Specific Glucosinolate Analysis Reveals Variable Levels of Epimeric

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Specific Glucosinolate Analysis Reveals Variable Levels of Epimeric Glucobarbarins, Dietary Precursors of 5‑Phenyloxazolidine-2-thiones, in Watercress Types with Contrasting Chromosome Numbers Niels Agerbirk,*,† Carl Erik Olsen,† Don Cipollini,‡ Marian Ørgaard,† Ib Linde-Laursen,† and Frances S. Chew§ †

Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark ‡ Department of Biological Sciences, Wright State University, 203 Biological Sciences Building I, 3640 Colonel Glenn Highway, Dayton, Ohio 45435, United States § Department of Biology, Tufts University, Medford, Massachusetts 02155, United States ABSTRACT: Watercress obtained in food stores in the United States contained significant levels of epiglucobarbarin [(R)-2hydroxy-2-phenylethylglucosinolate] and low levels of the 2S-epimer glucobarbarin identified by an HPLC+NMR+MS/MS approach. Typical combined levels were 4−7 μmol/g dry wt. The hydrolysis product, 5-phenyloxazolidine-2-thione (barbarin), was detected at similar levels as the precursor glucosinolates after autolysis of fresh watercress in water. Fragmentation patterns in MS2 of reference desulfoglucosinolates were side chain specific and suitable for routine identification. Watercress was of two main glucosinolate chemotypes: Material from U.S. food stores had a complex profile including glucobarbarins, gluconasturtiin, indole glucosinolates and high levels (6−28 μmol/g dry wt.) of long-chain methylsulfinylalkyl and methylthioalkyl glucosinolates. Material from European food stores had a simple profile dominated by gluconasturtiin, with low levels of epiglucobarbarin and moderate levels of indole glucosinolates. Some wild U.S. material was similar to the U.S. food store type. Both types were found to be Nasturtium officinale by floral parts morphology. Cytological analysis of one U.S. food store accession indicated that it represented a chromosome-doubled variant within N. off icinale. The nutritional consequences and invasive potential of the U.S. food store chemotype are discussed. KEYWORDS: watercress, chromosome number, glucosinolate, oxazolidine-2-thiones, polymorphism, NMR, MS/MS

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Oefelein),4,5 and some cultivated watercress in the United Kingdom belongs to the hexaploid form.5 Chromosome numbers of N. × sterile appear to be variable, and definite morphological characters may not exist.4 The natural distribution of both species of watercress (N. of f icinale and N. microphyllum) is similar and includes Europe, parts of Asia, and North Africa. They are also naturalized to most parts of the world including Africa, the Americas, and Australia.6 The distribution of the hybrid is uncertain, but it is reported from Europe and the United States.4,5,7 Watercress spreads mainly vegetatively and is regarded as a noxious or invasive weed.8−10 Hybridization may be a factor in the invasiveness of watercress.9,10 It is well established that watercress is rich in phenethylglucosinolate (3). This compound, like other glucosinolates, may be enzymatically converted to a mustard oil upon tissue disruption or by microbial metabolism in the gut.11 In the case of 3, the derived mustard oil is phenethyl isothiocyanate (Figure 1), which has received much scientific attention and may be involved in some health-promoting processes.12 Hence,

INTRODUCTION Glucosinolates are secondary metabolites in crops and wild plants of the order Brassicales with 132 individual structures documented by 2011.1 Together with the enzyme myrosinase (thioglucoside glucohydrolase, EC 3.2.1.147), glucosinolates provide a biochemical defense system that produces bioactive hydrolysis products upon tissue disruption. The glucosinolate glucobarbarin (1) is hydrolyzed to an unstable isothiocyanate that cyclizes to (R)-(−)-5-phenyloxazolidine-2-thione, (R)-4) (Figure 1).2 The epimer, (R)-2-hydroxy-2-phenylethylglucosinolate, giving the S stereoisomeric hydrolysis product (S)-4, is also known (Figure 1). A common name of the epimer was not proposed by the discoverers,3 but the name epiglucobarbarin (2) has been recommended (“glucosibarin” is also in use).1 Collectively, the two epimers can be termed “glucobarbarins”. Watercress is used as vegetable, salad, or garnish and belongs to the genus Nasturtium. Two species and a hybrid, each with characteristic chromosome numbers, are discussed here: the tetraploid (2n = 4x = 32) Nasturtium officinale W. T. Aiton (watercress) (syn. Rorippa nasturtium-aquaticum (L.) Hayek) and the octoploid (2n = 8x = 64) Nasturtium microphyllum (Boenn) Rchb. The key morphological character distinguishing the two species is the number of seed rows in the siliques, either two rows (N. off icinale) or one row (N. microphyllum).4 In addition, a partly sterile hexaploid (2n = 6x = 48) hybrid between the two is fairly common (N. × sterile (A. Shaw) © 2014 American Chemical Society

Received: Revised: Accepted: Published: 9586

July 9, 2014 August 19, 2014 September 5, 2014 September 16, 2014 dx.doi.org/10.1021/jf5032795 | J. Agric. Food Chem. 2014, 62, 9586−9596

Journal of Agricultural and Food Chemistry

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ybenzylglucosinolate (sinalbin) and 3-butenylglucosinolate (gluconapin) in addition to the dominating 3 and less abundant indoles.19 Both reports were inconclusive as definitive isomer distinction needs detailed comparison with authentic standards or exhaustive spectroscopic analysis.1 In the case of 1 and 2, seven isomers were known by 2014.1,22,23 Moreover, a number of the isomers were found to exhibit very similar HPLC retention times.1,23 The reports of contrasting glucosinolate profiles in nonEuropean watercress were interesting because structural types of glucosinolates and their hydrolysis products have complementary biological effects, such as different inductions of xenobiotic detoxification enzymes.12 In particular, isothiocyanates with methylsulfinylalkyl and methylthioalkyl side chains exert promising bioactivities.20 Furthermore, synergistic effects of different classes of hydrolysis products have been demonstrated.21 In the case of U.K. watercress, the longchain sulfinyl glucosinolates rather than dominant 3 were concluded to be mainly responsible for a phase II induction effect of the investigated watercress.17 The purpose of this work was to investigate glucosinolate profiles of watercress from a wide variety of sources, reflecting material typically used for human consumption around the world, to obtain a better basis for predicting and investigating nutritional and ecological effects of this vegetable. Due to the inconclusive nature of some previous reports of atypical profiles, we aimed at using isomer-specific methods including NMR and MS/MS analysis of HPLC peaks. During the investigation, after the discovery of a 2-hydroxylated glucosinolate and contrasting profiles in some U.S. material, it became relevant to identify the corresponding glucosinolate hydrolysis product and to check deviating material for the possibility of a deviating botanical identity.

