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Content of flavonols in Italian bean ( Phaseolus vulgaris L.) ecotypes

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Content of flavonols in Italian bean ( Phaseolus vulgaris L.) ecotypes
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  Content of flavonols in Italian bean ( Phaseolus vulgaris  L.) ecotypes Giovanni Dinelli  a,* , Alessandra Bonetti  a , Maurizio Minelli  a , Ilaria Marotti  a ,Pietro Catizone  a , Andrea Mazzanti  b a Department of Agroenvironmental Science and Technology (DiSTA), University of Bologna, V.le Fanin 44, 40127 Bologna, Italy b Department of Organic Chemistry ‘‘A. Mangini’’, University of Bologna, V.le Risorgimento 4, 40100 Bologna, Italy Received 18 February 2005; received in revised form 30 May 2005; accepted 21 July 2005 Abstract Methanol extracts of seeds from 23 accessions of 3  Phaseolus vulgaris  ecotypes (‘‘Sarconi’’, ‘‘Lamon’’, ‘‘Zolfino del Prato-magno’’), grown in different Italian regions (Basilicata, Veneto, Tuscany) were analyzed for their flavonoid content. Flavonoid gly-cosides were found in the seed coat from ten accessions of the ‘‘Zolfino’’ ecotype and in one accession of the ‘‘Sarconi’’ ecotype.From highest to lowest concentration these compounds were kaempferol 3- O -glucoside (compound  2 ), kaempferol 3- O -xylosylg-lucoside (compound  1 ) and a not completely identified kaempferol monoglucoside (compound  3 ). Total flavonol content variedfrom 0.19 to 0.84 g/kg of seed fresh weight. A great variability in the total flavonol content, being between 18% and 50%, and inthe relative abundance of different kaempferol derivatives was observed for the same genotypes sampled in the srcinal locationsin the 2001–2003 period. Fluctuation in flavonol content suggests that further researches are necessary for an exhaustive compre-hension of physiological mechanisms influencing the expression of these phenolic compounds. Obtained results evidenced that someItalian bean ecotypes may be an important source of functional compounds as kaempferol glycosides.   2005 Elsevier Ltd. All rights reserved. Keywords: Phaseolus vulgaris ; Italian bean ecotypes; Flavonols; Kaempferol; Nutraceutical compounds 1. Introduction Common bean had been recognized since a long timefor its protein content, but in general there have been rel-atively little research and discussion on its nutraceuticalvalue (Aragao et al., 1996; Bengtsson, 1991; Bollini, Carnovale, & Campion, 1999; Sharma, 1993). This cropis widely cultivated in Italy and nevertheless the relevantdecreasing in production in all Europe, Italy is withinEuropean Union a leader in bean production (Laghetti,Xhuveli,Perrino,Olita,&Hammer,1998).Tradenecessi-ties favored bean commercial cultivars, confining localecotypes in very small areas characterized by traditionalcropping practices (Limongelli, Laghetti, Perrino, &Piergiovanni, 1996). As regards Italian bean ecotypes,very few of them possess a high seed or pod qualitynegatively influencing the relative commercial value(Piergiovanni, Cerbino, & Gatta, 2000). These ecotypesarecroppedinfewfarmspreciselylocatedinmountainousorhillyareas:theyareknownbylocalnamesandsomeof them are labeled by Indication of Geographical Prove-nance (IGP). Some examples are ‘‘Fagiolo di Lamon’’,‘‘Fagiolo di Sarconi’’, ‘‘Fagiolo Zolfino del Prato-magno’’. ‘‘Lamon’’ bean comes from the mountainousarea of Lamon and Sovramonte (Veneto) and only fourpopulationsareknown:Spagnolet,Spagnolon,Calonegaand Canalin (Piergiovanni, Cerbino, & Brandi, 2000).