Article pubs.acs.org/JAFC
Distribution of Selenoglucosinolates and Their Metabolites in Brassica Treated with Sodium Selenate Adam J. Matich,* Marian J. McKenzie, Ross E. Lill, Tony K. McGhie, Ronan K.-Y. Chen, and Daryl D. Rowan The New Zealand Institute for Plant & Food Research Limited (Plant & Food Research), Private Bag 11600, Palmerston North 4442, New Zealand S Supporting Information *
ABSTRACT: In Brassica species, hydrolysis of (methylthio)glucosinolates produces sulfur-containing aglycons which have demonstrated anticancer benefits. Selenized Brassicaceae contain (methylseleno)glucosinolates and their selenium-containing aglycons. As a prelude to biological testing, broccoli, cauliflower, and forage rape plants were treated with sodium selenate and their tap roots, stems, leaves, and florets analyzed for selenoglucosinolates and their Se aglycons. Two new selenoglucosinolates were identified: glucoselenoraphanin in broccoli florets and glucoselenonasturtiin in forage rape roots. A new aglycon, selenoberteroin nitrile, was identified in forage rape. The major selenoglucosinolates were glucoselenoerucin in broccoli, glucoselenoiberverin in cauliflower, and glucoselenoerucin and glucoselenoberteroin in forage rape roots. In broccoli florets, the concentrations of selenglucosinolates exceeded those of their sulfur analogues. Fertilization with selenium slightly reduced (methylthio)glucosinolates and aglycons in the roots, but increased them in the florets, the leaves, and sometimes the stems. These discoveries provide a new avenue for investigating how consumption of Brassica vegetables and their organoselenides may promote human health. KEYWORDS: broccoli, cauliflower, forage rape, selenoglucosinolate, selenoberteroin, selenoerucin, selenoiberverin, glucoselenoraphanin, glucoselenonasturtiin
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temperature, and even the circadian clock.7 Broccoli (Brassica oleracea L. var. italica) predominantly produces the aliphatic GSL glucoraphanin,8 while in cauliflower (B. oleracea L. var. botrytis), the indole glucoside glucobrassicin dominates.8,9 In forage rape (Brassica napus) the major GSLs are the aromatic gluconasturtiin in the roots and the aliphatic GSLs progoitrin and glucobrassicanapin in the leaves and shoots.10 The Brassicaceae also display an interesting selenium metabolism. Selenium is a micronutrient chemically similar to sulfur, being immediately below it in the chalcogen group of the periodic table of elements. As with S, plant-derived Secontaining compounds play an important role in human health. Countries such as Finland, New Zealand, the United Kingdom, and some parts of China have low levels of Se in their soil, and Se deficiency can potentially be a health issue.11,12 In humans Se deficiency is associated with reduced male fertility and immune and cognitive function and with heart disease, hyperthyroidism, and an increased risk of certain types of cancer. There is evidence that these disorders can be suppressed by dietary organoselenide supplements.13,14 Plants accept Se into the S assimilation pathway as the enzymes of this pathway do not appear to distinguish between S- and Se-containing compounds. Se assimilation leads to production of the selenoamino acids selenomethionine (SeMet) and selenocysteine (SeCys). The latter compound is
INTRODUCTION Plants of the Brassica genus are recognized for their production of glucosinolates (GSLs) (Figure 1).1 Hydrolysis of these sulfur-rich secondary metabolites produces modified aglycons (referred to hereafter as aglycons) which have demonstrated anticarcinogenic benefits in mammals and therefore may play an important role in human health.1 Glucosinolate biosynthesis occurs by the multistep modification of eight possible precursor amino acids: alanine, leucine, isoleucine, valine, and methionine are the precursors for the aliphatic GSLs, phenylalanine and tyrosine are the precursors for the aromatic GSLs, and tryptophan is the precursor for the indolic GSLs. The three key steps involved in GSL biosynthesis are chain elongation, biosynthesis of the core structure, and side chain modification, resulting in the production of over 120 identified GSL compounds.2 Hydrolysis of the biologically inactive GSLs is catalyzed by the β-thioglucosidase myrosinase (EC 3.2.3.1) and results in the production of bioactive isothiocyanates.3 Plants that contain epithiospecifier proteins can also form nitriles and thiocyanates from GSLs at the expense of isothiocyanate production.4 These reactions occur during tissue disruption, as in herbivory, when the GSLs and enzymes are released from their cellular compartmentalization. The bioactive compounds formed act as feeding deterrents against generalist herbivorous insects.5 As a group, the isothiocyanates, nitriles, and thiocyanates can be considered as modified GSL aglycons. The isothiocyanates have been implicated in the reduced incidence and progression of cancer.6 Different Brassica species have differing GSL profiles, and the GSL content varies according to the cultivar, tissue, season, © XXXX American Chemical Society
Received: September 8, 2014 Revised: January 26, 2015 Accepted: January 27, 2015
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DOI: 10.1021/jf505963c J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. Structures of the glucosinolates and their modified aglycons (myrosinase enzyme hydrolysis products).
