Identification of hordatines and other phenolamides in barley (Hordeum vulgare) and beer by UPLC-QTOF-MS

Journal of Cereal Science 60 (2014) 645e652

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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs

Identification of hordatines and other phenolamides in barley (Hordeum vulgare) and beer by UPLC-QTOF-MS Juha-Matti Pihlava* MTT Agrifood Research Finland, Biotechnology and Food Research, 31600 Jokioinen, Finland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 March 2014 Received in revised form 2 July 2014 Accepted 9 July 2014 Available online 4 August 2014

Antifungal hordatines and their precursors, hydroxycinnamoylagmatines, were analyzed in dormant barley seeds and beer sample by ultra-performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight mass spectrometry (QTOF-MS) operating in MSE-mode. Three hordatine basic structures, namely A, B and C, were found in barley and beer samples. All of these hordatines had analogous structures containing either one (hordatines A1, B1 and C1) or two (hordatines A2, B2 and C2) hydroxylated agmatine residues. All hordatines and hydroxycinnamoylagmatines were also present as sugar conjugates. Furthermore, N6 emethylated form of hordatines A, B and C, as well as their precursors, coumaroyl-N6-methylagmatine and feruloyl-N6-methylagmatine, were tentatively identified from barley samples. In addition, a number of other phenolamides, namely hydroxycinnamic acids conjugated to spermidine, were identified from barley and beer. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Barley Beer Hordatine Phenolamides

1. Introduction Polyamides putrescine, spermidine, spermine and agmatine are cationic compounds ubiquitously distributed in the plant kingdom. Polyamides occur in higher plants in free forms, but they also conjugate with small molecules and proteins. They possess a critical role in plant development, response to abiotic stress and defense systems against pathogens and herbivores (Kuznetsov and Shevyakova, 2007; Kristensen et al., 2004; Bassard et al., 2010). When polyamides are conjugated with hydroxycinnamic acids, such as p-coumaric, ferulic and caffeic acid, they form a group called phenolamides, or hydroxycinnamic acid amides. The number of hydroxycinnamic acid residues in the polyamide backbone is usually one to three (Bienz et al., 2005). The accumulation of phenolamides has been described as a stress response against biotic or abiotic factors (Kristensen et al., 2004; Bassard et al., 2010; Moheb et al., 2011). It has also been suggested that, due their photochemical properties, polyhydroxycinnamoylamides, which

Abbreviations: Agm, agmatine; Cou, coumaric acid/coumaroyl; Fer, ferulic acid/ feruloyl; m/z, mass to charge ratio; MS, mass spectrometry; MSE, data independent acquisition, dynamically ramped MS/MS; OHAgm, hydroxyagmatine; QTOF, quadrupole-time of flight; UPLC, ultra performance liquid chromatography. * Tel.: þ358 29 5317 622. E-mail address: juha-matti.pihlava@mtt.fi. http://dx.doi.org/10.1016/j.jcs.2014.07.002 0733-5210/© 2014 Elsevier Ltd. All rights reserved.

are particularly accumulated in pollen, might act as a photoprotectant of DNA (Bienz et al., 2005). Hordatines are phenolamides typical to barley (Hordeum vulgare), although small amounts can also be found in wheat (Nomura et al., 2007). In the first step of hordatine biosynthesis, agmatine coumaroyltransferase (ACT) catalyzes agmatine conjugates from pcoumaroyl-CoA and feruloyl-CoA. The hydroxycinnamic acid agmatines are further oxidatively dimerized to hordatines (Kristensen et al., 2004) (Fig. 1). Until recently, no other hordatine aglycon structures, besides hordatine A, a dimer of p-coumaroylagmatine, and hordatine B, a dimer of feruloylagmatine and pcoumaroylagmatine, had been reported since their discovery (Stoessl, 1965, 1967). The glycosylated forms of hordatine A and B have collectively been known as hordatine M (Stoessl, 1966, 1967). Gorzolka et al. (2014) reported a new hordatine structure, hordatine C, consisting of two feroloylagmatines, in germinated barley. They also presented a fourth possible hordatine aglycon structure (hordatine D), which consist of feruloyl- and sinapoylagmatine. In addition, Gorzolka et al. (2014) reported the hydroxyl-forms of hordatines A, B, C and D as well as their glycosylated and diglycosylated forms. Heuberger et al. (2014) reported glycosides of hordatine C (exact m/z not provided), hydroxy-hordatine B (759.37 m/ z, named here as hordatine B1), dihydroxy-hordatine B (775.363 m/ z, named here as hordatine B2) and dihydroxy-hordatine C (805.372 m/z, named here as hordatine C2) from a large study of barley varieties and their corresponding malts.

