Phytochemical fingerprinting of vegetable Brassica oleracea and Brassica napus by simultaneous...

Phytochemical Fingerprinting of VegetableBrassica oleracea and Brassica napus bySimultaneous Identification of Glucosinolatesand PhenolicsPablo Velasco,a* Marta Francisco,a Diego A. Moreno,b Federico Ferreres,b

Cristina García-Viguerab and María Elena Carteaa

ABSTRACT:Introduction – Brassica vegetables have been related to the prevention of cancer and degenerative diseases, owing to theirglucosinolate and phenolic content.Objective – Identification of glucosinolates, flavonoids and hydroxycinnamic acids in representative varieties of kale, cabbageand leaf rape.Methodology – One local variety of each crop was evaluated in this study using a multi-purpose chromatographic method thatsimultaneously separates glucosinolates and phenolics. Chromatograms were recorded at 330 nm for flavonoid glycosides andacylated derivatives and 227 nm for glucosinolates.Results – Eight glucosinolates were identified in kale and cabbage, which exhibited the same glucosinolate profile, and 11glucosinolates were identified in leaf rape. Furthermore, 20 flavonoids and 10 hydroxycinnamic acids were detected in kale andcabbage, while 17 flavonoids and eight hydroxycinnamic acids were found in leaf rape.Conclusions – This study has provided a deeper and comprehensive identification of health-promoting compounds in kale,cabbage and leaf rape, thus showing that they are a good source of glucosinolates and phenolic antioxidants. Copyright © 2011John Wiley & Sons, Ltd.

Keywords: Vegetable brassicas; secondary metabolites; anticarcinogenic; antioxidant; LC-PAD-ESI/MSn

IntroductionBrassica vegetables have been related to the prevention of degen-erative diseases and different types of cancer as well asto cardiovascular health promotion (Cartea and Velasco, 2008;Traka and Mithen, 2009). Compounds that appear to contribute tothese health-related properties of Brassica species and other foodplants include isothiocyanates and their cognate glucosinolates,phenolics, including flavonoids and other non-nutrients (Jahangiret al., 2009; Jeffery and Araya, 2009) showing a variety of physi-ological activities, such as antioxidants, enzyme modulation andapoptosis and cell cycle controlling activities (Duthie et al., 2000).

Glucosinolates are a large group of sulphur-containing sec-ondary plant metabolites that occur in all Brassica crops. There isa wide variety of glucosinolates, but all share a b-thioglucosideN-hydroxysulfate common structure, containing a b-D glucopy-ranosyl moiety and a variable side-chain derived from methion-ine, tryptophan or phenylalanine. Upon cellular disruption,glucosinolates are hydrolysed to various bioactive breakdownproducts by the endogenous enzyme myrosinase. Isothiocyan-ates and indole glucosinolate metabolites (in particular indol-3-carbinol) are the two major groups of autolytic breakdownproducts of glucosinolates. Both of them exhibit protectiveactivities against different types of cancer. Several studies havereported that these compounds may affect distinct stages of

cancer development, including the induction of detoxificationenzymes (phase II enzymes) and the inhibition of activationenzymes (phase I enzymes) (Mithen et al., 2003), as well as anti-proliferative mechanisms like cell cycle arrest or apoptosis(Clarke et al., 2008).

Phenolic compounds are a large group of phytochemicalswidespread in the plant kingdom. They can be classified intosimple phenols, phenolic acids, hydroxycinnamic acid deriva-tives and flavonoids, depending on their structure. The mostcommon are the flavonoids that share a C6–C3–C6 phenylchro-mane skeleton. Flavonoids and hydroxycinnamic acid deriva-tives are widely distributed in plants and are importantbiologically active constituents of the human diet. In Brassicafoods, flavonoids are complex, containing up to five sugar resi-dues, and these may be further substituted with hydroxycin-namic residues (Vallejo et al., 2004). Bioavailability and activity of

* Correspondence to: P. Velasco, Misión Biológica de Galicia (CSIC), PO Box 28,E-36080 Pontevedra, Spain. E-mail: [email protected]

a Misión Biológica de Galicia (CSIC), PO Box 28, E-36080 Pontevedra, Spain

b Department of Food Science and Technology, CEBAS-CSIC, CampusUniversitario de Espinardo-Edificio 25, PO Box 164, Espinardo, E-30100Murcia, Spain

