Food Chemistry xxx (2012) xxx–xxx

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Food Chemistry

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Flavonol content and biometrical traits as a tool for the characterizationof ‘‘Cipolla di Giarratana’’: A traditional Sicilian onion landrace

Ezio Riggi a, Giovanni Avola a, Laura Siracusa b,⇑, Giuseppe Ruberto b

a Consiglio Nazionale delle Ricerche-Istituto per i Sistemi Agricoli e Forestali del Mediterraneo (CNR-ISAFOM), Str.le V. Lancia, Blocco Palma I, Zona Industriale, 95121 Catania, Italyb Consiglio Nazionale delle Ricerche-Istituto di Chimica Biomolecolare (CNR-ICB), Via Paolo Gaifami 18, 95126 Catania, Italy

a r t i c l e i n f o

Article history:Available online xxxx

Keywords:Allium cepaOnion landrace‘‘Cipolla di Giarratana’’Flavonols profileChemotaxonomic markers

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.10.134

⇑ Corresponding author. Tel.: +39 0957338348; faxE-mail address: laura.siracusa@icb.cnr.it (L. Siracu

Please cite this article in press as: Riggi, E., et al.ditional Sicilian onion landrace. Food Chemistry

a b s t r a c t

‘‘Cipolla di Giarratana’’, a locally cultivated white onion landrace, is listed as an item in the ‘List of Tra-ditional Agro-food Products’ of the Italian Department for Agriculture and itemised as ‘slow food presid-ium’ by the Slow Food Foundation. Ten local accessions were investigated for their biomorphological andbiochemical characteristics in five experimental locations. High-performance liquid chromatographycoupled with diode array detection and electron spray-mass spectrometry (HPLC/DAD/ESI-MS) was usedto identify the phenolic profile and quantify phenolic content in bulbs: quercetin, quercetin 3,40 di-O-glucoside and quercetin 40-O-glucoside were detected as major components. The ‘Cipolla di Giarratana’landrace is characterised by a high bulb weight (436 g) and high diameter (11 cm). The total flavonolscontent ranged between 68 and 408 mg kg�1 bulb fresh weight in nine of the 10 collected accessions.The opportunity of considering flavonol patterns as chemotaxonomic descriptors in order to characteriseonion germplasm is also discussed.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Onion (Allium cepa L.) has been used as common food since an-cient times (1550 BC) (Lanzotti, 2006). Onion bulbs are rich in sev-eral phytonutrients recognised as important elements of theMediterranean diet (carbohydrates, vitamins and minerals) and ithas also been reported to have numerous medicinal and functionalproperties such as antimicrobial, antispasmodic, anticholesterolae-mic, anticancer, and hypotensive (Prakash, Singh, & Upadhyay,2007). The majority of these biological effects has been associatedwith the presence of particular molecules belonging both to its pri-mary and secondary metabolism. Among them, flavonoids, espe-cially flavonols, are undoubtedly the most abundant non volatilesecondary metabolites present in onion bulbs. Flavonoids haveraised huge attention in the last decade due to their role in the pre-vention of several diseases associated with the progressive oxida-tion of living organisms. As a part of the so called ‘‘defensivecharacters’’ of the plants, their profile and content depend on ge-netic factors but it is also influenced by environmental conditions(temperature and soil water content during growing season) andagronomic management (fertilisation, stage of ripening at harvest,biotic stress management), as extensively reported in recent liter-ature (Figueiredo-González et al., 2012; Li et al., 2010; Parvu, Toiu,

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: +39 0957338310.sa).

Flavonol content and biometri(2012), http://dx.doi.org/10.10

Vlase, & Parvu, 2010; Prinz, Ringl, Huefner, Pemp, & Kopp, 2007;Robles et al., 2003; Siracusa, Patanè, Avola, & Ruberto, 2012).

Among the different flavonoid classes, exclusively compoundsbelonging to the subclass of flavonols, anthocyanins, and di-hydro-flavonols have been detected in onion bulbs, with quercetin(3,30,40,5,7 pentahydroxyflavone) and its derivatives as major com-pounds detected (Rodríguez Galdón, Rodríguez Rodríguez, & DíazRomero, 2008; Slimestad, Fossen, & Vågen, 2007). Onion flavonolshave been demonstrated to be useful in varietal comparisonstudies (Lee & Mitchell, 2011; Pérez-Gregorio, García-Falcón,Simal-Gándara, Rogridues, & Almeida, 2010; Rodríguez Galdón,Peña-Méndez, Havel, Rodríguez Rodríguez, & Díaz Romero, 2010);compositional studies on onion flavonols were also carried out tomonitor the changes in product quality with different post-harvestpractises, storage conditions, light exposition and heating (Leeet al., 2008; Olsson, Gustavsson, & Vågen, 2010; Rodrigues,Pérez-Gregorio, García-Falcón, Simal-Gándara, & Almeida, 2010),cooking (Rodrigues, Pérez-Gregorio, García-Falcón, & Simal-Gándara, 2009) and to evaluate the best sanitising technologyand packaging system for onion storage (Pérez-Gregorio, García-Falcón, & Simal-Gándara, 2011a; Pérez-Gregorio, González-Barreiro,Rial-Otero, & Simal-Gándara, 2011b).