Figure 1. Structures of glucobarbarin, epiglucobarbarin, gluconasturtiin, and their natural 5-phenyloxazolidine-2-thione hydrolysis products after hydrolysis catalyzed by myrosinase (MYR), the endogenous glucosinolate-hydrolyzing enzyme in watercress. “Glc” in abbreviated structures indicates the β-D-glucopyranoside part of the structure as detailed in the uppermost full structure of glucobarbarin.

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MATERIALS AND METHODS

Chemicals. 5-Phenyloxazolidine-2-thione and desulfated derivatives of the glucosinolates 1, 2, 3, 11, 12, 13, sinalbin, gluconapin, phydroxy-2, p-hydroxy-3, and p-methoxybenzylglucosinolate were obtained as previously described,1,24,25 whereas standards of desulfated 7, 9, and 10 (isolated from Rorippa species and identified by NMR and MS) were from unpublished work. Sinigrin used as external standard was from Karl Roth, Karlsruhe, Germany. HPLC, MS, and NMR Equipment. HPLC with UV detection was carried out using an LC-10AT pump with an SPD-M10AVP PDA detector (Shimadzu, Kyoto, Japan) at a flow rate of 1 mL/min. Two column stationary phases were used as specified, either “C18” (250 mm × 4.6 mm i.d., 5 μm, Luna C18 (2)) or “phenylhexyl” (250 mm × 4.6 mm i.d., 5 μm, Luna Phenylhexyl) (both Phenomenex, Torrance, CA, USA). The HPLC-MS instrument was an Agilent 1100 series LC (Agilent Technologies, Waldbronn, Germany) coupled to a Bruker HCT-Ultra ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) fitted with a Zorbax SB-C18 column (Agilent; 1.8 μm, 2.1 × 50 mm). The oven temperature was maintained at 35 °C. The mobile phases were (A) water with 0.1% (v/v) HCOOH and 50 μM NaCl and (B) acetonitrile with 0.1% (v/v) HCOOH. The NMR spectrometer was a Bruker Avance 400 instrument operated at 400.1 MHz, using D2O as solvent and dioxane (δH = 3.75 ppm) as internal standard. Plants. Fresh commercial watercress for isolation of intact glucosinolates (July and November 2012), glucosinolate analysis (2004, 2005, 2012), and rooting followed by cytological investigation and glucosinolate analysis (November 2013) was obtained from food stores near Boston, Massachusetts (MA), USA. Fresh commercial watercress for rooting, hydroponics growth, glucosinolate analysis, and botanical identification by floral parts morphology was obtained in 2012 from a grocery store in Dayton, Ohio (OH), USA. In all cases of

watercress is not only attractive for its taste and culinary value but may also be a health-promoting vegetable. There are several reports of physiological effects of dietary watercress on markers of human disease.13−15 Although the physiological effects of watercress may well be due to many phytochemicals, glucosinolates are likely an important class of constituents and are the focus of this paper. We are aware of five reports of the glucosinolate profile of watercress foliage, of which three are considered European material,14,16,17 one is considered Japanese material,18 and one is considered U.S. material.19 In the reports of European material, it was found that 3 was dominant, whereas two indole glucosinolates, two long-chain methylsulfinylalkyl glucosinolates (7-methylsulfinylheptyl and 8-methylsulfinyloctyl glucosinolates), and the corresponding methylthioalkyl glucosinolates were less abundant constituents with some variation in the levels of the aliphatics.14,16,17 The reports on North American and Japanese material deviate from the reports on European material. Japanese material was reported to have a profile similar to that reported for European material, except for a quite prominent peak suggested to be 2 and a minor peak suggested to be 1 (common names were interchanged).18 The reported evidence for 2 and 1 was the exact mass and comparison with published retention times in another laboratory. The calculated and observed m/z values for both 2 and 1 differed by 3 Da.18 In contrast, U.S. material was reported to contain 4-hydrox9587

dx.doi.org/10.1021/jf5032795 | J. Agric. Food Chem. 2014, 62, 9586−9596


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washed with 2 mL of 70% aqueous MeOH, 6 mL of AcOH/EtOH/ H2O (1:1:3) to elute weak acids (eluate intensely red), and 2 mL of H2O. Bound glucosinolates were eluted with 10 × 1 mL of aqueous K2SO4 (saturated, ca. 0.5 M). Most K2SO4 in fractions was removed by the addition of 2 volumes of 96% EtOH, leaving the mix at −5 °C for 30 min, and centrifugation (4000g), discarding the pellet. EtOH was removed by evaporation of two-thirds of the volume under a gentle air stream, and remaining solvent was removed by freezing followed by lyophilization. To remove the remaining the K2SO4, glucosinolates in the yellowish residue were dissolved in 2 + 2 mL of pure MeOH for each batch, leaving insoluble K2SO4. The MeOH solution was removed from the insoluble crystals and placed under a gentle air stream for solvent evaporation. The residue was dissolved in a total of 1 mL of H2O (for the combined batches after testing each separately), and this solution was used for injection to the HPLC, injecting 150 μL six times. We used the analytical HPLC with the phenylhexyl analytical column described above, A = 0.1% aqueous CF3COOH and B = MeOH: isocratic A for 2 min, 48 min linear gradient to 60% B, 2 min linear gradient to 100% B, isocratic B for 5 min, 3 min linear gradient to 100% A, 7 min isocratic A. Fractions were collected manually (after the waste solvent line had been replaced with a short piece of HPLC tubing after the UV detector), guided by the PDA output followed in real time. To avoid hydrolysis of glucosinolates by CF3COOH, collected fractions were neutralized with 1% (v/v) of a 3 M aqueous potassium acetate solution at the end of the collection day. Neutralized fractions were dried under a gentle air stream and then in vacuo overnight and dissolved in D2O for NMR analysis and subsequent HPLC-MS carried out as previously reported for intact glucosinolates,1 except that MS detection was in positive mode (observing [M − H + 2Na]+ adducts) for logistic reasons. Although positive mode detection of glucosinolates is rarely done, both glucosinolates and other anions such as carboxylic acids form positively charged disodium adducts with good ionization efficiency when the eluent is doped with NaCl as here (unpublished observation). Autolysis of Fresh Watercress. The apex of individual branches, with about three small leaves and further primordia, was used. Fresh apex (one large or two small, combined wt 0.4−0.8 g) was either extracted immediately in boiling 70% aqueous MeOH for glucosinolate determination (n = 4) or subjected to autolysis (homogenized in a mortar and incubated as macerated material with 3 mL of H2O (n = 5) or without any liquid added (n = 5)). After autolysis for 3−4 h, the macerate was either used for glucosinolate determination (n = 2 + 2), using the same extract as for intact material, or subjected to determination of 4 (n = 3 + 3). As a control for the effect of autolysis (n = 2), freshly picked apex was boiled (1 min) before homogenization, then homogenized (including water from the initial boiling), and extracted for 4. Macerates for determination of 4 were extracted twice with a total of 15 mL of boiling water (2 × 1 min). The extraction volume included any previously added water. Each extract was brought to 15.0 mL; 8.00 mL was taken, mixed with 2.00 mL of MeOH, and allowed to settle at −20 °C overnight. From settled extracts, 1.2 mL was taken and centrifuged at 4000g, and the supernatant was transferred to an HPLC vial. Quantitative analysis for 4 was by HPLC-PDA set at 242 nm, bandwidth 8 nm (phenylhexyl column, injection volume = 100 μL, A = water, B = MeOH: isocratic A for 2 min, 48 min linear gradient to 70% B, 2 min linear gradient to 100% B, isocratic B for 5 min, 3 min linear gradient to 100% A, 7 min isocratic A). Glucosinolates in the studied material were quantitated by the desulfoglucosinolate method (see below). For HPLC-MS analysis of 4, the gradient program was as follows: 0−0.5 min, isocratic 6% B; 0.5−12.5 min, linear gradient from 6 to 55% B; 12.5−13.1 min, linear gradient from 55 to 95% B; 13.1− 15.5 min, isocratic 95% B; 15.6−20 min, isocratic 6% B. The flow rate was 0.2 mL/min but increased to 0.3 mL/min from 15.2 to 17.5 min. For extracted ion chromatograms (combined 202 + 180 + 146 + 120 Da), the width was set to ±0.5 Da. Glucosinolate Analysis. Each accession was extracted at least in duplicate, using around 100 mg of dried foliage (or 0.3−1 g of fresh foliage) per extraction. Extraction, enzymatic desulfation, and HPLCPDA was carried out as previously reported,1 except that the C18

