum 136 839 434 139
White hybridsMon times Sy
Mean 134 841 24 035Minimum 41 746 06 008Maximum 237 947 72 128
Mon times CSMean 200 748 51 008Minimum 168 689 09 002Maximum 259 784 127 018
monohydrox percentage of monohydroxylated flavonols dihydroxpercentage of dihydroxylated flavonols trihydrox percentage of trihy-droxylated flavonols Total total flavonol content (mg per g of skin)
and trihydroxylated (myricetin laricitrin and syringetin)flavonol glycosides (glucosides glucoronides and smallquantities of galactosides) In red grapes the monohydrox-ylated flavonols represented the lowest percentage especiallyin Barbera grapes (43) Monastrell grapes presented a veryhigh percentage of dihydroxylated flavonols (707) muchhigher than in the other varieties and therefore a lowerpercentage of trihydroxylated flavonols whereas BarberaSyrah and Cabernet Sauvignon grapes reached a percentageof trihydroxylated flavonols of around 45ndash50 This factindicates lower F3101584051015840H activity in Monastrell grapes as alsoobserved for the anthocyanins
As regards the flavonol content Cabernet Sauvignongrapes showed low concentrations of flavonols and Monas-trell and Syrah much higher concentrations while the highestvalue was found in red grapes from a hybrid plant fromMonastrell times Syrah (Table 2 Figure 4) reaching values as
high as 183mgg of skin In the hybrids bearing white grapesa lower concentration of flavonols was measured comparedwith that of the hybrids bearing red grapes Azuma et al[23 24] stated that Myb genes besides the regulation of theUFGT expression appear to enhance the expression of allthe genes involved in the anthocyanin biosynthesis pathwaybecause the transcription of all anthocyanin biosynthesisgenes appears to be slightly activated which would explainthe higher concentration of flavonols in red grapes Also inthese white skinned grapes trihydroxylated flavonols werebarely presentMattivi et al [25] studying the flavonol profileof several grape varieties did not detect trihydroxylatedflavonols in white grapes Bogs et al [26] did not findsignificant expression of F3101584051015840H and UFGT in white grapevarieties which suggests a related regulation of F3101584051015840H andUFGT during berry ripening and justifies the almost nullpresence of trihydroxylated flavonols in white grapes F3101584051015840Hwas detected in white grapes var Chardonnay but prior toveraison [26] since it is needed for flavanol biosynthesiswhich seems to be controlled differently indeed no differ-ences in the percentage of trihydroxylated flavanols wereobserved between the red and white grapes arising fromthe cross of Monastrell times Syrah [20] However other studiesstated that flavonol synthase (FLS) was not upregulated whenUFGT was expressed and that the increase in flavonols inred grapes was a consequence of an increase flux through theflavonoid pathway [27]
4 Conclusions
The study of the anthocyanin and flavonol profiles of thegrapes from the hybrid plants can be useful for a targetedinformative metabolomic analysis [28] a tool for selectingpromising grapes according to their profile andor content