In this view, the use of onion flavonols as a differentiating toolcould support the identification of the huge amount of local land-races that could counteract the globalisation of crop productionleading to a narrow batch of grown varieties that dramaticallythreats biodiversity maintenance and trigger genetic erosion.

cal traits as a tool for the characterization of ‘‘Cipolla di Giarratana’’: A tra-16/j.foodchem.2012.10.134

2 E. Riggi et al. / Food Chemistry xxx (2012) xxx–xxx

Moreover, all these efforts for landraces characterization, in thecontext of European agriculture can be addressed to obtain protec-tion of geographical indications (PGI) or protection of designationof origin (PDO) that strongly support the economic sustainabilityfor farmers adopting typical landraces, but that can only beawarded if a product’s differential value can be clearly demon-strated (EC Regulation, 2006). To delimit geographical origin objec-tively, it is fundamental to know the importance of genetic andenvironmental factors in the phenotypes of the products that reachconsumers (Florez et al., 2009). In this scenario, a Sicilian tradi-tional onion landrace, ‘‘Cipolla di Giarratana’’, characterised bywhite large bulbs and sweet taste, and registered as Slow Food Pre-sidium, need to receive scientific attention to avoid the extinctionrisk threatening this population such as many other niche productscultivated in restricted areas. The present study was carried out toasses the flavonol profile and biomorphological traits of ‘‘Cipolla diGiarratana’’ and to evaluate its homogeneity within the local land-race and between different experimental sites.

2. Material and methods

2.1. Plant material and experimental design

The ‘‘Cipolla di Giarratana’’ landrace is actually cultivated in arestricted area involving no more then 30 farmers, growing geneticmaterial self reproduced by ten of them in confined conditions toprevent cross pollination. During March 2010, seeds of all the tenaccessions of this local landrace have been collected. A genotypesbatch including the ten accessions, and a white onion standardvariety (Tonda Musona), adopted as control, was grown in fiveLocations (L): two located in the area where this population is tra-ditionally cultivated (Giarratana, SE Sicily, 450–600 m above sealevel – L1, L3), two in different areas with similar average altitude(Palazzolo Acreide – L6 and Chiaramonte Gulfi – L5, SE Sicily, 650and 450 m a.s.l, respectively), and one in the South East coast ofSicily (Scicli – L6, 5 m a.s.l.). The same randomised blocks design,three times replicated, was field applied in each location. The cropwas transplanted in March 2011 and harvested in July, when 90%of foliage had fallen over. Typical cultural practises, adopted foronion crops in the experimental area, was applied. At harvest 15plants per plot, randomly selected, were collected. Bulbs were thencured in field for 10 days before analysis. Biometric (bulb weight,and maximum diameter), and morphological traits (position ofmaximum diameter, width of neck, shape of the whole bulb, ofthe stem end, and of the root end, according to CPVO-TP/046,2009), have been measured in each harvested bulb.

2.2. Flavonol content determination

2.2.1. ChemicalsAll solvents used in this study were high purity spectroscopic

grade solvents by Carlo Erba (Milano, Italy), formic acid wasobtained from Fluka. Pure reference standards – quercetin 3-O-glucoside, rutin (quercetin 3-O-rutinoside), keampferol andisorhamnetin were purchased from Fluka, and submitted toexhaustive LC/UV–vis–DAD/MS analysis prior to use to verify theirpurity.

2.2.2. Sample preparationBulbs (three for each replicates) were washed with tap water to

remove dirt and dried thoroughly with absorbent paper. Each bulbwas skinned (to remove dry skin), cut longitudinally into quartersand a representative sample of each quarter (50 g) was immedi-ately finely grounded with an electric blender to a homogeneouswhitish puree. Aliquots (3.5 g) of the puree were put in a 8 mL am-

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ber sample vial and a 3 mL of a acidic-alcoholic solution (5% formicacid in methanol) was added. The samples were then maintainedat room temperature (20 �C) overnight, in the dark and under vig-orous shaking (350 rpm). The resulting heterogeneous mixtures,still containing solid residues, were centrifuged (20 min at4000 rpm) and then filtered with PTFE filters (13 mm diameter,0.45 lm pore size, Pall Life Science), thus obtaining ca. 2,5 mL ofclear pale yellow solutions, ready for analyses. When required,the above mentioned analytical samples were stored for shortsperiods (one week at the most) at �20 �C under nitrogen atmo-sphere. The whole analytical batch thus consisted of 165 samples.

