Occurrence of Isoflavonoids in Brazilian Common Bean Germplasm(Phaseolus vulgaris L.)Paula Feliciano de Lima,*,† Carlos Augusto Colombo,‡ Alisson Fernando Chiorato,§
Lydia Fumiko Yamaguchi,∥ Massuo Jorge Kato,∥ and Sergio Augusto Morais Carbonell§
†Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13084-971 Campinas, Sao Paulo, Brazil‡Centro de Pesquisa e Desenvolvimento de Recursos Geneticos Vegetais and §Centro de Pesquisa e Desenvolvimento de Graos eFibras, Instituto Agronomico de Campinas, CP 28, 13012-970 Campinas, Sao Paulo, Brazil∥Nucleo de Apoio a Pesquisas em Diversidade Molecular de Produtos Naturais, Instituto de Química, Universidade de Sao Paulo, CP26077, 05508-000 Sao Paulo, Sao Paulo, Brazil
ABSTRACT: Common bean (Phaseolus vulgaris) is present in the daily diet of various countries and, as for other legumes, hasbeen investigated for its nutraceutical potential. Thus, 16 genotypes from different gene pools, representing seven types of seedcoats and different responses to pathogens and pests, were selected to verify their isoflavone contents. The isoflavonoids daidzeinand genistein and the flavonols kaempferol, myricetin, and quercetin were found. Grains of the black type showed the highestconcentrations of isoflavonoids and were the only ones to exhibit daidzein. IAC Formoso, with high protein content and sourceof resistance to anthracnose, showed the greatest concentration of genistein, representing around 11% of the content present insoybean, as well as high levels of kaempferol. Arc 1, Raz 55, and IAC Una genotypes showed high content of coumestrol. Theresults suggest the use of IAC Formoso to increase the nutraceutical characteristics in common bean.
Phaseolus vulgaris, the common bean, is the most importantamong the 50 species of native Phaseolus present in theAmericas. This species has Mexican origin and contains twomajor gene pools whose plants differ mainly in relation to seedsize: Mesoamerican (small seeds) and Andean (large seeds).The nutraceutical importance of these beans as a protein
source is remarkable in several regions or the world, includingBrazil.1,2 Studies related to the nutritional and nutraceuticalpotential of legumes have gained prominence in recent years,especially for soybean and chickpea, since they have higherlevels of phytoestrogens such as isoflavonoids.3−5 Morerecently, common bean has received functional food status asit contains bioactive phenolic compounds and large amounts ofcomplex carbohydrates and fibers, in addition to being a sourceof iron, phosphorus, magnesium, manganese, zinc, andcalcium.3,6,7 However, there is still little information thatcombines genetic breeding of common bean with itsnutraceutical diversity, even in light of a growing number ofstudies on consumption of the phenolic compounds present incommon bean, such as flavonols, isoflavones, and anthocyanins,and their beneficial effects on health.Isoflavonoids have gained prominence because they may play
an important role in the prevention and treatment of chronicdiseases, as has already been seen in studies that evaluatedmodels in vitro, in vivo, and as interventions in humans. In allthese cases, the important biological action of these compoundswas observed in antioxidant, “antimutagenic”, and anticarcino-genic activities.3,8−11 A subclass of flavonoids, isoflavonoids, arephytoestrogens that exhibit pseudohormonal properties as aresult of their functional and structural similarity to the natural
estrogen 17β-estradiol and may interact with estrogen receptors(ER).12,13
Isoflavone content is variable among plant species and mayeven vary among genotypes of the same species. In addition,isoflavone content may also be affected by external factorsrelated to crop location, such as temperature, fertilization levels,occurrence of pests and diseases, time since harvest, farmingpractices, and processing and food preparation methods.14,15
The isoflavone content is significantly less in seeds developed athigh temperatures during the grain filling phase, and somestudies report a possible relationship between resistance topathogens and the presence of phytoestrogens, whereisoflavoids would act as phytoalexins in plants.16
In the class of coumestans, coumestrol is a metabolite foundin different legumes, occurring especially in soybean, alfalfa,clover sprouts, and beans.17 It is discussed mainly in regard toits high affinity for estrogen receptors, around 30−100 timesgreater than the isoflavonoids exhibiting antagonist functions bycompeting with ER.18 It also has been shown to decreaseovulation rates, increasing ovarian apoptosis in mammals.19 Anovel activity of coumestrol was reported recently and theauthors reported that coumestrol binding to human estrogenreceptor β inhibited microglia-mediated inflammation.20
In the context of an increasing number of studies on thenutraceutical potential of common bean and the preference ofBrazilian consumers for this food, the aim of the present study
Received: March 2, 2014Revised: September 12, 2014Accepted: September 18, 2014Published: September 18, 2014
was to assess the flavonoid and coumestrol content ingermplasm with a broad genetic base of the species, for thepurpose of introducing new nutraceutical parameters in thecrop breeding program.