‘‘Sarconi’’beanisalocallygrownvariationofthe Cannel-lino and Borlotto beanecotypes.Thespecialenvironmen-talconditionsandtheplentifulsupplyofwaterofwestern 0308-8146/$ - see front matter    2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2005.07.028 * Corresponding author. Tel.: +39 051 2096672; fax: +39 0512096241. E-mail address:  gdinelli@agrsci.unibo.it (G. Dinelli). www.elsevier.com/locate/foodchem Food Chemistry 99 (2006) 105–114 FoodChemistry  Basilicata combine to give the beans grown in Sarconi aqualityquitedistinctfromthosecultivatedinotherzones.Whether fresh or dried, the Sarconi beans are muchappreciated for their thin coat, short cooking time andgood taste (Piergiovanni et al., 2000). Finally, ‘‘Zolfinodel Pratomagno’’ bean is a dwarf bean, with a character-isticpaleyellowcolor.ItiscroppedinPratomagnoarea,ahillyregioninTuscanywhichencompassessevenvillages.Productionischaracterizedbyalowyield,limitedstoragecapacityandpoorresponsetotechnicalinputs.Thesefac-tors brought this bean on the verge of extinction in the1970s. This ecotype has benefited from EU agri-environ-mentsfundsaimedatincreasingcultivationofthreatenedspecies (Turchi, 2000). All cited Italian ecotypes arecropped with traditional methods preventing crop frompathogens attack and permitting to obtain seeds withoutany chemical treatment.Flavonoids, which are found in virtually all plants,encompass a wide range of structural classes and biolog-ical functions (Kuhnau, 1976): they include severalchemical classes such as flavanones, flavones, flavonols,anthocyanins, biflavonoids, isoflavones, coumestans,isoflavans, pterocarpans (Seigler, 1998). Plant flavonoidcontent can be influenced by different factors, such aslight, temperature, mineral nutrition, pathogens,mechanical damage, plant growth regulators (Jaakolaet al., 2002). They could act as antioxidants, enzymeinhibitors, pigments for light absorbance, visual attrac-tants for pollination, light screen, inhibitors of plantgrowth, chemical signals in nodulation gene inductionand phytoalexins (Beier & Nigg, 1992; Paolacci et al.,2001). In common bean a conspicuous number of flavonoids were isolated and identified (Feenstra, 1960;Kucera, Leubner-Metzger, & Wellmann, 2003).Researchers accept the fact that the pigments responsi-ble for seed coat color in  Phaseolus vulgaris  are flavo-noids (Beninger, Hosfield, & Nair, 1998). Many of theflavonoids pigments that give rise to seed coat color inbeans may also impart positive health benefits as antiox-idants (Amic, Davidovic-Amic, Beslo, & Trinajstic,2003; Hertog, Feskens, Hollman, Katan, & Kromhout,1993; Hertog, Hollman, Katan, & Kromhout, 1992).In particular several glycosidic forms of quercetin andkaempferol have been identified in seeds of   P. vulgaris (Beninger et al., 1998; Beninger & Hosfield, 1999; Clif- ford, 1996; Hertog et al., 1993; Hertog, Hollman, & Ka-tan, 1992; Hodges & Forney, 2000; Lioi, 1989; Romani et al., 2004; Scalbert & Williamson, 2000; Vinson, Hao,Su, & Zubik, 1998; Wells, 2004). In particular Romani et al. (2004) quantified the content of flavonols in somelandraces from Pratomagno area.Since there is still little knowledge about the flavo-noid content of Italian bean ecotypes, we undertookthe present investigation to identify flavonoid com-pounds in 23 bean ecotypes from three different Italianhilly areas (Sarconi, Pratomagno, Lamon) and to com-pare them with flavonoid content of four commercialbean cultivars. The specific objectives were to determinethe storage localization in the seed (coat, embryo, endo-sperm) and to evaluate the trend of the flavonoid con-tent in a three-year lasting period (2001–2003). 