2.2.3. HPLC/DAD/ESI-MS analysesFor a representative number of samples (one replicate for each

thesis = 55 samples), a variable aliquots (0.8–1 mL) of the abovementioned solutions were transferred into standard laboratoryvials and brought to dryness in vacuum with a rotary evaporator(Heidolph Laborota 400). The resulting yellowish residues werethen re-dissolved in 250 lL of the original extraction solutionand submitted to qualitative analyses. Onion extracts were ana-lysed by HPLC/DAD/ESI-MS using a Waters instrument (WatersItalia S.p.A., Milano, Italy) consisting of a 1525 Binary HPLC Pump,a PDA 996 Photodiode Array Detector (DAD) and a Micromass ZQMass Analyser equipped with a ESI Z-spray source. DAD analyseswere acquired in the range between 700 and 190 nm. Total ion cur-rent (TIC) chromatograms were acquired in negative mode, using acone voltage of �20 V in the mass range between 80 and 1000 m/zunits. The other parameters used for the acquisition of the TICswere the following: capillary voltage: 2.75 kV; source tempera-ture: 150 �C; desolvation temperature: 260 �C; gas flow (L/h):400 (desolvation) and 210 (cone). Chromatographic runs were per-formed using a reverse-phase column (Ascentis C18, 250 � 4.6 mm,5 lm particle size, Supelco, Sigma Aldrich, Italy); onion polyphe-nols were eluted with the following gradient of B (acetonitrile) inA (2.5% solution of formic acid in water): t = 0 min, B = 15%;t = 5 min, B = 20%; t = 10 min, B = 30%; t = 20 min, B = 50%, then att = 25 min: B = 100%, for a total 30 min run time. The solvent flowrate was 1 mL min�1, the temperature was kept at 22 �C with a col-umn oven (Hitachi L-2300, VWR International, Milano, Italy) andthe injector volume selected was 20 lL. Data coming from theLC/MS apparatus were analysed and processed through a MassLynx v. 4.0 standard software (Waters).

2.2.4. HPLC/DAD quantitative analysesSmall aliquots (1.5 mL) of the acidic-alcoholic solutions con-

taining onion polyphenols were put in 2 mL amber conic vialsand submitted to HPLC–UV–vis–DAD high throughput analyses.Quantitative analyses were carried out on a Dionex instrumentequipped with a P580 binary high pressure pump, a PDA-100 Pho-todiode Array detector, a TCC-100 Thermostatted Column Com-partment and an ASI-100 Automated Sample Injector. Collecteddata were processed through a Chromeleon Chromatography Infor-mation Management System v. 6.70. Chromatographic analyseswere run using the same conditions (solvents, elution program,column) described in the previous paragraph. After a preliminaryanalysis of the corresponding UV–vis spectra (see previous para-graph), quantification was carried out at 350 nm for the majorityof glycosilated flavonols using a calibration curve established withrutin (quercetin 3-O-rutinoside, R2 = 0.9998), whilst aglycones(quercetin, isorhamnetin and kaempferol) plus some derivativeswhose maximum absorption wavelengths fell in the range be-tween 370 and 364 nm were quantified using quercetin as refer-ence standard (R2 = 0.9999). All analyses were carried out intriplicate; results are reported in milligramme (mg) of flavonolper kilogramme (kg) of vegetable material fresh weight (FW).

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E. Riggi et al. / Food Chemistry xxx (2012) xxx–xxx 3

2.3. Statistical analysis

As the main scope of the research was to characterise ‘‘Cipolla diGiarratana’’ landrace firstly verifying the homogeneity of theaccessions adopted by the local farmers, we initially performedAnalysis of Variance (ANOVA) according to the linear model focus-ing only on the 10 collected accessions:

Xijr ¼ mþ Giþ Ljþ BrðLjÞ þ GLijþ eijr

where Xijr is the observation of the Genotype i, in the Location j, inthe Block r; m is the overall mean; G, L, and B are the effects ofGenotype, Location and Block, respectively, and e is the residual ef-fect associated to each individual analytical value. In the adoptedmodel both Genotype and Location were treated as fixed factorsso focusing on the comparison of genotypes that represented thewhole accessions pool of the studied local landrace. For the samereason the studied genotypes were firstly limited to the local acces-sions (excluding standard variety) aiming at mining potential differ-ences between them. The block effect was considered a randomeffect.