commercial Ohio and Massachusetts watercress, the same producer (B&W Quality Growers, FL, USA) was indicated, but material was in no case obtained directly from this producer, so the origin is not absolutely certain. Wild Danish watercress for rooting followed by cytological investigation and glucosinolate analysis was from St. Vejleaa, Taastrup (55°40′ N, 12°19′ E). Fresh watercress for the autolysis experiment, indicated to be from Santa Clara Valley, California (CA), USA, was obtained in a food store (Safeway, Willits) and immediately transported to Denmark by plane, in accordance with relevant Danish legislation. After the autolysis experiment, remains were autoclaved. Additional watercress was obtained for glucosinolate analysis with the aim of sampling as consumers around the world would typically acquire it: wild-collected, home-grown, or from a food store (fresh or in one case desiccated). Wild watercress was collected in nature in Massachusetts (near Lenox, 42°21′ N; 73°17′ W), Ohio (near Yellow Springs, 39°48′ N; 83°53′ W), and Denmark (Mors, 56° N, 8° E). Watercress seeds were obtained from three different Web-based seed suppliers: Chiltern Seeds (Ulverston, UK), Johnny’s Select Seeds (Winslow, ME, USA), and Kitazawa Seed Co. (Oakland, CA, USA). Commercial fresh watercress was obtained in food stores. Chinese desiccated watercress was obtained in a Chinese shop in Copenhagen. Watercress cultivated from seeds, U.S. food store material from Ohio, and U.S. wild material was grown in a hydroponics system: Tubs for hydroponic growth were filled to 200 L with distilled water. To each tub, 66 mL each of the fertilizer concentrates FloraGro (2−1−6), FloraBloom (0−5−4), and FloraMicro (5−0−1) (General Hydroponics) were added at the start of the planting and not replenished. The growth solution was under continuous aeration by an aquarium pump. Seeds or shoots were placed in glass wool suspended in holes punched in floating Styrofoam blocks, with roots hanging into the nutrient solution. Tubs were placed under fluorescent lights providing ∼75 μmol PAR/(m2 s) on a 16:8 light/dark cycle at 22 °C. This light intensity, comparable to reduced natural light intensity in forest streams, promoted abundant growth, flowering, and seed set and did not seem limiting in any way. Botanical Identification. Wild-collected watercress was tentatively identified as N. of f icinale by the collector (either F.S.C, M.Ø. or D.C.) on the basis of general morphology and growth site. For wild U.S. material from 2012, material grown from seeds, commercial watercress obtained on the root in Denmark, and commercial watercress obtained from a grocery store in Ohio, and rooted, the identity was further confirmed by cultivation to flowering and seed set for inspection of key morphological characters. Voucher specimens have been deposited at the Wright State University Herbarium and at CP in the case of Danish material. For cytological analysis, two accessions were rooted in tap water, and numbers of chromocenters in root tip cells were counted as previously described.26 Lyophilization/Drying and Transport to Analysis Laboratory. Watercress foliage from Denmark (except wild collection), Spain, Portugal, and Massachusetts was lyophilized soon after acquisition (without previous freezing to avoid glucosinolate breakdown during freeze−thaw cycles) until completely crisp. Watercress foliage from Ohio and from growth in our hydroponics system was airdried at ambient conditions in the laboratory and then placed in plastic bags with silica gel. Watercress foliage from California, Australia, and Argentina and the wild-collected sample from Denmark was dried domestically at ambient indoor conditions until crisp. Dried samples were shipped to the University of Copenhagen analysis laboratory by mail or plane travel. Upon arrival, all material was stored frozen (−20 °C) in closed plastic bags until extraction to prevent reabsorption of moisture. Variable drying conditions were due to lack of availability of freeze-drying at some origins and are discussed under Results and Discussion. Intact Glucosinolate Isolation and Identification. U.S. food store watercress was lyophilized, and two batches (in total 1.42 g) were briefly and repeatedly extracted in boiling 70% aqeuous MeOH in a total of 70 mL. The extracts were filtered (paper), centrifuged (4000g), and applied to four 100 mg (dry wt) DEAE Sephadex A-25 anion exchange columns.1 Each column with bound glucosinolates was 9588