Research Article

Received: 24 February 2010; Revised: 1 June 2010; Accepted: 2 June 2010 Published online in Wiley Online Library: 24 January 2011

(wileyonlinelibrary.com) DOI 10.1002/pca.1259

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Phytochem. Anal. 2011, 22, 144–152Copyright © 2011 John Wiley & Sons, Ltd.

different heterosides depend on their substituents (Cermaket al., 2003). For this reason, it is important to characterise andquantify the different derivatives of phenolic compounds. Thesecompounds have direct antioxidant and free radical-scavengingactivities, but they can also induce the expression of variousgenes by encoding metabolic enzymes thought to decrease therisk of various diseases and disorders (Halliwell et al., 2005;Manach et al., 2005).

The Brassicaceae family has been widely investigated for glu-cosinolate (Kushad et al., 1999; Padilla et al., 2007c; Cartea et al.,2008a, b) and phenolic composition (Llorach et al., 2003b; Vallejoet al., 2004; Ferreres et al., 2005, 2006; Romani et al., 2006; Sousaet al., 2008). Nowadays, the profile of different Brassica species onthese molecules is well established, although their analysisrequires different methods, therefore making it laborious andtime consuming. Both phenolic compounds and glucosinolateshave beneficial properties on human health and synergisticeffects may exist between them. Bennett et al. (2003, 2006) useda method for analysing both kinds of compounds on differentspecies, which was later applied to a set of turnip green andturnip top local populations (B. rapa var. rapa; Francisco et al.,2009). However, as far as we know, no similar studies have beenconducted for other Brassica crops, like B. oleracea acephala, capi-tata groups and B. napus.

In Galicia (northwestern Spain), different Brassica species areused as leaf vegetable products for human and also for animalconsumption. Kales (Brassica oleracea acephala group), cabbages(B. oleracea capitata group), leaf rape (B. napus pabularia group)and turnip tops and turnip greens (B. rapa rapa group) are themost important Brassica crops in this region.

A collection of local varieties of B. oleracea and B. napus is keptat the Misión Biológica de Galicia (MBG-CSIC, Pontevedra, Spain),as part of the Brassica genus germplasm bank. In previousreports, this collection was characterised based on morphologi-cal and agronomical traits (Picoaga et al., 2003; Rodriguez et al.,2005; Padilla et al., 2007a; Soengas et al., 2008; Vilar et al., 2008)and the profile of desulphoglucosinolates in leaves was studied(Cartea et al., 2008a, b; Velasco et al., 2008). To date, no informa-tion is available on the content of intact glucosinolates and phe-nolic compounds in these species. Therefore, the objective of thiswork was to identify glucosinolates, flavonoids and hydroxycin-namic acids in representative varieties of kale, cabbage and leafrape, in order to make a more comprehensive assessment of thefunctional value of these species. Identification was carried outby means of a high-performance liquid chromatography, usingboth photodiode array and electrospray ionisation mass spec-trometry detection (LC-PAD-ESI/MSn).

ExperimentalPlant material

One local variety of each crop was evaluated in this study: a kale varietynamed ‘MBG-BRS0468’ (B. oleracea acephala), a white cabbage varietynamed‘MBG-BRS0057’(B. oleracea capitata) and a leaf rape variety named‘MBG-BRS0063’ (B. napus pabularia). These varieties are in the germplasmcollection at the MBG-CSIC and were selected based on previous agro-nomic and nutritional evaluations (Rodriguez et al., 2005; Cartea et al.,2008a, b; Soengas et al., 2008; Vilar et al., 2008). Populations were plantedin multipot-trays and seedlings were transplanted into the greenhouse atthe five or six leaves stage. Greenhouse conditions were: light 16:8 h,maximum temperature 25°C and minimum temperature 10°C. The soiltype was acid sandy loam. The third leaf of each plant was collected 3

months after transplanting, when the plants were well developed, inorder to extract and analyse metabolites. After harvesting on dry ice, thematerial was immediately transferred to the laboratory and frozen at-80°C, prior to their lyophilisation. The dried material was powdered byusing an IKA-A10 mill (IKA-Werke GmbH & Co.KG, Staufen, Germany) andthe powder was analysed.