■ MATERIALS AND METHODSPlant Material. The study was carried out at the Campinas (SP)
(22° 54′ 20″ S, 47° 03′ 39″ W) during the rainy season (December−May) with 16 common bean genotypes from the two major gene poolswith different seed coats and grain sizes, and one soybean genotypeused as a reference (Table 1, Figure 1). Seeds of each cultivar weresown in a 5-m-length row (10 plants/m) for a total area of 105 m2.After 3 months, grains from all genotypes were collected and kept inan air-circulation laboratory oven at 30 °C for drying to constantweight, and afterward ground in a mill and stored with protectionagainst moisture.Standards. Authentic standard daidzein, daidzin, genistein, and
genistin (isoflavonoids); quercetin, kaempferol, and myricetin(flavonols); coumestrol (coumestan); and chrysin were purchasedfrom Sigma Chemical Co. (St. Louis, MO). Stock solutions wereprepared by dissolving the standards in MeOH−H2O (1:1) for a finalconcentration of 10 mg/mL.Solvents. All solvents used were HPLC-grade and were purchased
from Sigma Chemical Co. (St. Louis, MO).Extraction and Purification of Flavonoids. Portions (2 g) of
dry and ground grains were extracted with 60 mL of 70% ethanol (pH2.0, adjusted with formic acid) for 24 h at ambient temperature.21 Theextracts were partitioned with 60 mL of hexane, and the aqueous phasewas evaporated under reduced pressure to dryness. The residue ofeach extract was resuspended in MeOH−H2O (1:1), filtered through a13 mm PTFE membrane filter of 0.45 μm, and kept at −20 °C up tothe time of analysis. Each extract was prepared and analyzed intriplicate.Identification and Quantification of Flavonoids. Quantifica-
tion of all compounds was conducted from construction of acalibration curve with chrysin as internal standard. Individualidentification of the flavonoids (negative mode) was performed onthe basis of retention times, spectroscopic data, and mass-to-chargeratio (m/z) of each standard by use of 10 points of concentration,0.005−5 μg/mL, containing the internal standard at a final
concentration of 4 μg/mL (Table 2). Results were expressed asmilligrams per gram dry weight of seed flour.