the only selenoamino acid incorporated into animal selenoproteins under direct genetic control. Selenoproteins are important in the control of cellular redox balance.15 Additionally, the Brassicaceae contain the Se-specific enzyme selenocysteine methyltransferase, which catalyzes methylation of SeCys to methyl selenocysteine (3-(methylseleno)-L-alanine) and the downstream production of γ-glutamylselenomethyl selenocysteine (γ-glutamyl-3-(methylseleno)-L-alanine). Both of these modified amino acids have reported anticancer properties in mammals, the latter compound not directly so but as methyl selenocysteine to which it hydrolyzes in the intestinal tract.14 At the cellular level it is proposed that the active cancer chemopreventative agent is actually methylselenol, which arises from the β-lyase hydrolysis of methyl selenocysteine.14 The Brassicaceae are therefore of increasing interest for health benefits associated with their production of S-containing glucosinolates and selenoamino acids. Recently, we reported several new Se-containing glucosinolates, and their aglycons, in the florets of broccoli and
cauliflower and the tap roots of forage rape plants treated with sodium selenate fertilizer.16 The selenoglucosinolates were of the methylseleno class (MeSe-GSLs), most likely derived from SeMet, and their downstream metabolites were methylseleno nitriles and isothiocyanates. In these experiments, the incorporation of Se into MeSe-GSLs, in the different tissues, appeared poorly correlated with the amounts of GSLs present. For example, forage rape tap roots had much higher concentrations of MeS-aglycons than the broccoli and cauliflower florets, yet the forage rape tap roots did not have correspondingly higher concentrations of the MeSe-aglycons.16 Furthermore, broccoli is reported to have its highest total and aliphatic GSL concentrations in the florets,17,18 but the highest concentrations of the major MeS-GSL glucoerucin16,19 were found in the tap roots.18 If incorporation of Se into glucoerucin is as efficient in the tap roots as it is in the florets, then the broccoli tap roots might produce higher concentrations of MeSe-GSLs. B
DOI: 10.1021/jf505963c J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
coffee grinder. The sample was combined with 2 volumes (v/w) of prechilled diethyl ether on ice in a Schott bottle, which was sealed and left at 1 °C for a week with daily mixing before the solid material was frozen at −20 °C and the solvent poured off. The solvent extracts were dried (MgSO4) and their volumes reduced to ca. 1.5 mL under a gentle stream of nitrogen. LC−MS Analysis of Glucosinolates. Separations were performed on an Ultimate 3000 rapid separation liquid chromatograph (Dionex, Germering, Germany) with a micrOTOF-Q II mass spectrometer (Bruker Daltonics, Bremen, Germany) fitted with an electrospray source operating in negative mode. Sample injections (1 μL) were made onto a 50 mm × 2.1 mm i.d., 1.8 μm, Zorbax SB-C18 (Agilent) analytical column maintained at 50 °C and with a flow of 400 μL/min. Solvent A was H2O/MeOH (10:90, v/v), and solvent B was HCO2H/ H2O (0.5:99.5, v/v). The solvent ramp was 1:99 (solvent A:solvent B) from injection to 0.5 min, followed by a linear gradient to 30:70 (0.5− 8 min), to 75:25 (8−13 min), and to 100:0 (13−15 min) and holding at 100:0 (15−17 min). The micrOTOF-Q II source parameters were as follows: 200 °C; drying N2 flow, 8 L/min; nebulizer N2, 4 bar (400 kPa); end plate offset, −500 V; capillary voltage, +3500 V; mass range, 100−1500 Da; 2 scans/s. Postacquisition internal mass calibration used sodium formate clusters with the sodium formate delivered by a syringe pump at the start of each analysis. MS data were processed by Compass DataAnalysis (Bruker Daltonics). The external