Seedlings with grapes presenting very high concentra-tions of anthocyanins and flavonols can be expected fromintraspecific crosses and these resulting grapes could leadto highly colored wines with increased health-related prop-erties In this way three plants arising from Monastrell timesSyrah (hybrids 8 37 and 71) presented anthocyanin andflavonol concentrations higher than 20 and 1mgg fresh skinrespectively (Figures 2 and 4) Also four plants arising fromMonastrell timesCabernet Sauvignon (hybrids 38 59 55 and 80)and two from Monastrell times Barbera (hybrid plants 120 121)showed anthocyanin values higher than 20mgg fresh skinaccompanied with high concentration of flavonols (Figures2 and 4) The hydroxylation pattern which also influenceswine color and its stability will be strongly influenced by theparentals pattern since values higher than that shown by thebest parental in this respect will be difficult to obtain Alsoin the case of the crosses between heterozygous parentals(Monastrell Cabernet Sauvignon and Syrah) hybrids bearingwhite grapes can be obtained some of them with a highconcentration of flavonols that could be of importance in thehealth properties of this fruit and its derived products suchas the wineThe information obtained in this study should behelpful for selecting parentals for breeding programs
8 Journal of Analytical Methods in Chemistry
0
500
1000
1500
2000
(120583g
g)
B-M
timesS
30B-
Mtimes
S 1
B-M
timesS
19M
timesS
28M
timesS
31B-
Mtimes
S 10
0M
timesS
20B-
Mtimes
S 59
B-M
timesS
118
B-M
timesS
9B-
Mtimes
S 14
B-M
timesS
94B-
Mtimes
S 82
B-M
timesS
15M
timesS
26M
timesS
0M
timesS
75B-
Mtimes
S 40
Mtimes
S 38
Mtimes
S 27
Mtimes
S 66
B-M
timesS
73B-
Mtimes
S 65
Mtimes
S 21
Mtimes
S 76
Mon
astre
llM
timesS
11M
timesS
3M
timesS
114
Mtimes
S 62
Mtimes
S 34
Syra
hM
timesS
56M
timesS
117
Mtimes
S 47
Mtimes
S 97
Mtimes
S 4
Mtimes
S 42
Mtimes
S 46
Mtimes
S 71
Mtimes
S 37
B-M
timesS
91M
timesS
8M
timesS
57
(a)
0
200
400
600
800
1000
(120583g
g)
B-M
timesCS
17
B-M
timesCS
13
B-M
timesCS
93
B-M
timesCS
171
B-M
timesCS
146
Mtimes
CS 9
9M
timesCS
27
Mtimes
CS D
Mtimes
CS 1
84M
timesCS
135
Mtimes
CS 4
B-M
timesCS
98
Mtimes
CS 1
07Ca
bern
etS
Mtimes
CS 9
1M
timesCS
7M
timesCS
26
Mtimes
CS 1
36M
timesCS
163
Mtimes
CS 9
6M
timesCS
84
Mtimes
CS 1
58M
timesCS
100
Mtimes
CS 1
99M
timesCS
72
Mtimes
CS E
Mtimes
CS 4
9M
timesCS
BM
timesCS
37
Mtimes
CS 1
9M
timesCS
16
Mtimes
CS 8
0M
timesCS
55
Mon
astre
llM
timesCS
38
Mtimes
CS 9
0M
timesCS
CM
timesCS
59
Mtimes
CS 5
6M
timesCS
209
Mtimes
CS A
(b)
0
200
400
600
800
1000
1200
1400
1600
Mtimes
B 55
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 66
Mtimes
B 75
Mtimes
B 10
9
Barb
era
Mtimes
B 69
Mtimes
B 12
1
Mtimes
B 12
0
Mon
astre
ll
Mtimes
B 70
Mtimes
B 84
(120583g
g)
Mtimes
B 94
Mtimes
B 12
2
(c)
Figure 4 Concentration of flavonols in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c) hybridgrapes (yellow bars indicate white grape bearing hybrids)
Journal of Analytical Methods in Chemistry 9
Acknowledgment
This work was made possible by financial assistance of theMinisterio de Ciencia e Innovacion Project AGL2006-11019
References
[1] F Alen-Ruiz M S Garcıa-Falcon M C Perez-Lamela EMartınez-Carballo and J Simal-Gandara ldquoInfluence of majorpolyphenols on antioxidant activity in Mencia and Brancellaored grapesrdquo Food Chem vol 113 pp 53ndash60 2008