The incidence of each factor on total observed variation hasbeen estimated as strength of association defined classical g2 andcalculated as the ratio between SSfactor and SStotal (Cohen, Cohen,West, & Aiken, 2003). When significant effects were observed,means were separated by Tukey’s HSD (Honestly Significant Differ-ence) test. When GxL significant interactions emerged, principalcomponent analysis (PCA) was used to test the principal effectsof Location and Genotypes and their combination, adopting acovariance matrix. Components with eigenvalues greater thanone were used. Correlations between traits significantly affectedby any of the factors, have finally performed. ANOVAs were per-formed with the DSAASTAT, a statistical analysis package that runsas an add-into Windows versions of Microsoft Excel (Onofri, 2007).For the PCA the statistical package StatistiXL1.5 (StatistiXL Ltd.)was applied. Correlations were performed using CoStat version6.003 (CoHort Software, Monterey, CA, USA).

3. Results and discussion

3.1. Biometrical and morphological traits

Significant differences emerged between Genotypes, Locations,but any regarding GxL interaction for biometrical traits (Table 1).This suggests that genotypes responded similarly across environ-ment sites for all the studied characteristics. Differences amongstaccessions ranged from �8% to +12% and from �6% to +4% whencomparing extreme and average values for bulb weight and maxi-mum diameter, respectively (Table 2). A wider range of variationwas observed when mean value for Locations were regarded andthe extreme significantly differing values reported in L3 and L4for bulb weight (534 and 293 g, respectively) and for maximumdiameter (11.8 and 9.4 cm, respectively) ranged between �33% to+23% and �15% to +7% when compared to overall means. The

Table 1Analysis of variance on biometrical traits of ‘‘Cipolla di Giarratana’’ bulbs.

Factors DF Bulb weight Bulb diameter

SS P SS P

Blocks 2 20449 0.4Location 4 1160987 <0.001*** 112 <0.001***

Genotype 9 111267 0.023* 14 0.017*

G � L 36 194154 0.404n.s. 24 0.414n.s.

Residual 90 504149 63

n.s. = not significant.* Significant at P 6 0.05.*** Significant at P 6 0.001.

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strength of association (classical g2) ascertained for Location mea-sured 0.58 and 0.52, respectively for bulb weight and maximumdiameter resulting 8- to 9-fold higher then what observed forGenotype (0.06 and 0.07, respectively).

No significant differences emerged for any of the morphologicalstudied traits and the shape of the landrace resulted with maxi-mum diameter located at the middle of the bulb, narrow neck,transverse medium elliptic shape, and flat ends of stem and root.Moreover, although we did not specifically measured the base col-our of dry skin, a slight difference was observed for the G5 acces-sion that showed a brighter white colour of the external skin.