2. Materials and methods  2.1. Plant materialsPhaseolus vulgaris  L. seeds of three Italian ecotypes(Sarconi, Lamon, Zolfino del Pratomagno) weredirectly sampled from local farmers or producer asso-ciations. Ten, three and ten populations were collectedfor Sarconi, Lamon and Zolfino del Pratomagnoecotypes, respectively (Table 1). The Italian bean Table 1List of analyzed Italian ecotypes and cultivars of   P. vulgaris  L.Ecotype/cultivar Code Original location Local nameSarconi S13 Sarconi (PZ, Italy) Riso biancoS14a Sarconi (PZ, Italy) TuvaglieddabiancaS14b Sarconi (PZ, Italy) TuvaglieddarossaS15 Sarconi (PZ, Italy) NasiedduS17 Sarconi (PZ, Italy) MunacheddaS18 Sarconi (PZ, Italy) CiuotoS19 Sarconi (PZ, Italy) Cannellino biancoS20 Sarconi (PZ, Italy) Verdolino nanoS21 Sarconi (PZ, Italy) Tabacchino nanoS22 Grumento Nova(PZ, Italy)San Micheleo   CiuotoLamon L1 Lamon (BL, Italy) SpagnolonL2 Lamon (BL, Italy) SpagnoletL3 Lamon (BL, Italy) CalonegaZolfino delPratomagnoZ1 Loro Ciuffenna(AR, Italy) – Z2 Loro Ciuffenna(AR, Italy) – Z3 Loro Ciuffenna(AR, Italy) – Z4 Loro Ciuffenna(AR, Italy) – Z5 Reggello (AR, Italy) – Z6 Terranova Bracciolini(AR, Italy) – Z7 Terranova Bracciolini(AR, Italy)Z8 Terranova Bracciolini(AR, Italy) – Z9 Terranova Bracciolini(AR, Italy) – Z10 Terranova Bracciolini(AR, Italy) – Contender C – – Tendergreen T – – Sanilac S – – Lingua di fuoco LDF – – 106  G. Dinelli et al. / Food Chemistry 99 (2006) 105–114  ecotypes were sampled for three years (2001–2003)from the same farmers or producer associations. Inaddition, four commercial bean cultivars (Sanilac,Tendergreen, Contender, Lingua di fuoco) were ana-lyzed. Seeds of ‘‘Zolfino del Pratomagno’’ ecotypesand ‘‘Sanilac’’ cultivar were characterized by a uni-form, light yellow seed coat pigmentation, while beanseeds of all the other samples showed different seedcoat pigmentation.  2.2. Standards and chemicals Authentic standard of flavonols (kaempferol,kaempferol 3- O -xylosylglucoside, kaempferol 3- O -glu-coside) were purchased by Indofine Co., Hillsborough,USA. The chemical structures of the three flavonols isreported in Fig. 1. Methanol, hydrochloric acid, aceticacid and all solvents used for HPLC were of analyticalor HPLC grade from Carlo Erba, Milan, Italy.  2.3. Standard solutions, calibration curves and calculationof flavonol content Flavonoids stock solutions were prepared by dissolv-ing the crystalline standards first in 1000 ppm stocksolutions. Subsequently, stock solutions were dilutedto 5 ppm with 80% methanol (v/v). Calibration curveswere obtained for each standard with high linearity( r  > 0.996) by plotting the standard concentrations asa function of the peak area obtained from HPLC anal-ysis with 40  l l injections. For this purpose, the stocksolutions of the standards were diluted with 80% meth-anol to five different concentrations ranging from 1 to20 mg l  1 . Each concentration was analyzed by tripli-cate injections.  2.4. Extraction of flavonols As regards whole seed extraction, 500 mg of grounddry seeds were dispersed in a mixture of 2 ml of acetoni-trile and 0.5 ml of 0.1 N HCl by stirring for 3 h (Barnes,Kirk, & Coward, 1994; Wang & Murphy, 1994). After3 h, the mixtures were centrifuged at 15,000 rpm for10 min at 10   C. Clear supernatant was filtered througha 0.45  l m nylon filter (Millipore Co., USA) and dried bya vacuum pump. Samples were reconstituted with 2.5 mlof 80% methanol.For the differential analysis of seed coat, endo-sperm and embryo, 100 g (fresh weight) of beans werewashed and then placed in enough distilled water tosoak the beans. After 10 min of soaking the seed coatswere separated from cotyledons and embryos. Seedcoats, endosperms and embryos were freeze-dried.The water exudates from the soaked bean were alsofreeze-dried and stored. Seed coats, endosperms, em-bryos and water exudates were lyophilized, groundto fine powder and flavonols were extracted as previ-ously reported.Since the extraction method has been mainly em-ployed for the analysis of isoflavones, it was validatedfor the purpose of the present research. Amounts of external standards (kaempferol 3- O -xylosylglucoside,kaempferol 3- O -glucoside and kaempferol at the con-centration of 10, 50 and 100 ppm) in a methanol solu-tion were added to 500 mg samples (ground dry seedof Z1 accession). The samples were mixed and left atroom temperature until the methanol had evaporated.The HPLC analysis method is described below. Theaverage