Sample preparation

An adaptation of the method described by Bennett et al. (2006) for thesimultaneous extraction and analysis of glucosinolates and phenolics wasused. Fifty milligrams of each sample were extracted in 1.5 mL 70%methanol at 70°C for 30 min with vortex mixing every 5 min to facilitatethe extraction. Samples were centrifuged (13000 g, 15 min, 4°C). Thesupernatants were collected and methanol was completely removedusing a rotary evaporator under vacuum at 37°C. The dry materialobtained was redissolved in 1 mL of ultrapure water and filtered through0.20 mm syringe PTFE filters (AnotopTM, Whatman International Ltd,Kent, UK).

Alkaline hydrolysis

In order to study acyl flavonoid derivatives, an alkaline hydrolysis wascarried out to eliminate acid moieties like p-coumaroyl (m/z 146) andcaffeoyl (m/z 162), which coincide with those of rhamnosyl and hexosylresidues respectively and may, therefore, provide a misidentification inMS analysis. Hydrolysis was performed as follows: 1 mL of the extract plus1 mL of 2 M sodium hydroxide (up to pH 9–10) for 12 h at room tempera-ture in a stoppered test tube under nitrogen atmosphere. The alkalinehydrolysis products were acidified with concentrated hydrochloric acid(up to pH 1–2) and directly analysed by LC-PAD-ESI/MSn.

LC-PAD-ESI/MSn analyses

Chromatographic analyses were carried out on a Luna C18 column (250 ¥4.6 mm, 5 mm particle size; Phenomenex, Macclesfield, UK). The mobilephase was a mixture of (A) 0.1% trifluoroacetic acid (TFA) and (B)acetonitrile–TFA (99.9:0.1) in a linear gradient starting with 0% B at0–5 min, reaching 17% B at 15–17 min, 25% B at 22 min, 35% B at 30 min,50% B at 35 min, 99% B at 50 min and at 55–65 min 0% B. The flow ratewas 1 mL/min and the injection volume was 20 mL. Chromatograms wererecorded at 330 nm for flavonoid glycosides and acylated derivatives andat 227 nm for glucosinolates. The LC-PAD-ESI/MSn analyses were carriedout in an Agilent HPLC 1100 series equipped with a photodiode arraydetector and a mass detector in series (Agilent Technologies, Waldbronn,Germany). The equipment consisted of a binary pump (model G1312A),an autosampler (model G1313A), a degasser (model G1322A) and a pho-todiode array detector (model G1315B). The HPLC system was controlledby ChemStation software (Agilent, version 08.03). The mass detector wasan ion trap spectrometer (model G2445A) equipped with an electrosprayionisation interface and was controlled by LCMSD software (Agilent,version 4.1). The ionisation conditions were adjusted at 350°C and 4 kV forcapillary temperature and voltage, respectively. The nebuliser pressureand flow rate of nitrogen were 65.0 psi and 11 L/min, respectively. Thefull-scan mass covered the range from m/z 100 up to m/z 1500. Collision-induced fragmentation experiments were performed in the ion trapusing helium as the collision gas, with voltage ramping cycles from 0.3 upto 2 V. Mass spectrometry data were acquired in the negative ionisationmode. MSn was carried out in the automatic mode on the more abundantfragment ion in MS(n-1).

For a quantitative analysis of glucosinolates and phenolic com-pounds, 20 mL of lyophilised extracts were analysed using the samecolumn and conditions mentioned in the previous paragraph, in anHPLC system (Waters Cromatografía SA, Barcelona, Spain), consisting ofa W600E multisolvent delivery system, an in-line degasser, a W717Plusautosampler and a W2996 photodiode array detector. Chromatograms

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were recorded at 227 nm for glucosinolates and 330 nm for phenoliccompounds.

Results and DiscussionIdentification of glucosinolates

The MS product ions obtained from glucosinolates were domi-nated by intense [M - H]-ions and other fragments. Glucosinolatefragmentation revealed two groups of typical fragments; one isassociated with the common moiety of glucosinolates (aglycone)and the other provides useful diagnostic ions for the identifica-tion of the variable side chain. As described by other authors(Fabre et al., 2007; Rochfort et al., 2008), the MS2 fragmentation ofthe aglycone side chain produces specific ions at m/z 195, 241,259 and 275. The MS3 fragmentation of the majority ion at m/z259 gives rise to fragments at m/z 139 and 97 (corresponding tothe sulphate group [SO4H]-) and m/z 81 ([SO3H]-). The m/z 97fragment ion is formed with high abundance in ESI negativemode (Mellon et al., 2002). Therefore, fragments at m/z 259 andm/z 97 constitute a very useful preliminary screening method fordetermining the presence of glucosinolates in plant extracts(Millán et al., 2009).