3.2. Flavonols profile and content

Onion bulbs are among the richest sources of dietary flavonoids.As previously mentioned, only highly hydroxylated flavonols(flavonoids bearing an hydroxyl group in the 3-position, locatedin the B ring), some di-hydroflavonols and anthocyanins have beenreported to occur in onions, for a total number of more than 50 dif-ferent metabolites. Quercetin, kaempferol, isorhamnetin and theirvariously glycosylated derivatives are almost the sole metabolitesdetected in onion extracts; particularly quercetin is present in 14of the 25 molecules reported, with an unusual high abundance of40-O-b-glucosides (Bonaccorsi, Caristi, Gargiulli, & Leuzzi, 2005;Rodríguez Galdón et al., 2008; Slimestad et al., 2007). Quantita-tively speaking, literature reports that the total amount of flavo-nols in onion bulbs strongly depends on their colour, being redonions generally the richest in these metabolites and white onionsthe poorest (Arabbi, Genovese, & Lajolo, 2004; Gennaro et al., 2002;Lin & Tang, 2007; Rodrigues, Pérez-Gregorio, García-Falcón,Simal-Gándara, & Almeida, 2011; Slimestad et al., 2007). Particularlywhite onions, according to the recent review of Slimestad et al.(2007), showed a total flavonol content of few mg kg�1 freshweight in 13 over the 15 varieties considered. Other authors, suchas Rhodes and Price (1996), Lee et al. (2008), and Rodrigues et al.(2011) reported higher values (from ca. 50 to 186 mg kg�1 freshweight). The polar extracts from ‘‘Cipolla di Giarratana’’ bulbs wereexhaustively analysed by means of HPLC/DAD/ESI-MS; the chro-matograms obtained showed from 13 to 18 different signals, whicheluted in the range from 6 to 22 min (Fig. 1). A deep analysis of theUV–vis (in terms of spectrum shape and absorption maximum) andmass spectra (pseudomolecular ions and fragmentations) helpedus in unambiguously identifying 10 of the flavonol derivatives de-tected (Table 3); peak assignments were further confirmed bycomparison with literature data (Bonaccorsi et al., 2005; Lee &Mitchell, 2011; Lee et al., 2008) and co-injection with pure refer-ence standards. Quercetin is indeed the most represented flavonol,being present in 50% of the metabolites identified, followed byisorhamnetin and kaempferol. Notably, all the extracts contain freequercetin (aglycon) in considerably high amounts, ranging from4.1 to 177.8 mg kg�1 fresh weight. In fact, we found free quercetinas the third compound in order of abundance (Table 4), right afterquercetin 3,40 di-O-glc (ranging from 19.8 to 129.3 mg kg�1 freshweight) and quercetin 40-O-glc (from 18.7 to 116.6 mg kg�1 freshweight); in our opinion, this can be considered a characterising fea-ture of the population studied, as this metabolite is generally re-ported in literature to occur as minor component or even belowdetection limits, especially in white onions. In particular Patil, Pike,and Yoo (1995) found high content of quercetin in red and yellowonions, ranging from 54 to 286 and from 117 to 202 mg kg�1 freshweight, respectively, and virtually not-detectable amount in whiteonions (<1 mg kg�1 fresh weight); this is perfectly in agreementwith other authors (Lee et al., 2008; Perez-Gregorio et al., 2010;Rodrigues et al., 2011). When converting our results in milli-gramme of quercetin (Q) kg�1 for comparison purposes, we alsofound a reasonably high total amount of flavonols in our ‘‘Cipolla

cal traits as a tool for the characterization of ‘‘Cipolla di Giarratana’’: A tra-16/j.foodchem.2012.10.134

Table 2Mean values of the biometrical and morphological studied traits in the different genotypes and locations (see text for details).

Bulb weight (g) Bulb Ø (cm) Bulb characteristica

16 17 18 19 20

Genotype 1 389.3 b 10.3 b 2 2 8 3 22 414.1 ab 10.8 ab 2 2 8 3 34 398.2 b 10.7 ab 2 2 9 2 25 460.9 ab 11.4 a 2 2 9 2 36 437.6 ab 11.0 ab 2 3 8 3 37 449.0 ab 11.1 ab 2 2 8 3 39 455.0 ab 11.4 a 2 2 8 2 212 488.5 a 11.4 a 2 2 8 2 213 424.2 ab 10.8 ab 2 2 8 2 315 437.9 ab 11.1 ab 2 2 8 2 3

Location L1 365.9 c 10.5 b 2 2 9 2 2L3 534.4 a 11.8 a 2 3 8 3 3L4 293.0 d 9.4 c 2 3 8 3 2L5 500.9 ab 11.7 a 2 1 8 2 2L6 483.2 b 11.5 a 2 2 8 2 3

Average 435.5 11.0 2 2 8 2 3

a Bulb characteristics according to CPVO-TP/046, 2009: 16 = position of maximum diameter; 17 = width of neck; 18 = shape of the whole bulb; 19 = shape of the stem end;20 = shape of the root end. Within each studied factor, values with different letters differ for p < 0.05.

Fig. 1. Representative chromatogram of a ‘‘Cipolla di Giarratana’’ extract, visualised at 350 nm (black line) and 370 nm (grey line). Peaks 1–10 were identified as follows: 1,quercetin 3,40 di-O-glucoside; 2, isorhamentin 3,40 di-O-glucoside; 3, rutin; 4, quercetin 3-O-glucoside; 5, quercetin 40-O-glucoside; 6, kaempferol 40-O-glucoside; 7,isorhamnetin 40-O-glucoside; 8, quercetin; 9, kaempferol; 10, isorhamnetin. UV–vis and MS data of the identified signals are given in Table 3.

4 E. Riggi et al. / Food Chemistry xxx (2012) xxx–xxx

di Giarratana’’ samples (from 44 to 318 mg Q kg�1 fresh weight,average 125 mg Q kg�1 fresh weight), which is also a good resultconsidering the generally low flavonol content characterisingwhite onions, as mentioned previously. Moreover, the use of theflavonols identified in our extracts as chemotaxonomic markers,thus accurately monitoring their variations all along the analyticalbatch, allowed us to highlight differences and/or similaritiesamong the genotypes and the different environments considered.