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J.-M. Pihlava / Journal of Cereal Science 60 (2014) 645e652

O

The phenylpropanoid pathway

OH NH

H2N

NH

L-Arginine NH2

Arginine decarboxylase R1

COSCoA NH

H2N

HO

NH

Agmatine

NH2

p-Hydroxycinnamoyl-CoA Other polyamides Agmatine coumaroyltransferase (ACT)

NH2

H2N

Putrescine O R1

NH

NH

NH

NH2 HO

NH

H2N

p-Hydroxycinnamoylagmatine

NH2

Spermidine

Peroxidase

NH

NH

H2N

NH2

Spermine O R1

NH

NH

NH NH2

NH2

O NH R2

NH

NH

O HO

Hordatine Fig. 1. Biosynthesis of polyamines, p-hydroxycinnamic acid agmatines and hordatines (adopted from Kristensen et al., 2004; Bassard et al., 2010). In monomeric forms R1 ¼ H, pcoumaroyl-CoA and p-coumaroylagmatine, and R1 ¼ OCH3, p-feruloyl-CoA and feruloylagmatine. In hydroxycinnamoylagmatines glycosylation is via the OH-group in position 4 of the aromatic ring. For hordatine A, R1 ¼ R2 ¼H, for hordatine B, R1 ¼ OCH3 and R2 ¼ H (or R1 ¼ H and R2 ¼ OCH3) and for hordatine C R1 ¼ R2 ¼OCH3.

Hordatines possess strong antifungal activities (Ludwig et al., 1960; Stoessl and Unwin, 1969) as well as the p-coumar€penack et al., 1998; oylagmatine and its hydroxylated form (von Ro Jin et al., 2003). They can act as preformed defense compounds in barley seedlings or as inducible accumulating defense chemicals in older plants after pathogen attack (Batchu et al., 2006; Kristensen et al., 2004). The accumulation of hordatines seems to be more a genetic property, rather than the result of direct influence of abiotic factors (Batchu et al., 2006). Considering barley as part of our diet, hordatine A glucoside has been identified from unmalted barley by Kohyama and Ono (2013) at concentrations of 103e254 nmol/g dry weight (73e181 ug/g dw). Based on the pearling tests, Kohyama and Ono (2013) concluded that hordatine A glucoside is localized in the aleurone layer of the

grain. Gorzolka et al. (2014) used a novel MALDI MS imaging technique to study the localization of hordatines in germinated barley seeds and were able to identify 8 hordatine aglycon structures and 12 hexose conjugates of hordatines. Interestingly, localization of hordatines varies, e.g. the amount of hordatine A was highest in the shoots, while the hordatine B was highest in the roots of germinated grain. Fuji et al. (2002) set up a study to investigate why certain beer congener stimulated gastrointestinal motility. The underlying mechanism was shown to be the ability of this congener to bind to muscarinic M3 receptor, which is present in smooth muscles of the upper gastrointestinal tract. Yokoo et al. (2004) were able to isolate these active components from beer and named them as compounds A and B. These compounds were later identified and characterized