All glucosinolates also showed an intense, consistent andconstant neutral loss under the fragmentation conditionscorresponding to the combined loss of sulphur trioxide andanhydroglucose ([M - H - 242]-). Other MS fragmentation path-ways were the losses of glucose radical [M - H - 162]- and/orthioglucose moiety [M - H - 196]- after the H-rearrangement ofthe side chain to the sulphur atom in thioglucose moiety

(Kokkonen et al., 1991). Indolic glucosinolates were also charac-terised by means of the examination of characteristic productions from their specific R. This group contains two nitrogenatoms and the m/z values of their deprotonated moleculeswere thus at odd mass numbers. Neoglucobrassicin and4-methoxyglucobrassicin, which exhibit identical molecularmasses and fragmentation ions, were differentiated by compari-son with reported elution sequence during reversed-phaseHPLC (Kushad et al., 1999; Francisco et al., 2009).

Therefore, the molecular ion [M - H]- of glucosinolates, theirfragment ion pattern and the retention times allowed the iden-tification of eight glucosinolates in kale and cabbage, whichexhibited the same glucosinolate profile, and 11 glucosinolatesin B. napus (Fig. 1): m/z 422, glucoiberin; m/z 388, progoitrin; m/z358, sinigrin; m/z 436, glucoraphanin; m/z 402, gluconapoleif-erin; m/z 372, gluconapin; m/z 463, 4-hydroxyglucobrassicin;m/z 386, glucobrassicanapin; m/z 447, glucobrassicin; m/z 422,gluconasturtiin; m/z 477, 4-methoxyglucobrassicin; and m/z477, neoglucobrassicin. The mass spectral information of theglucosinolates identified is summarised in Table 1. The GSprofile found in these three crops was similar to the profilesreported by other authors in B. oleracea and B. napus leaves(Cartea et al., 2008a, b). Cartea et al. (2008a, b) studied the glu-cosinolate content of the collection of kale, cabbage and leafrape kept at the MBG by HPLC-DAD and some differences werefound regarding the current work. Eight glucosinolates wereidentified in the B. napus collection by HPLC-DAD. In the presentwork, 4-methoxyglucobrassicin was not detected and four otherglucosinolates (4-hydroxyglucobrassicin, glucoiberin, sinigrinand glucoraphanin) were identified in trace quantities (Table 1,

Figure 1. Glucosinolate profile of leaf rape (B. napus pabularia group), cabbage (B. oleracea capitata group), and kale crops (B. oleracea acephala group).GIB, glucoiberin; PRO, progoitrin; SIN, sinigrin; GRA, glucoraphanin; GNL, gluconapoleiferin; GNA, gluconapin; OHGBS, 4-hydroxiglucobrassicin; GBN,glucobrassicanapin; GBS, glucobrassicin; GST, gluconasturtiin; MGBS, 4-methoxyglucobrassicin; NGBS, neoglucobrassicin.

P. Velasco et al.

Phytochem. Anal. 2011, 22, 144–152Copyright © 2011 John Wiley & Sons, Ltd.View this article online at wileyonlinelibrary.com

146

Fig. 1). With regard to kales and cabbages, Cartea et al. (2008b)found and quantified 10 and 15 glucosinolates, respectively. Inthis work, the most abundant glucosinolates (i.e. glucoiberin,sinigrin or glucobrassicin) were identified in both crops, butother minor glucosinolates like progoitrin, glucoiberverin, glu-coalyssin and glucobrassicanapin were not found in kale andcabbage (Table 1, Fig. 1). Concentrations of the main glucosino-lates are shown in Table 2.

Identification of flavonoids

The HPLC-DAD chromatogram of Brassica vegetable extractsrevealed the existence of glycosylated derivatives of three fla-vonoids with substitution in position 3, that is, kaempferol (267,300sh and 349 nm), quercetin (255, 267sh and 355 nm) andisorhamnetin (255, 268sh, 294sh and 354 nm). In addition, acy-lated flavonoids were detected in the extract, and their UVspectra, characterised by a high maximum absorption at 330 nmand a little maximum between 255 and 268 nm, suggested thatthe flavonoid-glycoside molecules were linked to hydroxycin-namic acid derivatives, in which sinapic, ferulic, caffeic andp-coumaric acids were the most abundant. Results are sum-marised in Table 3. The chromatographic profiles, recorded at330 nm, of the naturally occurring phenolic compounds in leafrape, kale and cabbage extracts and the deacylated phenoliccompounds, obtained after alkaline hydrolysis, are shown inFig. 2.