[2] S C Forester and A L Waterhouse ldquoMetabolites are key tounderstanding health effects of wine polyphenolicsrdquo Journal ofNutrition vol 139 no 9 pp 1324Sndash1831S 2009
[3] A Ortega-Regules I Romero-Cascales J M Lopez-Roca J MRos-Garcıa and E Gomez-Plaza ldquoAnthocyanin fingerprint ofgrapes environmental and genetic variationsrdquo Journal of theScience of Food and Agriculture vol 86 no 10 pp 1460ndash14672006
[4] V Cheynier and J Rigaud ldquoHPLC separation and characteri-zation of flavonols in the skins of Vitis vinifera var CinsaultrdquoAmerican Journal of Enology and Viticulture vol 37 pp 248ndash252 1986
[5] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero andI Hermosın-Gutierrez ldquoFlavonol profiles of Vitis vinifera redgrapes and their single-cultivar winesrdquo Journal of Agriculturaland Food Chemistry vol 55 no 3 pp 992ndash1002 2007
[6] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero M VGomez A H Velders and I Hermosın-Gutierrez ldquoFlavonol 3-O-glycosides series of Vitis vinifera Cv Petit Verdot red winegrapesrdquo Journal of Agricultural and Food Chemistry vol 57 no1 pp 209ndash219 2009
[7] P Sarni H Fulcrand V Souillol JM Souquet andV CheynierldquoMechanisms of anthocyanin degradation in grape must likemodel solutionsrdquo Journal of the Science of Food and Agriculturevol 69 no 3 pp 385ndash391 1995
[8] C Malien-Aubert O Dangles andM J Amiot ldquoColor stabilityof commercial anthocyanin-based extracts in relation to thephenolic composition Protective effects by intra- and inter-molecular copigmentationrdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 170ndash176 2001
[9] H Chang H C Eun and S C Hyang ldquoQuantitative Structure-Activity Relationship (QSAR) of antioxidative anthocyanidinsand their glycosidesrdquo Food Science and Biotechnology vol 17 no3 pp 501ndash507 2008
[10] Z Liang C Yang J Yang et al ldquoInheritance of anthocyaninsin berries of Vitis vinifera grapesrdquo Euphytica vol 167 no 1 pp113ndash125 2009
[11] A Bayo-Canha J I Fernandez-Fernandez A Martınez-Cutillas and L Ruiz-Garcıa ldquoPhenotypic segregation andrelationships of agronomic traits in Monastrell times Syrah winegrape progenyrdquo Euphytica vol 186 pp 393ndash407 2012
[12] A F Adam-Blondon C Roux D Claux G Butterlin DMerdinoglu and P This ldquoMapping 245 SSR markers on theVitis vinifera genome a tool for grape geneticsrdquoTheoretical andApplied Genetics vol 109 no 5 pp 1017ndash1027 2004
[13] A R Walker E Lee J Bogs D A J McDavid M R Thomasand S P Robinson ldquoWhite grapes arose through the mutationof two similar and adjacent regulatory genesrdquo Plant Journal vol49 no 5 pp 772ndash785 2007
[14] P K Boss C Davies and S P Robinson ldquoAnalysis of theexpression of anthocyanin pathway genes in developing Vitis
vinifera L cv Shiraz grape berries and the implications forpathway regulationrdquo Plant Physiology vol 111 no 4 pp 1059ndash1066 1996
[15] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002
[16] D Lijavetzky L Ruiz-Garcıa J A Cabezas et al ldquoMoleculargenetics of berry colour variation in table graperdquo MolecularGenetics and Genomics vol 276 no 5 pp 427ndash435 2006
[17] M C de Vicente and S D Tanksley ldquoQTL analysis of transgres-sive segregation in an interspecific tomato crossrdquo Genetics vol134 no 2 pp 585ndash596 1993