The results accounted from the identification and quantificationof flavonols were able to focus relevant differences in the accession5 (G5) and suggested to sort it out of the batch of the accessionbelonging to ‘‘Cipolla di Giarratana’’ population (Table 4). Accord-

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ing to the biometrical and morphological traits, previously de-scribed, the discussed differences slightly emerged only for thebase colour of the dry skin, underling the potential discriminatingrole of flavonols profile as biomarkers. With this in mind, the AN-OVA analyses showed that the homogeneity of the accessions werelimited to the nine remaining genotypes. In spite of what reportedfor biometrical traits, for all the identified flavonols significantinteractions emerged between the studied factors, but, also forflavonols content, the strength of association measured as classicalg2 ascertained for Location ranged between 2- and 7-fold higherthen what observed for Genotype excepting only for quercetin 3-O-glc (Table 5).

cal traits as a tool for the characterization of ‘‘Cipolla di Giarratana’’: A tra-16/j.foodchem.2012.10.134

Table 3Identified signals and chromatographic, mass-spectrometric and UV–vis data obtained from ‘‘Cipolla di Giarratana’’ extracts analyses.

Entrya Peak name Rtb (min) MW Diagnostic MS datac (m/z) UV–vis datac (nm)

1 Quercetin 3,40 di-O-glucoside 7.8 626.5 625* (M�H)�, 463 (M�glc�H)�, 301 (M�glc�glc�H)� 253, 264, 3442 Isorhamnetin 3,40 di-O-glucoside 8.3 640.4 639 (M�H)�, 477* (M�glc�H)�, 315 (M�glc�glc�H)� 251, 265, 3433 Rutin (quercetin 3-O-rutinoside)d 10.8 610.5 609* (M�H)�, 463 (M�rha�H)�, 301 (M�rha�glc�H)� 256, 3544 Quercetin 3-O-glucosided 11.0 464.4 463* (M�H)�, 301 (M�glc�H)� 256, 3515 Quercetin 40-O-glucoside 13.0 464.4 463 (M�H)�, 301* (M�glc�H)� 252, 264(sh), 3646 Kaempferol 40-O-glucoside 13.5 448.4 447⁄ (M�H)�, 285 (M�glc�H)� 265, 3627 Isorhamnetin 40-O-glucoside 13.8 478.4 477* (M�H)�, 315 (M�glc�H)� 252, 3658 Quercetind 17.6 302.2 301* (M�H)� 255, 3729 Kaempferold 20.8 286.3 285* (M�H)� 268, 368

10 Isorhamentind 21.0 316.3 315* (M�H)�, 301(M�Me�H)� 254, 370

a Peak numbers refer to Fig. 1.b Retention time (average of 450 injections).c Base peaks are marked with an asterisk. Glc = glucose, rha = rhamnose, Me = methyl (see materials and methods for details on MS and UV–vis acquisition parameters).d Co-injection with pure reference standard.

* Significant at P 6 0.05.

Table 4Flavonols content (mg kg�1 FW); values measured in the nine accessions of ‘Cipolla di Giarratana’ landrace, in G5 genotype, and in Tonda Musona variety.

Flavonolsa Cipolla di Giarratana accessions (nine genotypes) G5 Tonda Musona

Average Min Max Average Average

Q 3,40 DIGLC 59.8 19.8 129.3 5.8 <0.1I 3,40 DIGLC 3.3 1.0 8.3 0.4 <0.1RUT 0.6 0.1 1.7 0.1 <0.1Q 3GLC 1.3 <0.1 3.2 0.2 <0.1Q 40GLC 59.3 18.7 116.6 7.0 0.7K 40GLC 1.3 0.1 4.2 0.2 <0.1I 40GLC 3.9 0.8 7.8 0.5 <0.1Q 43.8 4.1 177.8 4.3 0.2K 1.0 0.1 5.5 0.1 <0.1I 2.6 0.2 11.4 0.2 <0.1Total flavonols 176.9 67.7 407.5 18.7 1.0

a Q 3,40 DIGLC = quercetin 3,40 di-O-glucoside; I 3,40 DIGLC = isorhamnetin 3,40 di-O-glucoside; RUT = rutin; Q 3GLC = quercetin 3-O-glucoside; Q 40GLC = quercetin 40-O-glucoside; K 40GLC = kaempferol 40-O-glucoside; I 40GLC = isorhamnetin 40-O-glucoside; Q = quercetin; K = kaempferol; I = isorhamnetin.

Table 5Analysis of variance on the 10 flavonols identified and discussed in the text. Acronymsare given according to Table 4.