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column was used. Peak overlap or coelution of either 3 and 13 or 3 and 10 occurred on two other reversed phase columns1 tested. The elution order in HPLC-PDA, with typical tR in minutes, was 11 (17.0), 8 (17.7)*, 2 (19.8), 1 (21.5), 9 (23.8), 11 (26.6), 10 (30.3), 3 (31.6), 13 (32.54), 5 (39.7)*, 6 (46.9)*, 7 (53.1). Peaks labeled * were only tentatively identified on the basis of MS2 but not compared with authentic standards. Quantitation was based on UV detection at 229 nm, relative to an external standard of sinigrin desulfated in parallel, using the following response factors: 0.95 for 1, 2, and 3, 1.00 for aliphatic side chain glucosinolates, 0.29 for 11, 0.28 for 12, and 0.25 for 13. Peak identification was based on UV spectra and comparison of tR with those of authentic desulfoglucosinolate references. HPLC-MS of one sample from each accession was also carried out to support the peak identification. The gradient program was as follows: 0−0.5 min, isocratic 2% B; 0.5−7.5 min, linear gradient from 2 to 40% B; 7.5−8.5 min, linear gradient from 40 to 90% B; 8.5−11.5, isocratic 90% B; 11.60−17 min, isocratic 2% B. The flow rate was 0.2 mL/min but increased to 0.3 mL/min from 11.2 to 13.5 min. The mass spectrometer was run in positive electrospray mode. The elution order was similar in the more condensed HPLC-MS gradient, except that 3 and 13 coeluted. Peak identity inferred from quantitative HPLC with UV detection was confirmed by observation of expected m/z values for sodium adducts in HPLC-MS and inspection of MS2 spectra. MS2 spectra were acquired in automatic mode using SmartFrag with the instrument default settings (isolation width, 4 m/z; MS/MS fragmentation amplitude, 1 V; start amplitude, 30%; end amplitude, 200%; acquisition time, 40 ms). Statistical Evaluation. Levels of phytochemicals were logtransformed to ensure variance homogeneity (tested and confirmed by F test) and compared statistically using a two-tailed t test. All procedures were carried out in Excel 2010.

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RESULTS AND DISCUSSION Identification of Epiglucobarbarin as a Major U.S. Watercress Glucosinolate by NMR and MS. To unequivocally identify glucosinolates in watercress obtained in the Massachusetts area, we extracted two independent batches obtained in separate months and separate stores and analyzed the intact glucosinolate fraction by HPLC. As the same eight peaks were detected in both chromatograms, we pooled the fractions and isolated the glucosinolates by preparative HPLC (Figure 2). Three major peaks were identified by NMR and MS: an early-eluting peak was 2, not previously documented from watercress, whereas the other two peaks proved to be well-established watercress constituents, 3 and 7-methylsulfinylheptylGSL (9) (Figure 2). The identification of 2 was unequivocal: not only did the mass, coupling pattern, and chemical shifts agree with the structure of 2, but the NMR spectrum also closely matched the previously reported spectrum.24 From this latter comparison it was evident that the R-epimer (2) was present, as the S-epimer (1) exhibits slightly different chemical shifts and coupling constants at the 1-, 2-, 1′-, and 6′-positions. The S-epimer 1 (Figure 2) was also identified at low levels on the basis of a very weak NMR spectrum24 and UV and mass spectra, and four minor peaks (Figure 2) were tentatively identified as previously reported17 watercress constituents by UV and mass spectra (results not shown). Formation of 5-Phenyloxazolidine-2-thione during Autolysis of U.S. Watercress. After crushing fresh watercress from a U.S. food store to allow autolysis catalyzed by endogenous myrosinase, we found the oxazolidine-2-thione type product 4 at similar levels as the combined levels of 1 and 2 in control experiments without autolysis (Table 1). The identification of 4 was by retention time in two different HPLC systems and UV and mass spectra as compared with an

Figure 2. Analysis of the total glucosinolate preparation from U.S. food store watercress (Boston, MA) by HPLC followed by 1H NMR. HPLC was carried out in (A) analytical scale and (B) preparative scale using the same instrument, conditions, and glucosinolate preparation, but with different injection volumes (10 and 150 μL, respectively). (C−E) 1H NMR spectra of individual peaks after neutralization with acetate and solvent change to D2O. (F) Detail of the NMR spectrum of 2 (C).

authentic standard (Figures 3 and 4).1 Four main ions were observed in HPLC-MS from 4: 202, 180, 146, and 120 (corresponding to [M + Na]+, [M + H]+, [M − H2S + H]+, and [M − CSO + H]+); this fragmentation and the UV maximum are both characteristic of the oxazolidine-2-thione moiety.1,23 The products formed from 1 and 2, (R)-4 and (S)-4), constitute an enantiomeric pair (Figure 1), but the enantiomers are indistinguishable using achiral HPLC columns and were not distinguished in the analysis. The detected product, probably a mixture of enantiomers dominated by the product of 2, is for this reason simply termed 4 without specification of enantiomer and compared with the sum of the epimeric precursors. Interestingly, levels of 4 were higher when water was immediately added to the macerated tissue than when the 9589



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Table 1. Levels of Glucobarbarins (1 + 2) and 5Phenyloxazolidine-2-thione (4) in Macerated Watercress Shoot Apex after Autolysis for about 3 ha 1+2

4

(μmol/g fresh wt)

(μmol/g fresh wt)

condition

mean

SD

nb

mean

SD

n

signif

control autolysis − water autolysis + water

3.6 0.03 0.03

0.2 0.02 0.02

4 2 2

0.5c 1.0 3.5

0.02 0.4 1.2

2 3 3

n/a ns **d

a

Mean levels of other major glucosinolates in the analyzed material (Santa Clara Valley, CA, USA) were as follows: 3, 6.1; 9, 3.9; 11, 0.8; 6, 0.6. The extent of hydrolysis was in all cases >98%. Statistical significance (signif) relates to levels of 4 and is relative to the control: ∗∗, P < 0.01; ∗, P < 0.05; ns, P ≥ 0.05. bThe number of replicates n generally differs for glucosinolates and hydrolysis products because the values resulted from independent experiments and extractions. c Maximum estimate including minor coeluting flavonoid-like matrix component. dLevel of significance relative to autolysis without water: ∗, the F test for variance inhomogeneity after log transformation was insignificant but close to significance (P = 0.10). However, a t test assuming variance inhomogeneity was still significant (∗, P = 0.010).

Figure 4. Identification by HPLC-MS of 5-phenyloxazolidine-2-thione (4) in U.S. food store watercress either (A) extracted directly or (B) subjected to autolysis with water. The trace is the combined extracted ion chromatogram of m/z 202, 180, 146, and 120. (C) Chromatogram of authentic 4 (35 μM) analyzed in parallel. (D−G) Mass spectra of the POAT peak at 9.1 min or an unidentified peak at 9.4 min as indicated.

Figure 3. Identification and quantitation by HPLC with UV detection at 242 nm of 5-phenyloxazolidine-2-thione (4) in U.S. food store watercress either (A) extracted directly or (B) subjected to autolysis. The asterisk indicates a peak containing 4 and an unknown metabolite. (Insets) Photodiode array UV spectra at the peak apex of the 4 peak. (C) Chromatogram of authentic 4 (35 μM) analyzed in parallel.