overall recovery in the 5–100 ppm range was98.3 ± 1.8%, 98.6 ± 1.5% and 95.3 ± 0.3% for kaempf-erol 3- O -xylosylglucosid, kaempferol 3- O -glucosideand kaempferol, respectively (Table 2). For kaempferol3- O -xylosylglucoside and kaempferol 3- O -glucoside themean recovery was slightly higher in the 100 ppm forti-fied samples, but the differences with respect to the otherfortifications were not significant. Since compound  3 Fig. 1. Chemical structure of kaempferol (a), kaempferol 3- O -gluco-side (b) and kaempferol 3- O -xylosylglucoside (c). G. Dinelli et al. / Food Chemistry 99 (2006) 105–114  107  was not clearly identified, the relative recovery was de-duced by comparing the present extraction method withthe extraction procedure (70% ethanol) proposed by Ro-mani et al. (2004) for the analysis of bean flavonols. Sixgrams of intact seeds (Z1 accession) were ground andthe resulting flour was divided in 12 aliquots (500 mgeach). Six aliquots were extracted with 70% ethanol (Ro-mani et al., 2004), while the remaining six aliquots wereextracted with acetonitrile (Barnes et al., 1994; Wang &Murphy, 1994). On the basis of the comparison betweenthe two extraction procedures, the mean recovery of compound  3  was 97.3 ± 2.2%. The mean recovery val-ues, experimentally determined, were used to correctthe flavonol content in the bean accessions sampled inthe srcinal location in the period 2001–2003.  2.5. Acid hydrolysis Bean extract solutions (whole seed, seed coat, endo-sperm, embryo) (2 ml) was mixed with concentratedHCl (37%, 0.5 ml) and heated in a sealed vials at 80– 85   C for 2 h. The acid hydrolysis procedures was shownto completely convert glycosides to aglycones by usingauthentic flavonols standards (data not shown). Subse-quently, flavonols were extracted with ethylacetate,evaporated in vacuum and suspended in methanol 80%(2 ml). The solutions were re-filtered prior to HPLCinjection.  2.6. Preparative HPLC  The seed coats of 50 g of beans were removed, ex-tracted and hydrolyzed as previously described. Thepurification of compound  4  (Fig. 2) for NMR analysiswas achieved on a Waters Xterra MS reverse-phaseC18 column (10  ·  150 mm, 5  l m) using a Beckman Sys-tem Gold 126 multisolvent pump, photodiode arraydetector Beckman 168 and a Spark Holland autosam-pler. The solvent system was (A) H 2 O/CH 3 COOH(99.9:0.1) and (B) ACN/H 2 O/CH 3 COOH (80:19.9:0.1).Separations were done in isocratic mode with a flow rateof 2 ml/min. The detection was at 260 nm. Each injec-tion volume was 1.5 ml. If the peak collected was notpure, it was re-chromatographed under the above condi-tions and the compound  4  collected pure after one ormore purification runs. All chromatograms were ana-lyzed using System Gold software (9.0). Table 2Mean recovery (MR, %) and coefficient of variation (CV, %)of kaempferol compounds (compound  1  = kaempferol 3- O -xylosylg-lucoside; compound  2  = kaempferol 3- O -glucoside; compound  4  =kaempferol) in six replicates of Zolfino (Z1) dry ground seed fortifiedwith standard concentrations (5, 50, 100 ppm)Compound 5 ppm 50 ppm 100 ppmMR(%)CV(%)MR(%)CV(%)MR(%)CV(%) 1  96.2 3.2 98.9 2.8 99.8 2.8 2  97.1 3.3 98.6 2.9 100.2 2.5 4  95.3 3.2 95.1 3.1 95.6 3.2Fig. 2. HPLC–DAD ( k  = 260 nm) profiles of methanol extracts fromaccession Z2 (Zolfino bean ecotype) collected in the 2001. (a) seed coatextract, (b) endosperm extract, (c) embryo extract, (d) acid hydrolysisof seed coat extract. Compound  1  = kaempferol 3- O -xylosylglucoside,compound  2  = kaempferol 3- O -glucoside, compound  3  = unidentifiedkaempferol monoglucoside, compound  4  = kaempferol.108  G. Dinelli et al. / Food Chemistry 99 (2006) 105–114   2.7. Analytical techniques and equipmentHPLC–DAD analysis . Analyses of flavonols werecarried out using a Beckman liquid chromatograph con-sisting of a Gold 126 multisolvent pump, photodiode ar-ray detector