Analytical Techniques and Equipment. The LC-ESI-QTOF-MS MicroTOF-QII Bruker equipped with an autosampler, binarypump, thermostated column compartment, and multiple-wavelengthdetector was used. Separations of flavonoids were achieved on a 150 ×2.0 mm i.d, 5 μm C18 Luna column (Phenomenex, Torrence, CA) at30 °C. Injection volume was 20 μL at a flow rate of 0.25 mL/min.Linear gradient elution was performed with solvent A (methanol/formic acid, 95:5 v/v) and solvent B (5% formic acid in water, v/v) at
Table 1. Flavonoid Content in 16 Genotypes of Common Bean by LC-qMSa
μg/g dry weight of seed flour
genotypetype ofgrain origin DA GE KA M Q CM
DA +GE
Arc 1 B M 6.82 ± 0.95 6.38 ± 0.78 2.28 ± 0.19 nd 2.56 ± 0.44 7.75 ± 0.33 13.20IAC Diplomata B M nd 1.10 ± 0.80 0.78 ± 0.06 nd 1.09 ± 0.17 2.32 ± 0.09Raz 55 B M 8.05 ± 1.83 5.98 ± 0.89 5.40 ± 0.78 26.15 ± 22.86 8.34 ± 0.91 17.67 ± 4.40 14.03TU B M nd 0.98 ± 0.17 4.85 ± 3.53 78.94 ± 13.94 15.85 ± 3.57 0.75 ± 0.14IPR Uirapuru B M nd 1.45 ± 0.06 2.82 ± 0.39 nd 4.37 ± 3.80 2.60 ± 0.21IAC Una B M 2.30 ± 0.28 2.95 ± 0.13 1.58 ± 0.74 nd 2.80 ± 0.94 5.95 ± 0.40 5.25IAC Alvorada C M nd nd 1.64 ± 1.18 nd nd ndIAC Carioca Comum C M nd 1.17 ± 0.15 35.09 ± 2.55 nd nd 1.19 ± 0.16IAC Formoso C M nd 8.93 ± 0.67 60.07 ± 3.17 nd 9.88 ± 3.04 16.85 ± 0.81IAC Perola C M nd 0.82 ± 0.65 16.37 ± 11.53 nd nd 3.31 ± 2.87IAC Harmonia S A/M nd nd 0.40 ± 0.32 nd nd ndIAC Boreal S A/M nd nd 2.69 ± 0.40 nd nd ndBrancao Argentino W A nd nd nd nd nd ndIAC Jabola Bo A/M nd 1.39 ± 0.09 117.65 ± 5.09 nd 2.79 ± 0.65 4.50 ± 1.04IAC Esperanca Bo A/M nd 2.16 ± 0.29 72.13 ± 1.06 nd nd 2.53 ± 0.36Flor de Mayo R A nd 1.93 ± 0.33 6.47 ± 0.59 nd nd ndSoja 62.66 ± 2.68 77.80 ± 8.38 nd nd nd nd 140.46aValues shown are means of three repetitions. Type of grain: B, black; C, carioca; S, striped; W, white; Bo, bolinha; R, red. Origin: A, Andean; M,Mesoamerican. Isoflavonoids: DA, daidzein, and GE, genistein. Flavonols: KA, kaempferol, M, myricetin, and Q, quercetin. CM, coumestrol. nd, notdetected.
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30% A/70% B from 0 to 4 min, 100% A from 4 to 50 min, and 30% A/70% B from 50 to 65 min. Chromatograms were recorded by UVdetection at 254, 270, and 330 nm and by ESI-MS in negative-ion scanmode (100−600 m/z) with nitrogen as nebulizer gas at 58 psi and 4.5kV capillary voltage at 350 °C. Identification of flavonoids andcoumestan was based on the following ions ([M − H]−, m/z) for eachanalyte: 253 for daidzein, 269 for genistein, 415 for daidzin, 431 forgenistin, 301 for quercetin, 285 for kaempferol, 317 for myricetin, 267for coumestrol, and 253 for chrysin.Data Analysis. Descriptive statistics were used to characterize the
data overall and across factors. To identify statistical differences inflavonoid content among factors, ANOVA and canonical analysismethods were employed. Statistical analyses were performed by use ofthe Genes program.22
■ RESULTS AND DISCUSSIONIdentification of Bean Flavonoids and Coumestrol.