HPLC-PDA is reliable for quantitative determination of 4 af ter autolysis. Whether the minor level observed in control experiments was a natural constituent in intact tissue or due to slight incidental tissue damage is unknown. A baselineseparated peak in HPLC-MS (tR = 9.5 min) shared the m/z 202 ion, but in this case it was interpreted as [M + H]+ (Figure 4E) and appeared to be unrelated to glucosinolate hydrolysis (Figure 4). Identification of Desulfated Glucosinolates by HPLC and MS/MS. Separation of all watercress glucosinolates (Figures 1 and 5) after desulfation was accomplished in HPLC-PDA. To obtain additional certainty, analyses were complemented by HPLC-MS/MS. The initial peak identification was based on retention times, UV spectra, and m/z values of [M + Na]+ adducts in HPLC-MS. In an attempt to

autolysis happened in macerated tissue alone, even though glucosinolate hydrolysis was essentially complete at both conditions (Table 1). An unexpected result of the autolysis experiment was the apparent detection of low levels of 4 in fresh watercress believed not to have been subject to autolysis (Table 1). Moderate interference from coeluting matrix components was likely in both HPLC-PDA and HPLC-MS, but the correct λmax and diagnostic MS fragments were observed (Figures 3 and 4D). Despite the modest background signals, we suggest that 9590



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methylthio and methylsulfinylalkyl glucosinolates yielded specific fragmentations that could be rationalized from sidechain structures. Authentic references of both desulfo 1 and 2 showed a fragment at m/z 364 indicating a characteristic loss of water (fragment type f). In addition, a fragment at m/z 276 indicating the loss of benzaldehyde (fragment type e) was observed for both desulfo 1 and 2. Interestingly, loss of water and benzaldehyde was not observed for available isomers of the desulfoglucobarbarins, desulfated p-hydroxy-3 and p-methoxybenzylglucosinolate, whereas loss of water and hydroxybenzaldehyde was observed for desulfo p-hydroxy 2.23 Hence, loss of water and benzaldehyde provided a useful signature of 1 and 2 specifically reflecting the presence of the 2-hydroxy-2-phenyl substituents. Likewise, the MS2 spectra of Na+ adducts of the two methylsulfinylalkyl desulfoglucosinolates available as authentic references, 9 and 10, were highly characteristic. Besides apparent loss of [CH4SO] resulting in [M + Na − 64] fragments (type g), the characteristic feature of this homologous series was the absence of the usually observed desulfoGSL fragments at m/z 185 and 219 (corresponding to Na+ adducts of C6H10O5 and thioglucose, fragment types a and b, respectively) and the presence of a major fragment ion corresponding to a Na+ adduct of the aglucone after loss of thioglucose (loss of 196, fragment type h) (Figure 6). On the basis of similar MS2 fragmentation and reasonable retention time and UV spectrum, the lower homologue 8 (not available as authentic desulfo reference) was tentatively identified in a variety of watercress samples. Methylthioalkyl desulfoglucosinolates exhibited a less intense [M + Na − 196]+ fragment ion (m/z 210 in the case of desulfo 6), whereas the remaining tested desulfoGSLs did not produce this fragment type (type h) (Figure 6). Only one desulfoglucosinolate from this homologous series was available as authentic reference (desulfo 7), but the two lower homologues (5 and 6) were tentatively identified

Figure 5. Structures, names, and abbreviations of aliphatic and indole glucosinolates detected in watercress. Three of them, 5, 6, and 8, were only tentatively identified on the basis of MS2, whereas the remaining were identified with certainty. “GSL” indicates the constant part of glucosinolates in structures and is an abbreviation of “glucosinolate” in names.

better distinguish isomers, we characterized the known peaks in MS2 of sodium adducts. A universal fragment ion was [M + Na − 162]+ representing an expectable loss of C6H10O5 (fragment type c) (Figure 6). However, desulfo glucobarbarins and

Figure 6. Fragments of sodium adducts of desulfated glucosinolates from U.S. food store watercress observed in MS2 after HPLC separation. Types of fragments are indicated with letters as follows: a, [C6H10O5 + Na]+; b [thioGlc + Na]+; c, loss of 162 [M + Na − C6H10O5]+; d, [Glc + Na]+; e, loss of 106, benzaldehyde [M + Na − C7H6O]+; f, loss of 18, water [M + Na − H2O]+; g, loss of 64, CH3(SO)H [M + Na − CH4SO]+; h, loss of 196 [M + Na − thioGlc]+; i, loss of 180 [M + Na − Glc]+. 9591



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Table 2. Glucosinolate Levelsa in Watercress of Different Originsb glucosinolates (μmol/g dry wt) benzenic accession

1

2

wild-collected Lenox, MAc 0.02 0.34 Lenox, MA (2005) 0.02 0.45 Yellow Springs, OHc 0.01 0.06 Mors, DKc 0.02 0.05 home-grown from seeds or wild plants Johnny’s Select Seedsc 0.00 0.01 Kitazawa Seedsc 0.00 0.01 Chiltern Seedsc 0.00 0.01 wild, MAc 0.01 0.29 obtained fresh in shop Boston, MA 0.52 6.27 Boston, MA (2004/5) 0.35 4.40 Dayton, OHc 0.25 3.92 San Francisco, CA 0.48 5.66 Buenos Aires, AR 0.00 0.03 Melbourne, AU 0.01 0.02 Peralta, ES 0.00 0.01 Pamplona, ES 0.00 0.01 Odemira, PT 0.00 0.04 Copenhagen, DK (A)c 0.00 0.03 Copenhagen, DK (B) 0.00 0.02 obtained dried in shop DK/CN 0.03 0.52 seeds, not germinated Chiltern Seeds 0.03 0.23