[18] L H Rieseberg M A Archer and R K Wayne ldquoTransgressivesegregation adaptation and speciationrdquoHeredity vol 83 no 4pp 363ndash372 1999
[19] Z Ju C Liu Y Yuan YWang andG Liu ldquoColoration potentialanthocyanin accumulation and enzyme activity in fruit ofcommercial apple cultivars and their F1 progenyrdquo ScientiaHorticulturae vol 79 no 1-2 pp 39ndash50 1999
[20] A Hernandez-Jimenez E Gomez-Plaza A Martınez-Cutillasand J A Kennedy ldquoGrape skin and seed proanthocyanidinsfrom Monastrell times Syrah grapesrdquo Journal of Agricultural andFood Chemistry vol 57 no 22 pp 10798ndash10803 2009
[21] H Kobayashi S Suzuki F Tanzawa andT Takayanagispi ldquoLowexpression of flavonoid 3rsquo5rsquo-hydroxylase (F3rsquo5rsquoH) associatedwith cyanidin-based anthocyanins in grape leafrdquo AmericanJournal of Enology and Viticulture vol 60 no 3 pp 362ndash3672009
[22] E Gomez-Plaza R Gil-Munoz A Hernandez-Jimenez JM Lopez-Roca A Ortega-Regules and A Martınez-CutillasldquoStudies on the anthocyanin profile ofVitis vinifera intraspecifichybrids (Monastrell times Cabernet Sauvignon)rdquo European FoodResearch and Technology vol 227 no 2 pp 479ndash484 2008
[23] A Azuma S Kobayashi N Mitani et al ldquoGenomic and geneticanalysis ofMyb-related genes that regulate anthocyanin biosyn-thesis in grape berry skinrdquoTheoretical and Applied Genetics vol117 no 6 pp 1009ndash1019 2008
[24] A Azuma S Kobayashi H Yakushiji M Yamada N Mitaniand A Sato ldquoVvmybA1 genotype determines grape skin colorrdquoVitis vol 46 no 3 pp 154ndash155 2007
[25] FMattivi R Guzzon U VrhovsekM Stefanini and R VelascoldquoMetabolite profiling of grape flavonols and anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7692ndash7702 2006
[26] J Bogs A Ebadi D McDavid and S P Robinson ldquoIdentifi-cation of the flavonoid hydroxylases from grapevine and theirregulation dining fruit developmentrdquo Plant Physiology vol 140no 1 pp 279ndash291 2006
[27] F Quattrocchio A Baudry L Lepiniec and E GrotewoldldquoThe regulation of flavonoid biosynthesisrdquo in The Science ofFlavonoids E Grotewold Ed pp 97ndash122 Springer New YorkNY USA 2008
[28] J M Cevallos-Cevallos J I Reyes-De-Corcuera E EtxeberriaM D Danyluk and G E Rodrick ldquoMetabolomic analysis infood science a reviewrdquo Trends in Food Science and Technologyvol 20 no 11-12 pp 557ndash566 2009
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
8 Journal of Analytical Methods in Chemistry
0
500
1000
1500
2000
(120583g
g)
B-M
timesS
30B-
Mtimes
S 1
B-M
timesS
19M
timesS
28M
timesS
31B-
Mtimes
S 10
0M
timesS
20B-
Mtimes
S 59
B-M
timesS
118
B-M
timesS
9B-
Mtimes
S 14
B-M
timesS
94B-
Mtimes
S 82
B-M
timesS
15M
timesS
26M
timesS
0M
timesS
75B-
Mtimes
S 40
Mtimes
S 38
Mtimes
S 27
Mtimes
S 66
B-M
timesS
73B-
Mtimes
S 65
Mtimes
S 21
Mtimes
S 76
Mon
astre
llM
timesS
11M
timesS
3M
timesS
114
Mtimes
S 62
Mtimes
S 34
Syra
hM
timesS
56M
timesS
117
Mtimes
S 47
Mtimes
S 97
Mtimes
S 4
Mtimes
S 42
Mtimes
S 46
Mtimes
S 71
Mtimes
S 37
B-M
timesS
91M
timesS
8M
timesS
57
(a)
0
200
400
600
800
1000
(120583g
g)
B-M
timesCS
17
B-M
timesCS
13
B-M
timesCS
93
B-M
timesCS
171
B-M
timesCS
146
Mtimes
CS 9
9M
timesCS
27
Mtimes
CS D
Mtimes
CS 1
84M
timesCS
135
Mtimes
CS 4
B-M
timesCS
98
Mtimes
CS 1
07Ca