The maximum and minimum temperatures of the locationwhere the plant material was grown ranged from 17.6 to 27.5°C (Figure 2) with mean monthly rainfall of 7.14 mm
(December 2010−May 2011). Studies on genetic and environ-mental effects, performed on soybean in locations where meantemperatures ranged from 19 to 27 °C, manifested a stronginfluence of the environment on isoflavonoid contents.23,24
In all 16 genotypes assessed, we found the flavonoidsquercetin (Q), myricetin (M), and kaempferol (KA) and thenonglycosylated forms of isoflavonoids daidzein (DA) andgenistein (GE), both of greater occurrence among the legumes,as well as coumestrol, all with wide variation of occurrenceamong the genotypes of the study (Table 1).25
With the exceptions of IAC Alvorada (carioca seed coat),IAC Boreal and IAC Harmonia (striped seed coat), andBrancao Argentino (white seed coat), all the genotypesexhibited genistein (GE), ranging from 0.82 (IAC Perola) to8.93 (IAC Formoso) μg/g.Among the types of grains, the genotypes of black seed coat
and Mesoamerican origin stood out: in addition to containingGE, they were the only ones to also exhibit daidzein (DA), as inthe case of IAC Una (2.30 ± 0.28 μg/g), Arc 1 (6.82 ± 0.95μg/g), and Raz 55 (8.05 ± 1.83 μg/g). A similar result wasreported by Romani et al.,26 who observed the presence ofthese isoflavonoids in a local variety of black bean, and by Diaz-Batalla et al.25 in assessment of cultivated and wild Mexicancommon beans. Liggins et al.10 also reported levels of daidzeinand genistein in beans with red seed coat. IAC Una, Arc 1, andRaz 55 were the only ones to show DA and GE at higher levels.IAC Una is considered to be the main Brazilian cultivar forresistance to the anthracnose pathogen (Colletotrichumlindemuthianum).27 In Arc 1 and Raz55, the presence of theprotein arcelin was observed, which is considered to be themain source of resistance to Zabrotes subfasciatus, one of themain pests of stored grains that occurs in common bean.28
With variation from 0.82 to 14.03 μg/g isoflavonoids for thegenotypes assessed, the results obtained were similar to thosereported in common bean, where contents from 6 to 14 μg/gwere found for different landraces, from 8.3 to 13.4 μg/g forstriped common beans, and from 7.1 to 11.4 μg/kg ofisoflavonoids (daidzein and genistein) in haricot bean.26,27,29
In soybean, a microbial toxic action of the glycosides daidzeinand genistein was proposed in resistance to fungus infection ofseeds and seedlings.30 In the genus Phaseolus, Adesanya et al.31
tested isoflavones, isoflavonones, pterocarpans, and coumestanfrom five species against Aspergillus niger and Cladosporiumcucumericum and observed the importance of at least a phenolicfunction to activate the fungitoxic activity of these compounds,making assessment of the structure−fungitoxic activity relation-ships within these groups of isoflavonoids possible.Corroborating the results found by Beninger et al.,32
flavonols occurred in all the genotypes assessed, except forthe genotype Brancao Argentino (Table 1). The highestoccurrence of the flavonol kaempferol (KA) was seen in grainswith a light-colored seed coat like those of the bolinha andcarioca types. IAC Jabola and IAC Esperanca, both with bolinhaand carioca seed coat types, such as IAC Carioca Comum, IACFormoso, and IAC Perola (all of Mesoamerican origin),exhibited kaempferol contents that ranged from 16.37 to117.65 μg/g (Table 1). A similar profile was found by Ranilla etal.6 in assessment of 25 Brazilian common bean cultivars as afunction of color of the seed coat and of the cotyledon. Thisflavonol modifies a series of cell signaling pathways acting withless toxicity in comparison to standard chemotherapies.33
Quercetin (Q) and myricetin (M) occurred preferentially inblack grains, ranging from 1.09 to 15.85 μg/g and from 26.15 to78.94 μg/g, respectively. Diaz-Batalla et al.25 found a similaroccurrence of quercetin in black grains and kaempferol inlighter-colored grains. In fact, quercetin and kaempferol are themost abundant flavonols in foods and are antioxidant andchelating compounds with beneficial effects on health.34 Theyare also associated with the function of protecting seeds againstpathogens and predators.35 An explanation for the fact ofcommon bean being considered a source of flavonols was thehigh occurrence of quercetin and kaempferol in the cultivar
Table 2. LC-qMS and Calibration Parameters of Flavonoids
Figure 2. Average maximum and minimum temperature during thegrowth biological assay.