Flavonols Factors

Blocks Location Genotype GxL Errors

DF 2 4 8 32 88Q 3,40 DIGLC SS 926 34754 7424 36419 22482

P 0.169n.s. <0.001*** 0.001** <0.001***

I 3,40 DIGLC SS 9 124 40 209 225P 0.173n.s. <0.001*** 0.056n.s. 0.001***

RUT SS 0.3 16 3 9 9P 0.263n.s. <0.001*** <0.001*** <0.001***

Q 3GLC SS 1 3 10 33 40P 0.283n.s. 0.141n.s. 0.012* 0.001**

Q 40GLC SS 760 27646 15242 32153 30100P 0.334n.s. <0.001*** <0.001*** <0.001***

K 40GLC SS 0.7 46 19 40 48P 0.506n.s. <0.001*** <0.001*** <0.001***

I 40GLC SS 1.8 122 65 269 286P 0.757n.s. <0.001⁄⁄⁄ <0.016* <0.001⁄⁄⁄

Q SS 225 105061 16159 107980 52871P 0.829n.s. <0.001⁄⁄⁄ 0.002** <0.001⁄⁄⁄

K SS 6 95 24 134 122P 0.128n.s. <0.001⁄⁄⁄ 0.03* <0.001⁄⁄⁄

I SS 12 306 58 489 265P 0.132n.s. <0.001⁄⁄⁄ 0.021* <0.001⁄⁄⁄

Totalflavonols

SS 1645 432766 70224 165446 132484P 0.581n.s. <0.001⁄⁄⁄ <0.001⁄⁄⁄ <0.001⁄⁄⁄

n.s. = not significant.* Significant at P 6 0.05.** Significant at P 6 0.01.*** Significant at P 6 0.001.

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Please cite this article in press as: Riggi, E., et al. Flavonol content and biometriditional Sicilian onion landrace. Food Chemistry (2012), http://dx.doi.org/10.10

3.3. Multivariate analysis

Due to the interactions emerged for all the identified flavonols, aprincipal component analysis (PCA) was performed. It reduced the10 identified flavonols, to two principal components (PC) explainingthe whole variance (99%) in the original data. The PC scores of PC1were strongly and significantly correlated with all studied traits.The two locations, that were the furthest apart in the plane deter-mined by the two PC, were L1 and L4 (Fig. 2); the main part of geno-types cultivated in the two above mentioned environmentsclustered on the right of the ordination along PC1 and were charac-terised by higher amount of quercetin, quercetin 3,40 di-O-glc andquercetin,40 O-glc. For the opposite reason, the genotypes G5 andthe control ‘‘Tonda Musona’’, apart from the growing site, tendedto cluster separately on the left along PC1 due to the low contentof the studied components. All the remaining combinations GxL,clustered in a cloud where no clear difference due to accessionsand environments emerged, suggesting a substantial homogeneityof the accessions of ‘‘Cipolla di Giarratana’’.

3.4. Correlations

Highly significant and negative relationships emerged betweenbiometrical traits and all of the identified flavonols but isorhamne-tin 40-O-glucoside (Table 6). Adverse environmental conditionsreducing bulb size had, probably, stimulated flavonols accumula-tion. Dixon and Paiva (1995) reviewed experimental papers report-ing effects of biotic and abiotic stresses on polyphenols

cal traits as a tool for the characterization of ‘‘Cipolla di Giarratana’’: A tra-16/j.foodchem.2012.10.134

Table 6Correlations between studied characters (biometrical and biochemical traits) in ‘‘Cipolla di Giarratana’’ bulbs. Acronyms are given according to Table 4.

Diam Q 3,40 DIGLC I 3,40 DIGLC RUT Q 3GLC Q 40GLC K 40GLC I 40GLC Q K I Total flavonols

Bulb weight 0.93*** �0.27** �0.30*** �0.49*** �0.15n.s. �0.36*** �0.46*** 0.02n.s. �0.48*** �0.34*** �0.43⁄⁄⁄ �0.52⁄⁄⁄

Bulb diameter �0.30*** �0.27** �0.43*** �0.12n.s. �0.40*** �0.46*** �0.04n.s. �0.40*** �0.26** �0.36⁄⁄⁄ �0.50⁄⁄⁄

Q 3,40 DIGLC 0.51*** 0.16n.s. 0.36*** 0.77*** 0.40*** 0.64*** 0.13n.s. 0.02n.s. 0.15n.s. 0.72⁄⁄⁄

I 3,40 DIGLC 0.43*** 0.15n.s. 0.42*** 0.41*** 0.52*** 0.44*** 0.35*** 0.69⁄⁄⁄ 0.64⁄⁄⁄