Alkaline hydrolysis was performed to reduce the complexity ofthe naturally occurring compounds present in the plant extracts

Table 1. List of glucosinolates identified with their corresponding retention times and MS data in extracts of leaf rape (B. napuspabularia group), cabbage (B. oleracea capitata group) and kale crops (B. oleracea acephala group)

Code Compound Rt

(min)m/z

[M - H]-MS2 [M - H]- m/z (%) Leaf

rapeKale Cabbage

GIB Glucoiberin 4.8 422 358(100), 342(1), 291(2), 275(3), 259(5), 195(2),180(6)

¥ ¥ ¥

PRO Progoitrin 5.9 388 332(29), 308(12), 301(17), 275(40), 259(100),240(18) 210(78), 195(14), 192(24), 154(16),136(42), 130(30)

¥ — —

SIN Sinigrin 6.6 358 278(2), 275(7), 259(100), 241(28), 195(4), 180(3),162(8), 135(2), 116(5)

¥ ¥ ¥

GRA Glucoraphanin 7.4 436 420(6), 372(100), 356(1), 291(2), 275(1), 259(3),194(2), 162(4)

¥ ¥ ¥

GNL Gluconapoleiferin 12.3 402 306(53), 275(20), 259(100), 240(7), 225(10), 215(24),195(10), 163(13), 160(9), 145(18), 140(23), 120(9)

¥ — —

GNA Gluconapin 12.5 372 292(4), 275(30), 259(100), 241(37), 227 (9), 195(25),176(8), 139 (11), 130(10)

¥ — —

OHGBS 4-Hydroxiglucobrassicin 13.1 463 403(2), 383(10), 365(6), 300(6), 285(73), 267(100),259(17), 240(25), 220(17), 169(30), 160(23),132(5)

¥ ¥ ¥

GBN Glucobrassicanapin 15.7 386 306(4), 275(21), 259(100), 241(33), 208(12), 195(6),163(4), 144(18) 139(8)

¥ — —

GBS Glucobrassicin 16.8 447 367(22), 291(6), 275(34), 259(100), 251(17), 241(11),224(3), 205(28), 195(11), 144(9)

¥ ¥ ¥

GST Gluconasturtiin 19.5 422 342(9), 275(24), 259(100), 244(7), 229(8), 195(13),180(24), 169(1), 163(5), 145(4), 140(6), 119(4)

¥ ¥ ¥

MGBS 4-Methoxyglucobrassicin 20.4 477 397(25), 299(14), 291(69), 275(98), 259(62), 241(80),235(100), 198(80), 195 (16), 144(70)

— ¥ ¥

NGBS Neoglucobrassicin 23.5 477 447(68), 446(100), 429(16), 385(5), 273(12), 259(16),241(26), 224(4), 205(5)

¥ ¥ ¥

Table 2. Concentration (mm/g d.w.) of the major glucosino-lates and phenolics found in extracts of leaf rape (B. napuspabularia group), cabbage (B. oleracea capitata group) andkale crops (B. oleracea acephala group)

Compound Leaf rape Kale Cabbage

Glucoiberin — 1.99 2.66Progoitrin 3.05 — —Sinigrin — 1.64 1.4Gluconapin 3.53 — —Glucobrassicanapin 2.05 — —Glucobrassicin 2.51 4.11 5.81Kaempf-3-O-(methoxycaffeoyl)-

soph-7-O-glc2.88 2.45 1.90

Kaempf-3-O-(caffeoyl)-soph-7-O-glc

2.10 2.78 2.17

Kaempf-3-O-(feruloyl)-soph-7-O-glc + Kaempf-3-O-(p-coumaroyl)-soph-7-O-glc

2.95 3.05 2.69

3-Caffeoyl quinic acid 0.71 0.85 0.643-p-Coumaroyl quinic acid 0.41 0.55 0.50Sinapic acid 10.41 2.42 1.801-Sinapoyl-2-

feruloylgentiobioside2.33 4.51 3.26


147


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due to the release of the hydroxycinnamic acids by cleavage ofthe ester linkage between the acids and the glycosides (Martens,2002). Apart from several hydroxycinnamic acids, after alkalinehydrolysis the chromatogram showed various flavonoid glyco-sides and the disappearance of the acylated derivatives (Fig. 2).