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IAC Formoso, exhibiting contents from 9.88 ± 3.04 to 60.07 ±3.17 μg/g.11
Genotypes of Mesoamerican origin with black and cariocaseed coats and of Andean origin with bolinha seed coatmanifested coumestrol (Table 1). Coumestrol is an importantcompound which contains high levels of estrogenic activity,30−100 times greater than that of the isoflavones, and it wasidentified in all the black grains (Table 1), ranging from 0.75(TU) to 17.67 (Raz 55) μg/g.18 Among the grains with cariocaseed coat, IAC Formoso presented the greatest content of thiscoumestan (16.85 ± 0.81 μg/g). Konar36 assessed commonbean and lentils through LC-MS/MS and found this coumestanin common bean (18.5 μg/kg) and a mean value of 16.9 μg/kgin yellow, green, and red lentils. Moreover, by LC-MS/MS andwith standards marked with 13C, Kuhnle et al.37 found contentsranging from 1 to 10 μg/100 g for common bean, valuesconsidered very inferior to ours, even when it is taken intoaccount that the authors expressed their results by fresh weight.IAC Formoso exhibited the greatest concentration of
genistein among all the genotypes assessed (8.93 ± 0.67 μg/g), around 11% of the total presented by the soybean control.Genistein is considered to be the isoflavone with greatestbiological activity and is furthermore classified as an induciblephytoalexin.21 In addition, IAC Formoso exhibited the secondgreatest concentration of coumestrol (16.85 ± 0.81 μg/g), acompound that has been reported as an estrogen receptor twiceas powerful as genistein and, furthermore, a modulator inhormone production of the thymus.38
Grouping Common Bean According to Seed CoatColor. Canonical variable analysis (CVA) was applied to thevalues of flavonoid content of the genotypes assessed. Thismultivariate analysis is similar to principal component analysis,and it is especially used in discriminating analyses withreplication of observations because it is based on the variancesof these replications.The first three canonical variables explained 97.5% of the
total variance among the 16 genotypes and showed theformation of three groups in accordance with the flavonoid andcoumestrol content (Figure 3). A first group gathered thegenotypes Arc 1 and Raz 55, both with a black seed coat and ofMesoamerican origin and with the greatest isoflavonoidcontents found in the study. A second group was composedof IAC Jabola and IAC Esperanca, both with bolinha typegrains and the greatest concentrations of the flavonolkaempferol, which occurred preferentially in light-coloredgrains. All the other genotypes were in a third group.Canonical variable 1 (VC1), with 78.8% of the total variance,
differentiated the genotypes of the study in accordance withdaidzein and genistein content. Thus, Arc 1 and Raz 55 weredifferentiated from IAC Jabola and IAC Esperanca, bypresenting the highest and lowest contents of daidzein andgenistein, respectively (Table 1). Canonical variable 2 (VC2)represents 13.2% of the variation and showed the separation ofthe genotypes in two groups with contrasting values ofconcentration of total flavonoids. In VC2, the first groupgathered IAC Carioca comum, IAC Jabola, IAC Esperanca, IACFormoso, IAC Una, TU, Arc 1 and Raz 55. A second group wasformed by IAC Diplomata, IPR Uirapuru, IAC Alvorada, IACPerola, IAC Harmonia, IAC Boreal, Flor de Mayo, and BrancaoArgentino.The variability found for flavonoid content in the grains of 16
genotypes assessed under field conditions may be explained byhow these materials arose. Thus, factors such as seed coat type
and selection for tolerance to biotic and abiotic stresses mayexplain the different concentrations of metabolites found.Related studies reinforce this hypothesis, where differences inthe variability of isoflavonoids and flavonoids in accordancewith the type of seed coat, genotype, and genotype−environmental interaction were cited by various authors.23,39,40
Important levels of flavonoids were observed in thegermplasm of common bean grown under the same environ-mental conditions and high variability between groups withdifferent seed coat colors. Among the beans with carioca-typeseed coat, which represents nearly all of the common beansgrown, IAC Formoso, a cultivar recently introduced forplanting on a commercial scale, showed high values offlavonoids and coumestrol. This result suggests that thiscultivar may be used in the genealogy of future geneticmaterials with a view toward adoption of nutraceuticalcharacteristics in common bean.
■ ACKNOWLEDGMENTSThis work was financially supported by FAPESP. C.A.C.,A.F.C., M.J.K., and S.A.M.C. thank the Conselho Nacional deDesenvolvimento Cientifico e Tecnolo gico (CNPq) forgranting fellowships.