standard used for quantitation (using [M − H]− current peak areas) was glucoerucin, 2a ([4-(methylthio)butyl]glucosinolate) (Cfm Oskar Tropitzsch, Marktredwitz, Germany). The external standard and the plant extracts were spiked with an internal standard, epicatechin (Sigma-Aldrich, Auckland, New Zealand), to enable correction for sample volumes and for changes in LC−MS system responses. Identification of the S- and Se-GSLs by high-resolution LC−MS/MS has been described previously.16 Total Elemental Se Present as GSLs in the Plant Tissues. The total concentration of elemental Se present as MeSe-GSLs in each plant part was calculated by summing the concentrations of elemental Se in each of the individual MeSe-GSLs, using the molecular weight of the GSLs and the percentage of that MW that was constituted by elemental Se. GC−MS Analysis of Glucosinolate Hydrolysis Products (Modified Aglycons). Separations were performed on an Agilent 6890N gas chromatograph coupled to a Waters GCT time-of-flight (ToF) mass spectrometer with an EI energy of 70 eV and a scan time of 0.4 s. Splitless injections of 1 μL over 30 s were made at 220 °C onto a 20 m × 0.18 mm i.d. × 0.18 μm film, Stabilwax (Restek, Bellefonte, PA) capillary column with a He flow of 0.9 mL/min. The oven temperature program was 1 min at 35 °C, 3 °C/min to 100 °C, and 7 °C/min to 240 °C, which was held for 5 min. Compounds were quantitated (using M+ peak areas) against the synthetic16 external standard selenoerucin nitrile, 5b, CH3SeC4H8CN, at concentrations of 0.02, 0.08, and 0.5 μL/L, to provide a response calibration curve. To keep the concentrations of compounds in the solvent extracts in the linear range of the MS detector, samples were injected neat and at 10and 50-fold dilutions. All external standards and plant extracts were spiked with an internal standard (pentadecafluorooctanol) (Pierce Chemicals, Rockford, IL) to enable correction for extract sample volumes and changes in GC−MS system responses. GC−MS Data. 6-(Methylseleno)hexanenitrile, CH3SeC5H10CN (Figure 1). RI (Wax) 2109. HR-ToF-EIMS: m/z 191.0194 (−1.9 mDa), [M]+. ToF-EIMS (Figure 2): m/z (fragment) (rel intens) 193 ([M]+, Se82) (9),191 ([M]+, Se80) (55), 189 ([M]+, Se78) (27), 187 ([M]+, Se76) (11), 123 [CH3SeC2H4]+ (9), 109 [CH3SeCH2]+ (30), 96 [C5H10CN]+ (100) and [CH3SeH]+ (minor), 94 [CH2Se]+ (25), 69 [C4H7N]+ (70), 55 [C3H5N]+ (89), 41 [C2H3N]+ (39). LC−MS Data. Glucoselenoraphanin, C12H22NO10S2Se, [M − H]−, m/z 483.9856 (+0.6 mDa). MS/MS: C6H11O5S−, m/z 195.0281 (−4.6 mDa); C6H11O6SO3−, m/z 259.0104 (−2 mDa), C6H11O5SSO3−, m/z 274.9882 (−1.3 mDa); C11H18NO9S2−, m/z 372.0424 (+0.1 mDa). Statistical Treatment. Error values are standard errors of the mean (SEM), calculated using the Microsoft Excel 2007 “standard deviation” function and dividing the value obtained by the square root
Other workers have synthesized a number of isoselenocyanates: selenosulforaphane and several alkyl and phenylalkyl isoselenocyanates.20,21 These synthetic compounds were reported as more bioeffective than their S analogues at reducing the incidence and progression of carcinogenesis. However, in these synthetic organoselenides the Se atom would have been located in the glucose moiety (e.g., compound 11, R1 = Se) of the parent GSL, in contrast with the methylseleno compounds found in planta, where the Se atom has been found only in the position derived from methionine.16 We were therefore interested in investigating which Brassica plant tissues had the highest concentrations of GSL compounds capable of incorporating Se, whether they also had the highest concentrations of the newly discovered methylseleno compounds, and also whether Se-GSLs with the Se incorporated into the thioglucose group could also be produced in Brassica.