bern
etS
Mtimes
CS 9
1M
timesCS
7M
timesCS
26
Mtimes
CS 1
36M
timesCS
163
Mtimes
CS 9
6M
timesCS
84
Mtimes
CS 1
58M
timesCS
100
Mtimes
CS 1
99M
timesCS
72
Mtimes
CS E
Mtimes
CS 4
9M
timesCS
BM
timesCS
37
Mtimes
CS 1
9M
timesCS
16
Mtimes
CS 8
0M
timesCS
55
Mon
astre
llM
timesCS
38
Mtimes
CS 9
0M
timesCS
CM
timesCS
59
Mtimes
CS 5
6M
timesCS
209
Mtimes
CS A
(b)
0
200
400
600
800
1000
1200
1400
1600
Mtimes
B 55
Mtimes
B 10
1
Mtimes
B 11
3
Mtimes
B 66
Mtimes
B 75
Mtimes
B 10
9
Barb
era
Mtimes
B 69
Mtimes
B 12
1
Mtimes
B 12
0
Mon
astre
ll
Mtimes
B 70
Mtimes
B 84
(120583g
g)
Mtimes
B 94
Mtimes
B 12
2
(c)
Figure 4 Concentration of flavonols in Monastrell times Syrah (a) Monastrell times Cabernet Sauvignon (b) and Monastrell times Barbera (c) hybridgrapes (yellow bars indicate white grape bearing hybrids)
Journal of Analytical Methods in Chemistry 9
Acknowledgment
This work was made possible by financial assistance of theMinisterio de Ciencia e Innovacion Project AGL2006-11019
References
[1] F Alen-Ruiz M S Garcıa-Falcon M C Perez-Lamela EMartınez-Carballo and J Simal-Gandara ldquoInfluence of majorpolyphenols on antioxidant activity in Mencia and Brancellaored grapesrdquo Food Chem vol 113 pp 53ndash60 2008
[2] S C Forester and A L Waterhouse ldquoMetabolites are key tounderstanding health effects of wine polyphenolicsrdquo Journal ofNutrition vol 139 no 9 pp 1324Sndash1831S 2009
[3] A Ortega-Regules I Romero-Cascales J M Lopez-Roca J MRos-Garcıa and E Gomez-Plaza ldquoAnthocyanin fingerprint ofgrapes environmental and genetic variationsrdquo Journal of theScience of Food and Agriculture vol 86 no 10 pp 1460ndash14672006
[4] V Cheynier and J Rigaud ldquoHPLC separation and characteri-zation of flavonols in the skins of Vitis vinifera var CinsaultrdquoAmerican Journal of Enology and Viticulture vol 37 pp 248ndash252 1986
[5] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero andI Hermosın-Gutierrez ldquoFlavonol profiles of Vitis vinifera redgrapes and their single-cultivar winesrdquo Journal of Agriculturaland Food Chemistry vol 55 no 3 pp 992ndash1002 2007
[6] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero M VGomez A H Velders and I Hermosın-Gutierrez ldquoFlavonol 3-O-glycosides series of Vitis vinifera Cv Petit Verdot red winegrapesrdquo Journal of Agricultural and Food Chemistry vol 57 no1 pp 209ndash219 2009
[7] P Sarni H Fulcrand V Souillol JM Souquet andV CheynierldquoMechanisms of anthocyanin degradation in grape must likemodel solutionsrdquo Journal of the Science of Food and Agriculturevol 69 no 3 pp 385ndash391 1995
[8] C Malien-Aubert O Dangles andM J Amiot ldquoColor stabilityof commercial anthocyanin-based extracts in relation to thephenolic composition Protective effects by intra- and inter-molecular copigmentationrdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 170ndash176 2001
[9] H Chang H C Eun and S C Hyang ldquoQuantitative Structure-Activity Relationship (QSAR) of antioxidative anthocyanidinsand their glycosidesrdquo Food Science and Biotechnology vol 17 no3 pp 501ndash507 2008
[10] Z Liang C Yang J Yang et al ldquoInheritance of anthocyaninsin berries of Vitis vinifera grapesrdquo Euphytica vol 167 no 1 pp113ndash125 2009