RUT 0.06n.s. 0.22** 0.23** �0.13n.s. 0.71⁄⁄⁄ 0.60⁄⁄⁄ 0.67⁄⁄⁄ 0.58⁄⁄⁄

Q 3GLC 0.27** 0.30⁄⁄⁄ 0.07n.s. 0.36⁄⁄⁄ 0.53⁄⁄⁄ 0.23⁄⁄ 0.46⁄⁄⁄

Q 40GLC 0.56⁄⁄⁄ 0.59⁄⁄⁄ 0.23⁄⁄ 0.03n.s. 0.19⁄ 0.78⁄⁄⁄

K 40GLC 0.18⁄ 0.37⁄⁄⁄ 0.30⁄⁄⁄ 0.39⁄⁄⁄ 0.58⁄⁄⁄

I 40GLC �0.22⁄ �0.24⁄⁄ �0.01n.s. 0.34⁄⁄⁄

Q 0.77⁄⁄⁄ 0.83⁄⁄⁄ 0.75⁄⁄⁄

K 0.74⁄⁄⁄ 0.51⁄⁄⁄

I 0.66⁄⁄⁄

n.s. = not significant.⁄Significant at P 6 0.05.** Significant at P 6 0.01.*** Significant at P 6 0.001.

Principal Component Plot

L1 1L1 2

L1 4

L1 5L1 6

L1 7

L1 9L1 12

L1 13

L1 TM

L1 15

L3 1L3 2L3 4

L3 5

L3 6

L3 7

L3 9

L3 12

L3 13

L3 TM

L3 15

L4 1

L4 2

L4 4

L4 5

L4 6

L4 7

L4 9

L4 12

L4 13

L4 TM

L4 15

L5 1

L5 2

L5 4

L5 5

L5 6

L5 7L5 9

L5 12

L5 13

L5 TM

L5 15

L6 1

L6 2 L6 4

L6 5

L6 6

L6 7

L6 9

L6 12

L6 13

L6 TM

L6 15

Quercetin 3,4' diO-glc

Quercetin

Quercetin 4' O-glc

-80

-60

-40

-20

0

20

40

60

80

100

0040020002-

PCA 1 (91.9%)

PCA

2 (7

.3%

)

Fig. 2. Principal component analysis (PCA) based on the variation of flavonols content in the 11 Genotypes grown in the five Locations. TM = Tonda Musona standard variety.

6 E. Riggi et al. / Food Chemistry xxx (2012) xxx–xxx

accumulation, suggesting direct effects of insufficient nutrientslevels in the soil (mainly nitrogen, phosphorus), or direct effectsof UV-B, or biotic stress (insects). Nevertheless, despite the knowl-edge on the effect of environment on flavonol content, it is verydifficult to generate precise predictive relationships betweenenvironment and flavonol concentration (Mogren, Olsson, &Gertsson, 2006; Rodrigues et al. (2011)). High direct correlationhave been also reported between almost all the identified flavonolsand this can be explained because their common biosyntheticorigin (Rodríguez Galdón et al., 2008).

4. Conclusions

The present study assessed the flavonol profile and the mainbiomorphological traits of ‘‘Cipolla di Giarratana’’ onion landrace.

Please cite this article in press as: Riggi, E., et al. Flavonol content and biometriditional Sicilian onion landrace. Food Chemistry (2012), http://dx.doi.org/10.10

The results suggest that the quality of produced polyphenols is ge-netic controlled, whilst their amount is environment related. Theflavonol profile also allowed to individuate one suspect accession,that was reproduced as ‘‘Cipolla di Giarratana’’. It was completelyundifferentiated in terms of biometrical and morphological traits,slightly different in terms of skin colour, but dramatically differentin terms of amount of each of the identified flavonols. Furthermore,the landrace’s homogeneity was supported by the reducedstrength of association of variation due to Genotype when com-pared with Location.

The powerful of chemotaxonomic role of flavonols can be re-lated to the wide number of flavonols composing the whole iden-tified patterns, but also to the specific characteristics of some of theidentified flavonols. In particular, free quercetin (aglycon) contentappeared in unusually high amounts. This chemotaxonomic tool

cal traits as a tool for the characterization of ‘‘Cipolla di Giarratana’’: A tra-16/j.foodchem.2012.10.134

E. Riggi et al. / Food Chemistry xxx (2012) xxx–xxx 7

could help the differentiation of a landrace that has to be clearlydemonstrated when characterization is addressed to obtain protec-tion of geographical indications (PGI) or protection of designationof origin (PDO).

Acknowledgements

The experimental activities were financial supported by the Lo-cal Government of Giarratana and technically supported by the‘‘Associazione Cipolla di Giarratana’’. The authors are grateful toMs. Chiara Ippolito for her skilful technical assistance.

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