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MATERIALS AND METHODS
Plant Growth and Harvesting. The two broccoli (B. oleracea L. var. italica) cultivars chosen were a high-GSL-producing cultivar (‘Booster’, hereafter referred to as high-GSL broccoli) and a moderateto low-GSL-producing cultivar (‘Triathlon’, hereafter referred to as low-GSL broccoli). Cauliflower (B. oleracea L. var. botrytis cv. ‘Liberty’) and forage rape (B. napus cv. ‘Maxima’) were also selected. Plants were grown from seed in separate 8 L bags of potting mix in a glass house at Plant & Food Research, Palmerston North, New Zealand. Plants were watered once daily via watering spikes for 1 min. Selenium feeding was commenced for broccoli and cauliflower following the emergence of immature floret material from the meristem and for forage rape once the plants had a well-established stem (ca. 16 cm from the soil to the top of the meristem) and approximately 10 fully expanded leaves. Plants were fed via the soil with 20 mL of 5.0 mM sodium selenate twice weekly, for 4 weeks, after watering. This Se application is equivalent to ca. 15 kg/ha, which is considerably higher than the 3−76 g/ha applied to agricultural and forage crops.11 Broccoli produced at this high rate of Se application contained at least 500-fold more Se than control samples22 but suffered no adverse effects upon its growth. Following the Se feeding, the leaves, stems, and lower stem/tap root (3−4 cm long) were harvested from all of the plants, after four months of growth, as well as mature floret tissue from the broccoli and cauliflower plants. For the forage rape roots, the outer fibrous ring was removed and only the inner pith was sampled. All material was immediately frozen in liquid nitrogen before storage at −80 °C until extraction. Extraction of Glucosinolates for LC−MS Analysis. Triplicate extractions were performed on the tissue from selenized and duplicate extractions from control (nonselenized) plants, with each replicate comprising tissue from a separate plant. In the case of the leaves, for each replicate the two most recently expanded leaves were harvested from each of two separate plants (four leaves per replicate). For each replicate frozen tissue (2−10 g FW) was accurately weighed, ground with dry ice to a powder in a coffee grinder, and poured into boiling 70% ethanol (10 volumes (v/w)), which was refluxed for 5 min to inactivate the myrosinase.8 Samples were cooled to 5 °C and concentrated in vacuo at 40 °C to volumes of ca. 5 mL. Pigments were removed from the concentrates by washing with an equal volume of dichloromethane (2× for the leaf and floret extracts), and solids were removed by centrifugation at 1000g for 1 min in a Hema-C centrifuge (Jouan, Saint Herblain, France). A cold (ice−water) acidic (0.5% AcOH) methanol extraction of GSLs was also performed on the high-GSL broccoli florets, based upon the method of Hsu et al.23 The extracts were colorless, so pigment removal was not necessary. The amounts of glucoselenoraphanin in two replicates were compared with those in two parallel hot ethanolic extracts. Extraction of Glucosinolate Hydrolysis Products (Modified Aglycons) for GC−MS Analysis. The same number of replicates was used as above for the glucosinolate extractions. For each replicate 3−5 g FW was accurately weighed and ground with dry ice to a powder in a C
DOI: 10.1021/jf505963c J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
florets we analyzed. Despite these very high levels of glucoraphanin, we did not identify its selenoxide analogue 10b in any of the selenized Brassica in our previous study in which a low-GSL broccoli was selenized.16 Selenoxides are generally considered to be thermally unstable;25 hence, it was surprising to identify glucoselenoraphanin. Thermal instability may however explain the extremely low levels of selenization (