Deacylated flavonoids. The MS ion trap analysis of the saponi-fied extracts of the leaves showed mainly the presence of severalkaempferol derivatives, but quercetin and isorhamnetin werealso determined. The presence of the ion at m/z 285/284 [Agl -H/2H]- as base peak for compounds 2, 3, 4, 7, 8 and 9 shows themas kaempferol derivatives (3,5,7,4’-tetrahydroxyflavone), whilein compounds 1 and 6 this ion was m/z 301/300, indicativeof quercetin derivatives (3,5,7,3’,4’-pentahydroxyflavone pen-tahydroxyflavone) and m/z 315 for 5 isorhamnetin derivatives(3,5,7,4’-tetrahydroxy-3’-methoxyflavone; Table 3). The MS2 [M -H]- fragmentation analysis of compounds 1–5 showed ions [M -H - 162]- as the base peak, which indicated a loss of glycosidicresidue at position 7 (Ferreres et al., 2004). According to previousstudies (Llorach et al., 2003a; Ferreres et al., 2004; Vallejo et al.,2004), the fragmentation pattern and the relative abundance ofthe ions obtained indicated that compounds 1–3 are glycosy-lated with a hexoside in position 7 and a di- or trihexoside inposition 3. The first fragmentation of the deprotonated molecularion [M - H]- in this compounds is expected to be always due tothe breakdown of the O-glycosidic bond at position 7 (Ferreres

et al., 2004). The remaining glycosyl moieties of the flavonoidmolecule are expected to be linked to the hydroxyl at position 3on the flavonol aglycone. The fragmentation MS3 [(M - H) →(M - H - 162)]- of 1–3 showed losses from interglycosidicfragmentations at position 3 of the ring that, with theprevious Brassica works mentioned above, suggest the (1 → 2)interglycosidic linkage between the disaccharide moieties ofthe flavonoids (mainly sophorosides). These compounds weretentatively characterised as (1) quercetin-3-O-sophoroside-7-O-glucoside; (2) kaempferol-3-O-sophorotrioside-7-O-glucoside;and (3) kaempferol-3-O-sophoroside-7-O-glucoside. Compounds4 and 5 were characterised as flavonoids with two sugar moietieslinked to different phenolic hydroxyl (di-O-glycosidics). Accord-ing to Ferreres et al. (2004), in these 3,7-di-O-glucosides, a basepeak ion at [M-H - 162]- in the MS2[M-H]- mode is alwaysobserved (Table 3). On the other hand, for compounds 6–8 thefragment ion [M - H - 180]- and the appearance of [Agl-H]- as thebase peak in the MS2[M - H]-, together with the fragmentations,show them to be flavonol-O-diglycosidics. The UV spectra andthe MS fragmentation for compounds 7 and 8 show that theyare kaempferol-3-O-dihexosides isomers. The [M - H - 180]- ionwas not observed in the fragmentation of 7, while this ion isvery important for compound 8, indicating a interglycosidiclinkage (1 → 2) for this compound. Thus, they were identifiedas (4) kaempferol-3,7-di-O-glucoside, (5) isorhamnetin-3,7-di-O-glucoside, (6) quercetin-3-O-sophoroside, (7) kaempferol-3-O-