Figure 3. Multivariate exploratory analysis for 16 genotypes ofcommon bean. Three groups were formed after canonical analysis: theyellow group contained IAC Diplomata (1), IAC Una (2), IPRUirapuru (3), IAC Alvorada (4), IAC Perola (5), IAC Carioca comum(6), TU (7), IAC Harmonia (8), IAC Boreal (9), Brancao Argentino(10), Flor de Mayo (13) and IAC Formoso (16) genotypes; The redgroup contained IAC Jabola (11) and IAC Esperanca (12) genotypes;and the green group contained Arc1 (14) and Raz 55 (15) genotypes.
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■ ABBREVIATIONSANOVA, analysis of variance; DA, daidzein; GE, genistein;HPLC, high-performance liquid chromatography; KA, kaemp-ferol; LC-ESI-QTOF-MS, liquid chromatography−electrosprayionization quadrupole time-of-flight mass spectrometry; M,myricetin; PTFE, poly(tetrafluoroethylene); Q, quercetin
■ REFERENCES(1) Angioi, S. A.; Rau, D.; Attene, G.; Nanni, L.; Bellucci, E.;Logozzo, G.; Negri, V.; Spagnoletti-Zeuli, P. L.; Papa, R. Beans inEurope: Origin and structure of the European landraces of Phaseolusvulgaris L. Theor. Appl. Genet. 2010, 121, 829−843.(2) Carbonell, S. A. M.; Chiorato, A. F.; Carvalho, C. R. L.; Junior, E.U. R.; Ito, M. A.; Borges, W. L. B.; Ticelli, M.; Santos, N. C. B.; Gallo,P. B. IAC Formoso: New carioca common bean cultivar. Crop Breed.Appl. Biotechnol. 2010, 10, 374−376.(3) Espinosa-Alonso, L. G.; Lygin, A.; Widholm, J. M.; Valverde, M.E.; Paredes-Lopez, O. Polyphenols in wild and weedy Mexicancommon beans (Phaseolus vulgaris L.). J. Agric. Food Chem. 2006, 54,4436−4444.(4) Boniglia, C.; Carratu, B.; Gargiulo, R.; Giammarioli, S.; Mosca,M.; Sanzini, E. Content of phytoestrogens in soy-based dietarysupplements. Food Chem. 2009, 115, 1389−1392.(5) Kuhnle, G. G. C.; Dell’Aquila, C.; Runswick, S. A.; Bingham, S. A.Variability of phytoestrogen content in foods from different sources.Food Chem. 2009, 113, 1184−1187.(6) Ranilla, L. G.; Genovese, M. I.; Lajolo, F. M. Polyphenols andantioxidant capacity of seed coat and cotyledon from Brazilian andPeruvian bean cultivars (Phaseolus vulgaris L.). J. Agric. Food Chem.2007, 55, 90−98.(7) Broughton, W. J.; Hernandez, G.; Blair, M. W.; Beebe, S. Beans(Phaseolus spp.) - Model food legumes. Plant Soil 2003, 252, 55−128.(8) Dong, M.; He, X.; Liu, R. H. Phytochemicals of black bean seedcoats: Isolation, structure elucidation, and their antiproliferative andantioxidative activities. J. Agric. Food Chem. 2007, 55, 6044−6051.(9) Pascual-Teresa, S.; Hallundb, J.; Talbotc, D.; Schrootd, J.;Williamse, C. M.; Bugelb, S.; Cassidy, A. Absorption of isoflavones inhumans: effects of food matrix and processing. J. Nutr. Biochem. 2006,17, 257−264.(10) Liggins, L.; Bluck, L. J. C.; Runswick, S.; Atkinson, C.; Coward,W. A.; Bingham, S. A. Daidzein and genistein contents of vegetables.Br. J. Nutr. 2000, 84, 717−725.(11) Dinelli, G.; Bonetti, A.; Minelli, M.; Marotti, I.; Catizone, P.;Mazzanti, A. Content of flavonols in Italian bean (Phaseolus vulgaris L.)ecotypes. Food Chem. 2006, 99, 105−114.