[11] A Bayo-Canha J I Fernandez-Fernandez A Martınez-Cutillas and L Ruiz-Garcıa ldquoPhenotypic segregation andrelationships of agronomic traits in Monastrell times Syrah winegrape progenyrdquo Euphytica vol 186 pp 393ndash407 2012
[12] A F Adam-Blondon C Roux D Claux G Butterlin DMerdinoglu and P This ldquoMapping 245 SSR markers on theVitis vinifera genome a tool for grape geneticsrdquoTheoretical andApplied Genetics vol 109 no 5 pp 1017ndash1027 2004
[13] A R Walker E Lee J Bogs D A J McDavid M R Thomasand S P Robinson ldquoWhite grapes arose through the mutationof two similar and adjacent regulatory genesrdquo Plant Journal vol49 no 5 pp 772ndash785 2007
[14] P K Boss C Davies and S P Robinson ldquoAnalysis of theexpression of anthocyanin pathway genes in developing Vitis
vinifera L cv Shiraz grape berries and the implications forpathway regulationrdquo Plant Physiology vol 111 no 4 pp 1059ndash1066 1996
[15] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002
[16] D Lijavetzky L Ruiz-Garcıa J A Cabezas et al ldquoMoleculargenetics of berry colour variation in table graperdquo MolecularGenetics and Genomics vol 276 no 5 pp 427ndash435 2006
[17] M C de Vicente and S D Tanksley ldquoQTL analysis of transgres-sive segregation in an interspecific tomato crossrdquo Genetics vol134 no 2 pp 585ndash596 1993
[18] L H Rieseberg M A Archer and R K Wayne ldquoTransgressivesegregation adaptation and speciationrdquoHeredity vol 83 no 4pp 363ndash372 1999
[19] Z Ju C Liu Y Yuan YWang andG Liu ldquoColoration potentialanthocyanin accumulation and enzyme activity in fruit ofcommercial apple cultivars and their F1 progenyrdquo ScientiaHorticulturae vol 79 no 1-2 pp 39ndash50 1999
[20] A Hernandez-Jimenez E Gomez-Plaza A Martınez-Cutillasand J A Kennedy ldquoGrape skin and seed proanthocyanidinsfrom Monastrell times Syrah grapesrdquo Journal of Agricultural andFood Chemistry vol 57 no 22 pp 10798ndash10803 2009
[21] H Kobayashi S Suzuki F Tanzawa andT Takayanagispi ldquoLowexpression of flavonoid 3rsquo5rsquo-hydroxylase (F3rsquo5rsquoH) associatedwith cyanidin-based anthocyanins in grape leafrdquo AmericanJournal of Enology and Viticulture vol 60 no 3 pp 362ndash3672009
[22] E Gomez-Plaza R Gil-Munoz A Hernandez-Jimenez JM Lopez-Roca A Ortega-Regules and A Martınez-CutillasldquoStudies on the anthocyanin profile ofVitis vinifera intraspecifichybrids (Monastrell times Cabernet Sauvignon)rdquo European FoodResearch and Technology vol 227 no 2 pp 479ndash484 2008
[23] A Azuma S Kobayashi N Mitani et al ldquoGenomic and geneticanalysis ofMyb-related genes that regulate anthocyanin biosyn-thesis in grape berry skinrdquoTheoretical and Applied Genetics vol117 no 6 pp 1009ndash1019 2008
[24] A Azuma S Kobayashi H Yakushiji M Yamada N Mitaniand A Sato ldquoVvmybA1 genotype determines grape skin colorrdquoVitis vol 46 no 3 pp 154ndash155 2007
[25] FMattivi R Guzzon U VrhovsekM Stefanini and R VelascoldquoMetabolite profiling of grape flavonols and anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7692ndash7702 2006
[26] J Bogs A Ebadi D McDavid and S P Robinson ldquoIdentifi-cation of the flavonoid hydroxylases from grapevine and theirregulation dining fruit developmentrdquo Plant Physiology vol 140no 1 pp 279ndash291 2006