Figure 2. Phenolic profiles of leaf phenolics in rape (B. napus pabularia group), cabbage (B. oleracea capitata group), and kale (B. oleracea acephalagroup). 1, Querc-3-O-soph-7-O-glc; 2, Kaempf-3-O-triglc-7-O-glc; 3, Kaempf-3-O-soph-7-O-glc; 4, Kaempf-3,7-di-O-glc; 5, Isorhmnt-3,7-di-O-glc;6, Querc-3-O-soph; 7, Kaempf-3-O-diglc; 8, Kaempf-3-O-soph; 9, Kaempf-7-O-glc; 10, Querc-3-O(caffeoyl)-soph-7-O-glc; 11, Kaempf-3-O-(methoxycaffeoyl)-soph-7-O-glc; 12, Kaempf-3-O-(caffeoyl)-soph-7-O-glc; 13, Querc-3-O-(sinapyl)-soph-7-O-glc; 14, Kaempf-3-O-(sinapoyl)-soph-7-O-glc; 15, Kaempf-3-O-(feruloyl)-soph-7-O-glc; 16, Kaempf-3-O-(p-coumaroyl)-soph-7-O-glc; 17, Kaempf-3-O-(caffeoyl)-soph-7-O-glc (isomer); 18,Kaempf-3-O-(p-coumaroyl)-soph; 19, Kaempf-3-O-(methoxycaffeoyl)-soph; 20, Kaempf-3-O-(caffeoyl)-soph; 21, Querc-3-O-(feruloyl)-soph; 22,Kaempf-3-O-(sinapoyl)-soph; 3CQAc, 3-caffeoyl quinic acid; 3pCoQAc, 3-p-coumaroyl quinic acid; 4CQAc, 4-caffeoyl quinic acid; SG, sinapylglucoside;4FQAc, 4-feruloyl quinic acid; SA, sinapic acid; A, 1,2-disinapoylgentiobioside; B, 1-sinapoyl-2-feruloylgentiobioside; C, 1,2,2’-trisinapoylgentio-bioside; D, 1,2’-disinapoyl-2-feruloylgentiobioside


149


diglucoside and (8) kaempferol-3-O-sophoroside. Compound 9was identified as a monoglycoside kaempferol derivative withhydroxyl at free position 3: (9) kaempferol-7-O-glucoside.

In native extracts of kale, cabbage and leaf rape, we observedcompounds previously described by other authors in Brassicaspp. (Llorach et al., 2003a, b; Ferreres et al., 2004, 2005, 2006;Vallejo et al., 2004). Compound 5 was only found in B. napusextracts in trace quantities.

Acylated flavonoids. Several of the flavonoids in the Brassicasample extracts had UV spectra with a broad maximum absor-bance around 330–340 nm (Table 3), suggesting that they wereacylated with hydroxycinnamic acids (Fig. 2). Comparison of theHPLC-DAD chromatogram of the extracts with that of the saponi-fied extract (Fig. 2) indicated the existence of acylated com-pounds in high amounts. The MS study of these compoundsallowed detection of a total of 13 acylated flavonol glycosides(compounds 10–22). These compounds were acyl derivativesfrom compounds 1 (10, 13), 3 (11, 12, 14, 15, 16, 17), 6 (21) and8 (18, 19, 20, 22). Fragmentation of some of these acylatedderivatives showed a base peak in MS2 resulting from the loss ofsugar in position 7 [(M - H) - 162)]-. This fragmentation is typicalof flavonid-3-O-(acyl)glycoside-7-O-hexoside and has beenwidely described in different Brassicas (Llorach et al., 2003b; Fer-reres et al., 2005, 2006, 2008). Other important ion was alsodetected due to the loss of the acyl radical and/or the sugar andacids from the [M - H]-. The resulting fragmentation after the lossof sugar residues at position 7 {-MS3 [(M - H) → (M - H - 162)]-},showed that the acid loss is easily detected and that the acylationis always present on sugars at position 3 in these compounds.Losses of 162, 206, 176, 146 and 192 have been identified ascaffeic acid, sinapic acid, ferulic acid, p-coumaric acid and meth-oxycaffeic, respectively. The ion corresponding to the flavonoid3-O-glycoside was always the base peak in MS3 of these com-pounds. Thus, they had been characterised as acylated deriva-tives of quercetin-3-O-sophoroside-7-O-glucoside with caffeoyl(10) and sinapoyl (13) and kaempferol-3-O-sophoroside-7-O-glucoside with methoxycaffeoyl (11), caffeoyl (12 and 17),sinapoyl (14), feruloyl (15) and p-coumparoyl (16). Compounds18–22 presented a fragmentation MS2 [M - H]- similar to the MS3

of previous compounds (Table 3), which is expected of flavonoidswith glycosilation on a single phenolic hydroxyl. In addition, anion resulting from the loss of the acyl radical and fragment m/z180 (162 +18) [(M - H) - acyl - 180]- from the interglycosi-dic breakdown was observed in the fragmentation of 19–22,confirming the structure of flavonoid-O-diglycosides. Thesecompounds have been characterised as acyl derivatives ofkaempferol-3-O-sophoroside with p-coumaroyl (18), methoxy-caffeoyl (19), caffeoyl (20), sinapoyl (22) and quercetin-3-O-(feruloyl) sophoroside (21).