(12) Kuhnle, G. G. C.; Dell’Aquila, C.; Low, Y. L.; Kussmaul, M.;Bingham, S. A. Extraction and quantification of phytoestrogens infoods using automated solid-phase extraction and LC/MS/MS. Anal.Chem. 2007, 79, 9234−9239.(13) Boue, S. M.; Burow, M. E.; Wiese, T. E.; Shih, B. Y.; Elliott, S.;Carter-Wientjes, C. H.; McLachlan, J. A.; Bhatnagar, D. Estrogenic andantiestrogenic activities of phytoalexins from red kidney bean(Phaseolus vulgaris L.). J. Agric. Food Chem. 2004, 59, 112−120.(14) Wang, H.; Murphy, P. A. Isoflavone content in commecialsoybean foods. J. Agric. Food Chem. 1994, 42, 1666−1673.(15) Eldridge, A. C.; Kwolek, W. F. Soybean isoflavones: Effect ofenvironment and variety on composition. J. Agric. Food Chem. 1983,31, 394−396.(16) Morris, P. F.; Savard, M. E.; Ward, E. W. B. Identification andaccumulation of isoflavonoids and isoflavone glucosides in soybeanleaves and hypocotyls in resistance responses to Phytophthoramegasperma f.sp. glycinea. Physiol. Mol. Plant Pathol. 1991, 39, 229−244.(17) Hong, Y. H.; Wang, S. C.; Hsu, C.; Lin, B. F.; Kuo, Y. H.;Huang, C. J. Phytoestrogenic compounds in alfalfa sprout (Medicagosativa) beyond coumestrol. J. Agric. Food Chem. 2011, 59, 131−137.(18) Bacaloni, A.; Cavaliere, C.; Faberi, A.; Foglia, P.; Samperi, R.;Lagana, A. Determination of isoflavones and coumestrol in river water
and domestic wastewater sewage treatment plants. Anal. Chim. Acta2005, 531, 229−237.(19) Lee, Y.-H.; Yuk, H. J.; Park, K.-H.; Bae, Y.-S. Coumestrolinduces senescence through protein kinase CKII inhibition-mediatedreactive oxygen species production in human breast cancer and coloncancer cells. Food Chem. 2013, 141, 381−388.(20) Saijo, K.; Collier, J. G.; Li, A. C.; Katzenellenbogen, J. A.; Glass,C. K. An ADIOL-ERbeta-CtBP trans repression pathway negativelyregulates microglia-mediated inflammation. Cell 2011, 145, 584−595.(21) Romani, A.; Vignolini, P.; Galardi, C.; Aroldi, C.; Vazzana, C.;Heimler, D. Polyphenolic content in different plant parts of soycultivars grown under natural conditions. J. Agric. Food Chem. 2003,51, 5301−5306.(22) Cruz, C. D. Programa Genes - Analise multivariada e simulaca o.1st ed.; Editora UFV, Universidade Federal de Vicosa: Vicosa, Brazil,2006; Vol. 1.(23) Carrao-Panizzi, M. C.; Del Pino Beleia, A.; Kitamura, K.;Oliveira, M. C. N. Effects of genetics and environment on isoflavonecontent of soybean from different regions of Brazil. Pesqui. Agropecu.Bras. 1999, 34, 1787−1795.(24) Tsukamoto, C.; Shimada, S.; Igita, K.; Kudou, S.; Kokubun, M.;Okubo, K.; Kitamura, K. Factors affecting isoflavone content insoybean seeds: Changes in isoflavones, saponins, and composition offatty acids at different temperatures during seed development. J. Agric.Food Chem. 1995, 43, 1184−1192.(25) Díaz-Batalla, L.; Widholm, J. M.; Fahey, G. C.; Costano-Tostado, E.; Paredez-Lopez, O. Chemical components with healthimplications in wild and cultivated Mexican common bean seeds(Phaseolus vulgaris L.). J. Agric. Food Chem. 2006, 54, 2045−2052.(26) Romani, A.; Vignolini, P.; Galardi, C.; Mulinacci, N.;Benedettelli, S.; Heimler, D. Germplasm characterization of Zolfinolandraces (Phaseolus vulgaris L.) by flavonoid content. J. Agric. FoodChem. 