[27] F Quattrocchio A Baudry L Lepiniec and E GrotewoldldquoThe regulation of flavonoid biosynthesisrdquo in The Science ofFlavonoids E Grotewold Ed pp 97ndash122 Springer New YorkNY USA 2008
[28] J M Cevallos-Cevallos J I Reyes-De-Corcuera E EtxeberriaM D Danyluk and G E Rodrick ldquoMetabolomic analysis infood science a reviewrdquo Trends in Food Science and Technologyvol 20 no 11-12 pp 557ndash566 2009
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Analytical Methods in Chemistry 9
Acknowledgment
This work was made possible by financial assistance of theMinisterio de Ciencia e Innovacion Project AGL2006-11019
References
[1] F Alen-Ruiz M S Garcıa-Falcon M C Perez-Lamela EMartınez-Carballo and J Simal-Gandara ldquoInfluence of majorpolyphenols on antioxidant activity in Mencia and Brancellaored grapesrdquo Food Chem vol 113 pp 53ndash60 2008
[2] S C Forester and A L Waterhouse ldquoMetabolites are key tounderstanding health effects of wine polyphenolicsrdquo Journal ofNutrition vol 139 no 9 pp 1324Sndash1831S 2009
[3] A Ortega-Regules I Romero-Cascales J M Lopez-Roca J MRos-Garcıa and E Gomez-Plaza ldquoAnthocyanin fingerprint ofgrapes environmental and genetic variationsrdquo Journal of theScience of Food and Agriculture vol 86 no 10 pp 1460ndash14672006
[4] V Cheynier and J Rigaud ldquoHPLC separation and characteri-zation of flavonols in the skins of Vitis vinifera var CinsaultrdquoAmerican Journal of Enology and Viticulture vol 37 pp 248ndash252 1986
[5] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero andI Hermosın-Gutierrez ldquoFlavonol profiles of Vitis vinifera redgrapes and their single-cultivar winesrdquo Journal of Agriculturaland Food Chemistry vol 55 no 3 pp 992ndash1002 2007
[6] N Castillo-Munoz S Gomez-Alonso E Garcıa-Romero M VGomez A H Velders and I Hermosın-Gutierrez ldquoFlavonol 3-O-glycosides series of Vitis vinifera Cv Petit Verdot red winegrapesrdquo Journal of Agricultural and Food Chemistry vol 57 no1 pp 209ndash219 2009
[7] P Sarni H Fulcrand V Souillol JM Souquet andV CheynierldquoMechanisms of anthocyanin degradation in grape must likemodel solutionsrdquo Journal of the Science of Food and Agriculturevol 69 no 3 pp 385ndash391 1995
[8] C Malien-Aubert O Dangles andM J Amiot ldquoColor stabilityof commercial anthocyanin-based extracts in relation to thephenolic composition Protective effects by intra- and inter-molecular copigmentationrdquo Journal of Agricultural and FoodChemistry vol 49 no 1 pp 170ndash176 2001
[9] H Chang H C Eun and S C Hyang ldquoQuantitative Structure-Activity Relationship (QSAR) of antioxidative anthocyanidinsand their glycosidesrdquo Food Science and Biotechnology vol 17 no3 pp 501ndash507 2008
[10] Z Liang C Yang J Yang et al ldquoInheritance of anthocyaninsin berries of Vitis vinifera grapesrdquo Euphytica vol 167 no 1 pp113ndash125 2009
[11] A Bayo-Canha J I Fernandez-Fernandez A Martınez-Cutillas and L Ruiz-Garcıa ldquoPhenotypic segregation andrelationships of agronomic traits in Monastrell times Syrah winegrape progenyrdquo Euphytica vol 186 pp 393ndash407 2012
[12] A F Adam-Blondon C Roux D Claux G Butterlin DMerdinoglu and P This ldquoMapping 245 SSR markers on theVitis vinifera genome a tool for grape geneticsrdquoTheoretical andApplied Genetics vol 109 no 5 pp 1017ndash1027 2004