Beckman 168 and a Spark Hollandautosampler. For the separations a Waters XTerra MSreverse-phase C18 column (2.1  ·  150 mm, 5  l m) operat-ing at 25   C was employed. The eluent was composed of (A) H 2 O/CH 3 COOH (99.9:0.1) and (B) ACN/H 2 O/CH 3 COOH (80:19.9:0.1). A two-step linear solvent gra-dient system was used starting from 20% to 40% of solu-tion B for a 30-min period at a flow rate of 0.2 ml/min.The percentage of solution B reached 30% from 5 to9 min, then 40% from 10 to 20 min. The injection vol-ume was 10  l l. UV–Vis spectra were recorded in the200–500 nm range and the chromatograms were re-corded at 260 nm. HPLC–MS analysis . HPLC–MS analyses were car-ried out with a Finnigan Surveyor LC System linkedto a TSQ Quantum-Finnigan mass spectrometer withan electrospray interface (Finnigan). The separationswere performed using a Waters XTerra MS reverse-phase C18 column (2.1  ·  150 mm, 5  l m) operating un-der the same chromatographic conditions previously re-ported. Spectra were recorded in negative (fragmentor80) and positive (fragmentor 120) ion mode. The massspectrometer operating conditions were: capillary tem-perature 300   C; nebulizer pressure 45 psi, spray voltage3200 V, mass range of 50–1000 amu, quadrupole tem-perature 40   C. NMR analysis . NMR analysis of the purified flavonol 4  and of the standard kaempferol were carried out at600 MHz for  1 H spectra (Varian Inova 600 MHz spec-trometer, equipped with an indirect detection probe).A standard sample of authentic kaempferol was pre-pared by dissolving 1.0 mg of the compound in 0.7 mlof CD 3 OD. An analytical sample of purified flavonol  4 was prepared from 1.0 mg of compound dissolved in0.7 ml of CD 3 OD. Direct comparison between the pro-ton spectra of the two samples confirmed the structuralidentification of compound  4  as kaempferol (Fig. 3).  2.8. Identification and quantification of flavonoids Identification of flavonoids was based on the compar-ison of their retention times, diode array scans and massspectra with those of authentic standards. The com-pound  4  was purified and the chemical structure deter-mined by NMR analysis. Quantification of individualflavonoids was based on HPLC analyses using a five-point regression curve ( r 2 > 0.97). For each collectionsite in each year five different seed samples were ana-lyzed. Analytical results were expressed as means withtheir SDs. Data were elaborated using the SAS Institutestatistical package. Analysis of variance was carried outusing a general linear model procedure with Student– Newman–Keuls comparison of means to test for signif-icant differences between means. 3. Results and discussion HPLC–DAD analysis of bean samples collected inthe 2001 evidenced three flavonols, hereinafter namedcompounds  1  –  3 , in one Sarconi accession (S20) and inall the analyzed Zolfino populations (Z1–Z10). No fla-vonol was observed in all the other samples (remainingSarconi and Lamon accessions, bean cultivars). Forexample, the chromatographic profile of Z1 populationsat 260 nm, evidencing the qualitative composition of flavonols, is reported in Fig. 2. The three compoundswere characterized by similar absorption spectra, withabsorption peaks at 266 and 347 nm (data not shown).As evidenced by differential analysis of various seedparts (embryo, endosperm, seed coat), the three com-pounds are primarily stored in the seed tegument(Fig. 2). In fact only traces of the three flavonols werefound in the endosperm (less than 0.1% of the total con-tent) and no compound was found in the embryo. Thisresult is in agreement with the literature (Beninger Fig. 3. 1H NMR spectrum of purified compound  4  as compared tospectrum of kaempferol standard. (a) 1H NMR spectrum of kaempf-erol standard; (b) 1H NMR spectrum of compound  4 . G. Dinelli et al. / Food Chemistry 99 (2006) 105–114  109
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