aliphatic methylthioalkyl

aliphatic methylsulfinylalkyl

indolic

3

5

6

7

8

9

10

11

12

13

sum

n

20.64 14.86 34.34 41.10

0.06 0.01 0.01 0.00

1.02 0.04 0.40 0.00

0.16 0.00 0.17 0.00

0.44 0.31 0.09 0.12

6.02 4.91 2.91 2.38

1.30 0.85 1.91 1.08

0.54 0.64 0.26 0.39

0.27 0.00 0.01 0.00

0.05 0.00 0.37 0.16

30.79 22.09 40.52 45.30

3 1 4 2

22.82 19.56 24.42 8.66

0.00 0.00 0.01 0.01

0.05 0.03 0.11 0.00

0.00 0.01 0.02 0.00

0.03 0.03 0.03 0.32

0.76 0.42 0.74 5.40

0.35 0.26 0.35 1.23

0.30 0.24 0.41 0.14

0.00 0.00 0.00 0.00

0.10 0.11 0.14 0.00

24.44 20.66 26.23 16.05

2 2 2 3

16.41 11.21 7.99 15.76 13.67 20.20 32.75 25.04 27.47 21.23 9.59

0.03 0.01 0.01 0.10 0.00 0.00 0.00 0.00 0.07 0.00 0.00

0.36 0.51 0.01 1.58 0.01 0.00 0.00 0.00 1.68 0.11 0.25

0.04 0.05 0.01 0.20 0.00 0.00 0.00 0.00 0.94 0.05 0.12

1.47 0.82 0.49 1.82 0.02 0.02 0.02 0.00 0.04 0.03 0.01

11.81 7.80 5.31 21.11 0.10 0.23 0.01 0.00 0.97 0.65 0.07

1.42 0.98 0.63 4.08 0.31 0.06 0.00 0.00 0.54 0.32 0.04

0.78 0.80 0.51 1.58 0.50 0.21 1.03 0.96 0.46 0.13 0.20

0.00 0.00 0.00 0.47 0.05 0.04 0.00 0.00 0.13 0.00 0.02

0.17 0.16 0.06 0.24 0.68 0.53 0.44 0.10 1.14 0.16 0.44

39.25 27.09 19.19 52.99 15.36 21.31 34.27 26.11 33.41 22.72 10.75

2 2 3 2 4d 2 2 2 2 2 2

1.12

0.03

0.39

0.04

0.12

1.45

0.21

0.08

0.01

0.20

4.16

2

52.82

0.14

5.25

2.73

0.06

3.44

4.46

0.02

0.00

0.00

69.05

1

a

Levels (in leaves) are shown in bold if they are numerically larger than the average of all freshly obtained leaves for that glucosinolate. Accessions were from 2012 or 2013 unless otherwise indicated. bMA, Massachusetts, USA; OH, Ohio, USA; DK, Denmark; CA, California, USA; AR, Argentina; AU, Australia; ES, Spain; PT, Portugal; CN, China. cPlants were confirmed as Nasturtium of f icinale by observation of two rows of seeds in siliques. dMean of two similar accessions.

in a variety of watercress samples on the basis of similar MS2 fragmentation, UV, and reasonable retention time. In general, whether Na+ associates with the glucone or aglucone moiety of desulfoglucosinolates upon fragmentation in the ion trap seems to depend on the nature of the side chain. The highly polar sulfinyl group seems to have higher affinity for Na+ than the thioglucose residue, whereas affinities of the thioether and the thioglucose residue are roughly comparable. A terminal methylsulfinyl group can additionally be recognized by the loss of [CH4SO]. Also, desulfoglucosinolates with a combined 2-hydroxy-2-phenyl substitution seem to be recognizable by the loss of water and a benzaldehyde in a reaction that is somewhat similar to a retro-aldol reaction. Diversity of Watercress as Available to Consumers. We investigated whether consumers would likely obtain watercress containing glucobarbarins (1 and 2). Initial inspection of the distribution of high levels revealed an unexpected pattern: 2 was found at relatively high levels in all tested food store accessions from the United States, whereas only very low levels were found in accessions from Europe and in a few tested accessions from other continents (Table 2; Figure 7). The apparent grouping of the accessions was tested statistically in the relevant subset of data: material obtained fresh in food stores. The mean levels of 2 in four U.S. food store samples were significantly higher than the levels in the remaining eight food store samples (P < 0.001). Wild material from the United States was variable: watercress from

Figure 7. Contrasting glucosinolate chromatograms of a typical European accession of watercress from (A) Spain and (B) a typical U.S. food store watercress from California. Glucosinolates were enzymatically desulfated before analysis as indicated by “d” before glucosinolate numbers, and UV detection at 229 nm was employed.

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Introduction. The key morphological distinction between the two species of watercress discussed here, N. of f icinale and N. microphyllum, is based on mature siliques. All tested accessions were fertile and had seeds in two rows in siliques, including watercress foliage from the same grocery store in Dayton (Ohio) as used for glucosinolate analysis, wild-collected material from Ohio, Massachussets, and Denmark, material from a Danish food store, and all accessions grown from seed (Table 2). We concluded that all of these accessions belonged to N. off icinale as opposed to N. microphyllum. The liberal seed set would suggest that the partially sterile hybrid N. × sterile was not represented. Because of the unusual glucosinolate profile of the material from U.S. food stores, we also established chromosome numbers based on the number of chromocenters, defined as stainable regions in interphase nuclei that approximately reflect individual chromosomes.28 The result was striking: whereas the number of chromocenters in Danish material was close to the established chromosome number of N. off icinale, the U.S. food store material revealed approximately twice as many chromocenters (Figure 8), indicative of chromosome doubling. It was

Massachusetts showed moderate levels of 2, whereas wild material from Ohio was very low in 2. Plants grown from commercially obtained seeds were low in glucobarbarins irrespective of the supplier, whereas wild-collected plants grown in parallel retained approximately the same relative levels found in the wild-collected material. We considered whether the apparent trace peaks of 2 in European material and material grown from seeds could be confirmed with certainty by MS/MS. This was the case for seeds of a European type and for material from Denmark (wild), Argentina, and Portugal. Neither sinalbin nor gluconapin19 was detected. Interestingly, the four tested U.S. food store accessions were also remarkably high in methylthioalkyl and methylsulfinylalkyl glucosinolates, whereas the remaining eight food store accessions from Europe (n = 5), Argentina (n = 2), and Australia (n = 1) were lower in these kinds of glucosinolates (P < 0.001 for the sum of 5−10). Indeed, 34−55% of the glucosinolate content of U.S. food store samples consisted of these aliphatic glucosinolates (Table 2; Figure 7). Wild material appeared to be more homogeneous for aliphatics, with intermediate levels, whereas material grown from seeds had rather low levels (Table 2). Considering total glucosinolate levels, there was no obvious association of high or low levels with specific origins, except for a tendency for higher total levels in wild-collected watercress compared to watercress grown at our particular artificial light conditions. To obtain the widest possible temporal and geographical diversity of samples, we had to accept a range of drying methods and storage periods. Spontaneous air-drying was used for some samples, was used in classical qualitative natural product chemistry, and is reliable (N.A., unpublished results) for preparative purposes. However, it cannot be considered optimal for quantitative analysis because of the extended period in which physiological postharvest processes can occur. However, our use of spontaneous air-drying at some remote sampling sites and in the Wright State University laboratory was not a factor in the distinction of two chemotypes of watercress. Indeed, nearly the full range of 1, 2 and aliphatics (5−10) was observed in samples from Wright State University, Dayton (Ohio), where spontaneous drying at ambient conditions was used for logistic reasons. This range was comparable with the combined results from Copenhagen, Peralta, and Boston, where lyophilization was used (Table 2). Storage conditions and time were likewise not factors for the distinction of two chemotypes. First, analytical results of our 2004−2005 samples were essentially the same initially and after storage until this investigation (results not shown). Furthermore, the 2012 sample had essentially the same glucosinolate content after storage for a year, although glucosinolate hydrolysis occurred rapidly after the addition of water to the stored sample, so uptake of water during storage was apparently insignificant. Hence, we do not consider even extended frozen storage in closed plastic bags a problem for glucosinolate stability, with the possible exception of the oxidation-sensitive 12, which was not observed in old samples (Table 2). Indeed, a reliable glucosinolate profile of a 90-year-old herbarium specimen was recently reported with comparison with more recent material,27 indicating long-term stability. Botanical Identity of Watercress Accessions. Because of the observation of two distinct chemotypes of commercially available watercress, it was relevant to test by conclusive botanical identification whether the chemotypes reflected any of the established watercress taxa mentioned in the