[13] A R Walker E Lee J Bogs D A J McDavid M R Thomasand S P Robinson ldquoWhite grapes arose through the mutationof two similar and adjacent regulatory genesrdquo Plant Journal vol49 no 5 pp 772ndash785 2007
[14] P K Boss C Davies and S P Robinson ldquoAnalysis of theexpression of anthocyanin pathway genes in developing Vitis
vinifera L cv Shiraz grape berries and the implications forpathway regulationrdquo Plant Physiology vol 111 no 4 pp 1059ndash1066 1996
[15] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002
[16] D Lijavetzky L Ruiz-Garcıa J A Cabezas et al ldquoMoleculargenetics of berry colour variation in table graperdquo MolecularGenetics and Genomics vol 276 no 5 pp 427ndash435 2006
[17] M C de Vicente and S D Tanksley ldquoQTL analysis of transgres-sive segregation in an interspecific tomato crossrdquo Genetics vol134 no 2 pp 585ndash596 1993
[18] L H Rieseberg M A Archer and R K Wayne ldquoTransgressivesegregation adaptation and speciationrdquoHeredity vol 83 no 4pp 363ndash372 1999
[19] Z Ju C Liu Y Yuan YWang andG Liu ldquoColoration potentialanthocyanin accumulation and enzyme activity in fruit ofcommercial apple cultivars and their F1 progenyrdquo ScientiaHorticulturae vol 79 no 1-2 pp 39ndash50 1999
[20] A Hernandez-Jimenez E Gomez-Plaza A Martınez-Cutillasand J A Kennedy ldquoGrape skin and seed proanthocyanidinsfrom Monastrell times Syrah grapesrdquo Journal of Agricultural andFood Chemistry vol 57 no 22 pp 10798ndash10803 2009
[21] H Kobayashi S Suzuki F Tanzawa andT Takayanagispi ldquoLowexpression of flavonoid 3rsquo5rsquo-hydroxylase (F3rsquo5rsquoH) associatedwith cyanidin-based anthocyanins in grape leafrdquo AmericanJournal of Enology and Viticulture vol 60 no 3 pp 362ndash3672009
[22] E Gomez-Plaza R Gil-Munoz A Hernandez-Jimenez JM Lopez-Roca A Ortega-Regules and A Martınez-CutillasldquoStudies on the anthocyanin profile ofVitis vinifera intraspecifichybrids (Monastrell times Cabernet Sauvignon)rdquo European FoodResearch and Technology vol 227 no 2 pp 479ndash484 2008
[23] A Azuma S Kobayashi N Mitani et al ldquoGenomic and geneticanalysis ofMyb-related genes that regulate anthocyanin biosyn-thesis in grape berry skinrdquoTheoretical and Applied Genetics vol117 no 6 pp 1009ndash1019 2008
[24] A Azuma S Kobayashi H Yakushiji M Yamada N Mitaniand A Sato ldquoVvmybA1 genotype determines grape skin colorrdquoVitis vol 46 no 3 pp 154ndash155 2007
[25] FMattivi R Guzzon U VrhovsekM Stefanini and R VelascoldquoMetabolite profiling of grape flavonols and anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7692ndash7702 2006
[26] J Bogs A Ebadi D McDavid and S P Robinson ldquoIdentifi-cation of the flavonoid hydroxylases from grapevine and theirregulation dining fruit developmentrdquo Plant Physiology vol 140no 1 pp 279ndash291 2006
[27] F Quattrocchio A Baudry L Lepiniec and E GrotewoldldquoThe regulation of flavonoid biosynthesisrdquo in The Science ofFlavonoids E Grotewold Ed pp 97ndash122 Springer New YorkNY USA 2008
[28] J M Cevallos-Cevallos J I Reyes-De-Corcuera E EtxeberriaM D Danyluk and G E Rodrick ldquoMetabolomic analysis infood science a reviewrdquo Trends in Food Science and Technologyvol 20 no 11-12 pp 557ndash566 2009
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