Identification of hydroxycinnamic acids

According to previous studies, 10 hydroxycinnamic acids andderivatives (3CQAc, 3pCoQAc, SG, 4CQAc, 4FQAc, SA, A, B, Cand D) were detected in leaves of leaf rape, kale and cabbage(Table 4, Fig. 2; Llorach et al., 2003a; Ferreres et al., 2006; Franciscoet al., 2009). The most abundant in the three crops were3-caffeoyl quinic acid [3CQAc; Table 2; Rt 17.3 min; UV 295sh,325 nm; MS: 353, MS2 (353): 191(100), 179(62)], 3-p-coumaroylquinic acid [3pCoQAc; Rt 19.1 min; UV 311 nm; MS: 337, MS2 (337):191(7), 179(100)] and sinapoylglucoside [SG; Rt 20.5 min; UV

Tab

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(m/z

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m/z

(%)

MS3 [(

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224)

]-Le

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Kale

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P. Velasco et al.


150

329 nm; MS: 285, MS2 (285): 291(100), 223 (85)]. 4-caffeoyl quinic[4CQAc; Rt 19.6 min; UV 295sh, 326 nm; MS: 353, MS2 (353):191(16), 179(53), 173(100), 135(12)] and 4-feruloyl quinic [4FQAc;Rt 22.9 min; UV 325 nm; MS: 367, MS2 (367): 191(5), 173(3),163(100), 119(5)] were also identified in kale and cabbage, butnot in leaf rape. Derivatives formed from the interaction ofhydroxycinnamic acids with quinic acid and glucose have beenreported in kale, pak choi, Chinese leaf mustard, turnip greensand turnip tops (Rochfort et al., 2006; Ayaz et al., 2008; Ferrereset al., 2008; Francisco et al., 2009; Olsen et al., 2009). Sinapic acid[SA; Rt 27.3 min; UV 329 nm; MS: 223, MS2 (223): 208(35), 179(30),164(100)] was a compound present in high quantities in B. napusand detected in trace amounts in the two B. oleracea crops.

Other hydroxycinnamic acid derivatives identified were sinapicand ferulic acids, which were esterified carrying more than onehexose moiety (compounds A–D). In all cases, the loss of 224from the deprotonated molecular ion corresponded to sinapicacid (Table 4). Compounds A and D also presented ferulic acidand displayed the loss of this acid (194). By comparison with datareported earlier in other Brassicas (Llorach et al., 2003a; Ferrereset al., 2006), these compounds were tentatively identified as: 1,2-disinapoylgentiobioside (A), 1-sinapoyl-2-feruloylgentiobioside(B), 1, 2, 2’-trisinapoylgentiobioside (C) and 1,2’-disinapoyl-2-feruloylgentiobioside (D). These results are in accordance withcompounds detected in other Brassica species, like turnip tops(Romani et al., 2006; Francisco et al., 2009), tronchuda cabbage(Ferreres et al., 2006), broccoli (Vallejo et al., 2004) and now forthe first time, in kale and leaf rape.

This study shows that kale, cabbage and leaf rape are a goodsource of phenolic antioxidants. The main naturally occurringphenolic compounds identified were flavonols and hydroxycin-namic acids.The majority of the flavonoids found in these varietiesare kaempferol, glycosylated and acylated with different hydroxy-cinnamic acids. Quercetin and isorhamnetin derivatives were alsofound. Kaempferol and quercetin are the most prevalent fla-vonoids in the Brassicaceae family (Podsedek, 2007). Kaempferol isknown to be a strong antioxidant and quercetin a potent freeradical scavenger and is considered to be protective against car-diovascular diseases (Rice-Evans et al., 1996). Cabbage is a well-established crop like cauliflower or broccoli. Kale and leaf rape areminor crops in many parts of the world but they are a good sourceof nutritive compounds and, due to their rusticity (Rodriguez et al.,2005; Padilla et al., 2007b), they could be a good substitute fordifferent Brassica species under hard weather conditions.

Acknowledgements

This research was supported by the National Plan for Researchand Development (AGL2009-09922) and the Excma DiputaciónProvincial de Pontevedra. Marta Francisco acknowledges an I3PFellowship from the CSIC. The authors thank Rosaura Abilleiraand Susana Calvo for all their invaluable help in the laboratorywork.

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