Figure 8. Cytological analysis (DAPI-stained interphases) of (A) wild Danish watercress with 34 chromocenters and (B) U.S. food store watercress (Boston, Massachusetts) with ca. 68 chromocenters.

confirmed by glucosinolate analysis that the U.S. food store material with double chromosome number was relatively high in 2 (4.3% of total) and aliphatic glucosinolates (38%), whereas the investigated wild Danish material was low in 2 (0.2%) and had moderate levels of aliphatic glucosinolates (21%). Watercress Is Polymorphic for Glucosinolate Profiles and Chromosome Numbers and Is a Dietary Source of 5-Phenyloxazolidine-2-thione. Combining the morphological, cytological, and chemical evidence, we conclude that watercress, N. of f icinale, as available to consumers is polymorphic for glucosinolate profile and chromosome number. In the cases tested for both characters, they were correlated, but further research will be needed to test for a general association. Specifically, epiglucobarbarin (2) was a significant constituent of watercress from U.S. food stores and some wild accessions including chromosome-doubled material, in contrast to the remaining tested accessions mainly from Europe including diploid material. Furthermore, watercress from U.S. food stores exhibited a distinct profile of aliphatic glucosinolates, with considerable levels of long-chain methylsulfinylalkyl and methylthioalkyl glucosinolates in addition to the traditionally recognized glucosinolate in watercress, 3. These aliphatic glucosinolates were previously known from watercress, with some variation also within European material.14,16 In the following, we term the complex chemotype obtained in U.S. food stores “the U.S. food store type”, whereas 9593



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could also be the case for glucobarbarins.40,41 This general acceptance of an oxazolidine-2-thione-forming glucosinolate should perhaps be extended to 1 and 2 unless future research shows otherwise. Origins, Potentials, and Ecological Risks of U.S. Food Store Watercress. The difference between European and U.S. food store material suggests a non-European origin of U.S. food store watercress. As watercress is considered introduced in the United States, the ultimate origin must be overseas or by a recent evolutionary event such as hybridization or chromosome number mutation. A simple explanation of the apparent chromosome number difference would be artificial chromosome doubling.42 However, hybridization or chromosome number mutation is by no means a prerequisite for glucosinolate variation.23,43,44 The finding of wild material with significant levels of 2 and aliphatic glucosinolates at a natural habitat in United States in both 2005 and 2012 (Table 2) would suggest that the U.S. food store type of watercress occurs in nature to some extent and may have been naturalized for at least a decade. There is some evidence to suggest that the difference in glucosinolate profile between the two types could have ecological effects, and it is possible that variation in glucosinolate profiles may relate to variation in the prevalence of watercress in invaded habitats,9,10,44−49 including possible conversion of 1, 2, 3, and 4 to phytoalexins.50,51 A cultivar of broccoli with increased levels of another methylsulfinylalkyl glucosinolate, glucoraphanin, has been commercialized under the name “superbroccoli”.52 Perhaps a “super watercress” can be created or selected on the basis of the already available material, or the current U.S. material might in itself merit this designation. However, comparison of nutritional effects of watercress with different glucosinolate profiles is needed before one type of watercress can be recommended over another. Although we have discovered a new class of glucosinolates and glucosinolate products in watercress, associated with nutritional problems in animal feed at high doses35 and possibly in insect resistance in a wild crucifer,44 there is nothing in our results to suggest that current varieties constitute any nutritional risk at usual human intakes. However, the potentially different invasive properties of the U.S. food store variant with distinct glucosinolate profile and chromosome number should be considered before introduction of this high chromosome number chemotype for cultivation abroad.9,10

the simpler chemotype dominated by 3 and obtained from a number of European sources is termed “the European type”. Altered postharvest glucosinolate biosynthesis induced by storage conditions could in principle explain differences between freshly harvested and shelf-stored produce, but this was not what we observed. Rather, the two chemotypes were observed within both the wild-collected and commercially obtained watercress accessions and in commercial material cultivated and rooted for chromosome counts, suggesting that genetic factors were important for the observed chemical differences. We have also confirmed the conversion product 4 from U.S. food store watercress. This is the first unequivocal demonstration of 1, 2. and 4 in a regular food crop. The formation of 4 suggests that this hydrolysis product will also be formed when watercress is chewed and possibly also by microbial breakdown in the colon.11,12 The detection method for 4 (achiral HPLC) did not allow distinction of absolute configuration, but from the dominance of 2 in watercress the detected 4 can be inferred to consist mainly of (S)-4 (Figure 1). The common name “barbarin” has been suggested for (R)-429 and may be preferable for daily use. The (S)-4 formed from watercress should preferably be called (S)-barbarin in cases when precision is needed. Health Benefits and Hypothetical Risks of Watercress Glucosinolates. There seems to be a consensus regarding phenethyl isothiocyanate (from 3) and methylsulfinylalkyl and methylthioalkyl isothiocyanates as health-promoting.12,16,20,21,30,31 Indeed, an increase in the consumption of glucosinolate-containing vegetables seems to be advantageous and is recommended by public health authorities such as the National Cancer Institute.32−34 However, the detection of 1 and 2 as well as their product 4 in some watercress needs separate scrutiny because 4 is a different type of hydrolysis product. We are aware of a single investigation of nutritional effects (in rats at quite high levels) of 1 and a hydrolysis product assumed to be (R)-4.35 Significant detrimental effects of 1 and/or (R)-4 were observed on the palatability of the feed and on weights of liver, kidney, suprarenal glands, testicles, and thyroid gland. The detrimental effect on thyroid weight is in accordance with the general disturbance of iodine metabolism in the thyroid gland by oxazolidine-2-thiones.36 Facile conversion of these to mutagenic N-nitroso derivatives in the presence of nitrite at “stomach conditions” is also known.37 A different effect is inhibition of tyrosinase enzymes by (R)-4.38 This effect was not suggested to be toxic or antinutritional, but was suggested to be useful in preventing enzymatic browning of fruits and vegetables as well as in cosmetic and medical applications. It is not simple to predict whether the (S)-4 formed in U.S. food store type of watercress would have the same effects as (R)-4 formed from 1, but it seems relevant to expect physiological effects. It is relevant to consider the occurrence of a related oxazolidine-2-thione-forming glucosinolates in many cabbages: progoitrin, the precursor of goitrin ((S)-5-vinyloxazolidine-2thione). This glucosinolate causes negative effects of rapeseed meal in animal fodder and also showed antinutritional effects on rats and formed nitrosamides under stomach conditions.35−37 Nevertheless, we are not aware of any health risk of eating even considerable amounts of, for example, Chinese cabbage containing this glucosinolate at 2−5 μmol/g dry wt,39 whereas the contribution to bitterness is well established as