2004, 52, 3838−3842.(27) Carbonell, S. A. M.; Ito, M. F.; Pompeu, A. S.; Francisco, F. G.;Ravagnani, S.; Almeida, A. L. L. Racas fisiologicas de Colletotrichumlindemuthianum e reacao de linhagens e cultivares de feijoeiro noEstado de Sao Paulo. Fitopatol. Bras. 1999, 24, 60−65.(28) Ribeiro-Costa, C. S.; Pereira, P. R. V. S.; Zulovski, L.Desenvolvimento de Zabrotes subfasciatus (Boh.) (Coleoptera:Chrysomelidae, Bruchinae) em genotipos de Phaseolus vulgaris L.(Fabaceae) cultivados no estado do Parana e contendo arcelina.Neotrop. Entomol. 2007, 36, 560−564.(29) Konar, N.; Poyrazoglu, E. S.; Demir, K.; Artik, N. Determinationof conjugated and free isoflavones in some legumes by LC-MS/MS. J.Food Compos. Anal. 2012, 25, 173−178.(30) Graham, T. L.; Kim, J. E.; Graham, M. Y. Role of constitutiveisoflavone conjugates in the accumulation of glyceollin in soybeaninfected with Phytophthora megasperma. Mol. Plant-Microbe Interact.1990, 3, 157−166.(31) Adesanya, S. A.; O’Neill, M. J.; Roberts, M. F. Structure-relatedfungitoxicity of isoflavonoids. Physiol. Mol. Plant Pathol. 1986, 29, 95−103.(32) Beninger, C. W.; Hosfield, G. L.; Bassett, M. J. Flavonoidcomposition of three genotypes of dry bean (Phaseolus vulgaris)differing in seed coat color. J. Am. Hortic. Sci. 1999, 124, 514−518.(33) Zhang, Y.; Chen, A. Y.; Li, M.; Chen, C.; Yao, Q. Ginkgo bilobaextract kaempferol inhibits cell proliferation and induces apoptosis inpancreatic cancer cells. J. Surg. Res. 2008, 148, 17−23.(34) Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J. Flavonoidantioxidants: Chemistry, metabolism and structure-activity relation-ships. J. Nutri. Biochem. 2002, 13, 572−584.(35) Islam, F. M. A.; Rengifo, J.; Redden, R. J.; Basford, K. E.; Beebe,S. E. Association between seed coat polyphenolics (tannins) anddisease resistance in common bean. Plant Food Hum. Nutr. 2003, 58,285−297.(36) Konar, N. Non-isoflavone phytoestrogenic compound contentsof various legumes. Eur. Food Res. Technol. 2013, 236, 523−530.(37) Kuhnle, G. G. C.; Dell’Aquila, C.; Aspinall, S. M.; Runswick, S.A.; Joosen, A. M. C. P.; Mulligan, A. A.; Bingham, S. A. Phytoestrogen
Journal of Agricultural and Food Chemistry Article
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content of fruits and vegetables commonly consumed in the UK basedon LC-MS and 13C-labelled standards. Food Chem. 2009, 116, 542−554.(38) Ndebele, K.; Graham, B.; Tchouwou, P. B. Estrogenic activity ofcoumestrol, DDT, and TCDD in human cervical cancer cells. Int. J.Environ. Res. Public. Health 2010, 7, 2045−2056.(39) Lee, S. J.; Seguin, P.; Kim, J. J.; Moond, H. I.; Ro, H. M.; Kim, E.H.; Seo, S. H.; Kang, E. Y.; Ahn, J. K.; Chung, I. M. Isoflavones inKorean soybeans differing in seed coat and cotyledon color. J. FoodCompos. Anal. 2010, 23, 160−165.(40) Kim, J. K.; Kim, E. H.; Lee, O. K.; Park, S. Y.; Lee, B.; Kim, S.H.; Park, I.; Chung, I. M. Variation and correlation analysis of phenoliccompounds in mung bean (Vigna radiata L.) varieties. Food Chem.2013, 141, 2988−2997.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf5033312 | J. Agric. Food Chem. 2014, 62, 9699−97049704