■

AUTHOR INFORMATION

Corresponding Author

*(N.A.) Phone: +45 35 33 37 23. E-mail [email protected]. Funding

This work was supported by the Torben and Alice Frimodts Fond (N.A.), the Arabis Fund (F.S.C.), and the Ohio Plant Biotechnology Consortium (D.C.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank four anonymous reviewers for helpful comments and suggestions and the following individuals for contributing watercress samples: Francisco R. Badenes-Peréz (ES, PT), Marta A. Carballo (AR), Deyang Xu (CN), Maya and Marie Louise Mileck (CA), Elizabeth Neilson (AU). We also thank Birgitte Rasmussen and Cecilie Ida Cetti Hansen for skillful 9594



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(21) Bonnesen, C.; Eggleston, I. M.; Hayes, J. D. Dietary indoles and isothiocyanates that are generated from crucifererous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines. Cancer Res. 2001, 61, 6120−6130. (22) Fahey, J. W.; Zalcmann, A. T.; Tatalay, P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 2001, 56, 5−51. (23) Agerbirk, N.; Olsen, C. E.; Heimes, C.; Christensen, S.; Hauser, T. P.; Bak, S. Multiple hydroxyphenethyl glucosinolate isomers from a geographically structured glucosinolate polymorphism in the crucifer Barbarea vulgaris. Phytochemistry 2014, DOI: 10.1016/j.phytochem.2014.09.003, accepted for publication. (24) Agerbirk, N.; Olsen, C. E.; Nielsen, J. K. Seasonal variation in leaf glucosinolates and insect resistance in two types of Barbarea vulgaris ssp. arcuata. Phytochemistry 2001, 58, 91−100. (25) Agerbirk, N.; Warwick, S.; Hansen, P. R.; Olsen, C. E. Sinapis phylogeny and evolution of glucosinolates and specific nitrile degrading enzymes. Phytochemistry 2008, 69, 2937−2949. (26) Ørgaard, M.; Linde-Laursen, I. Cytogenetics of Danish species of Barbarea (Brassicaceae): chromocentres, chromosomes and rDNA sites. Hereditas 2007, 144, 159−170. (27) Mithen, R.; Bennett, R.; Marquez, J. Glucosinolate biochemical diversity and innovation in the Brassicales. Phytochemistry 2010, 71, 2074−2086. (28) Fransz, P.; Soppe, W.; Schubert, I. Heterochromatin in interphase nuclei of Arabidopsis thaliana. Chromosome Res. 2003, 11, 227−240. (29) Kjær, A. Naturally derived isothiocyanates (mustard oils) and their parent glucosides. Fortschr. Chem. Org. Naturst. 1960, 18, 122− 176. (30) Barillari, J.; Canistro, D.; Paolini, M.; Ferroni, F.; Pedulli, G. F.; Iori, R.; Valgimigli, L. Direct antioxidant activity of purified glucoerucin, the dietary secondary metabolite contained in rocket (Eruca sativa Mill) seeds and sprouts. J. Agric. Food Chem. 2005, 53, 2475−2482. (31) Armah, C. N.; Traka, M. H.; Dainty, J. R.; Defernez, M.; Janssens, A.; Leung, W.; Doleman, J. F.; Potter, J. F.; Mithen, R. F. A diet rich in high-glucoraphanin broccoli interacts with genotype to reduce discordance in plasma metabolite profiles by modulating mitochondrial function. Am. J. Clin. Nutr. 2013, 98, 712−722. (32) Zhang, X.; Shu, X. O.; Xiang, Y. B.; Yang, G.; Li, H.; Gao, J.; Cai, H.; Gao, Y. T.; Zheng, W. Cruciferous vegetable consumption is associated with a reduced risk of total and cardiovascular disease mortality. Am. J. Clin. Nutr. 2011, 94, 240−246. (33) Wu, Q. J.; Yang, Y.; Vogtmann, E.; Wang, J.; Han, L. H.; Li, H. L.; Xiang, Y. B. Cruciferous vegetables intake and the risk of coleorectal cancer: a meta-analysis of observational studies. Ann. Oncol. 2013, 24, 1079−1087. (34) National Cancer Institute. Cruciferous vegetables and cancer prevention, www.cancer.gov/cancertopics/factsheet/diet/cruciferousvegetables (accessed Nov 20, 2013). (35) Bille, N.; Eggum, B. O.; Jacobsen, I.; Olsen, O.; Sørensen, H. Antinutritional and toxic effects in rats of individual glucosinolates (± myrosinases) added to a standard diet. Z. Tierphysiol. Tierernaehr. Futtermitteilkd. 1983, 49, 195−210. (36) Vanderpas, J. Nutritional epidemiology and thyroid hormone metabolism. Annu. Rev. Nutr. 2006, 26, 293−322. (37) Lüthy, J.; Garden, B.; Friederich, U.; Bachmann, M. Goitrin − a nitrosatable constituent of plant foodstuffs. Experientia 1984, 40, 452− 453. (38) Seo, B.; Yun, J.; Lee, S.; Kim, M.; Hwang, K.; Kim, J.; Min, K. R.; Kim, Y.; Moon, D. Barbarin as a new tyrosinase inhibitor from Barbarea orthocerus. Planta Med. 1999, 65, 683−686. (39) Hong, E.; Kim, S.-J.; Kim, G.-H. Identification and quantitative determination of glucosinolates in seeds and edible parts of Korean Chinese cabbage. Food Chem. 2011, 128, 1115−1120. (40) Fenwick, G. R.; Griffiths, N. M.; Heaney, R. K. Bitterness in Brussels sprouts (Brassica oleracea L. var. gemmifera): the role of

glucosinolate analysis, Samantha Davis for assistance with watercress cultivation, Karen R. Munk for chromosome preparation, and M. S. C. Pedras for sharing a paper in press.

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