J.-M. Pihlava / Journal of Cereal Science 60 (2014) 645e652

as cis- and trans -isomer of hordatine A by Yamaji et al. (2007). Furthermore, hordatine A also exhibit antagonist activity against the a1A adrenoceptors, which are found in central and peripheral nervous systems. a1A-Adrenoceptors play an important role in contraction of smooth muscle tissues in the cardiovascular system and lower urinary tract (Wakimoto et al., 2009). Besides the physiological effects, hordatines or their glycosides, can also cause astringent oral sensations and thus contribute to beer aftertaste (Kageyama et al., 2012). The stereochemistry of hordatine A glucoside and diglucoside and hordatine B glucoside from the acrospires of barley malt, has been extensively studied by Kageyama et al. (2011, 2012). The polyamides per se, and especially agmatine, have also a wide variety of biological activities in mammals. They are required for the growth, the maintenance and the function of normal cells (Piletz et al., 2013; Moinard et al., 2005). The aim of this preliminary study was to screen and identify hordatine structures and other phenolamides in barley and beer, by ultra-performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight mass spectrometry (QTOF-MS) operated in data independent acquisition MSE-mode. 2. Materials and methods 2.1. Materials and reagents Methanol used for extraction was LC-MS-quality (VWR International, Leuven, Belgium). Deionized water was from a laboratory MilliQ system (Millipore, Bedford, MA, USA). Acetonitrile and water containing 0.1% formic acid were LC-MS ultra-quality (SigmaeAldrich, St. Louis, MO, USA). Malting type barley SW Makof and waxy type barley variety Cinnamon were from the field trials (year 2006, Hauho, Finland). Finnish lager beer (4.6% -alc.) was purchased from a local grocery store. 2.2. Sample preparation Samples were ground with a falling-number hammer mill, using a 1.0 mm sieve and stored in a freezer until analysis. A sample of ground barley (2.5 g) was extracted with 80% methanol (100 ml) with magnetic stirring overnight. The sample was then centrifuged (1500 rpm 10 min) and the supernatant was transferred to a rotary evaporation bottle. The remaining solid residue was mixed with methanol and centrifuged. The combined supernatants were evaporated to dryness in a rotary evaporator. The sample was dissolved in methanol (2 ml) and filtered through a 0.20 um PTFE membrane filter (Pall corporation, Port Washington, NY, USA) into an autosampler vial. An aliquot of the beer sample was filtered through a 0.20 um membrane filter into an autosampler vial. 2.3. Instrumentation An ultra-performance liquid chromatographic system (Acquity UPLC I-Class, Waters, Milford, MA, USA) was coupled to a Waters Xevo G2 QTOF mass spectrometer. Compounds were separated on a Waters Acquity BEH C18 (1.7 mm, 2.1 mm * 150 mm) column using a gradient of 0.1% formic acid in water (A) and of 0.1% formic acid in acetonitrile (B). The gradient programme was as follows: 2e60% of B in 24 min, 60e100% of B in 24e31 min, held at 100% of B for 2 min, 100-2% in 1 min and held at 2% of B for 4 min. The mobile-phase flow rate was 0.55 ml/min. The temperature of the column oven was 45  C. Sample injection volume was 1 ml.

647

The UPLC was connected to the mass spectrometer via an electrospray interface (ESI), using capillary voltage of þ0.5 kV. The sampling cone was set to 35 V and extraction cone to 4 V. The cone and desolvation nitrogen gas flows were 15 and 990 l/h, respectively. The desolvation temperature was 550  C. Source temperature was 150  C. Argon was used as the collision gas. The mass spectrometer was operated in high resolution positive ion mode. Sample analysis was done by data independent acquisition (MSE) centroid data mode in a full scan m/z 50e1200 with 0.2 s scan time. Acquisition of mass spectral data was accomplished in two continuous functions. In the first function, precursor data was collected using a constant low collision energy of 4 V in MSmode and in the second, MSE, function, the precursor ions were further fragmented using high collision energy ramped from 15 to 40 eV. Leucine-enkelphalin (m/z 565.2771) was used as a reference lock-mass compound with automatic mass correction enabled. The system was operated by Waters MarkerLynx 4.1 software. Data was handled by post targeted analysis technique i.e. filtering the data manually using exact masses [MþH]þ ± 2.5 mDa of a number of possible hordatine and agmatine candidates and creating ion chromatograms. The [MþH]þ -ion in the mass spectrum of the peaks in these chromatograms were then checked using the Elemental Composition function of the Waters MassLynx software. The molecular structures of certain compounds were processed using the MassFragment function of MassLynx, in order to assign the structural composition of the major fragment ions in the MSE spectrum. MSE data was in the end also crosschecked using the m/z-values of fragments typical to agmatine (C5H12N3 114.103 m/z), hydroxy-agmatine (C5H13N4 129.114 m/z) and methylagmatine (C6H14N3 128.119 m/z) structure (Supplemental material Figure S1). Agmatines, agmatine glycosides and the methylated forms of hordatine B and C, were further confirmed by separate MSeMS experiment using collision energy ramped from 15 to 50 eV. 3. Results and discussion Hydroxycinnamic acid agmatines and hordatines found by UPLC-QTOF analysis are listed in Tables 1 and 2. Since both barley varieties, SW Makof and Cinnamon, contained the same compounds, they will be referred later only as barley. 3.1. Hydroxycinnamic acid agmatines The precursors of hordatines, namely p-coumaroylagmatine, pcoumaroylhydroxyagmatine, coumaroyl-N6-methylagmatine, feruloylagmatine, feruloylhydroxyagmatine, feruloyl-N6-methylagmatine and sinapoylagmatine were identified from barley and beer (Table 1). Of these, coumaroyl-N6-methylagmatine and feruloyl-N6-methylagmatine and the hexose conjugates of all agmatines, have not been reported before. The MSE spectrum of p-coumaroylagmatine and p-coumaroylhydroxyagmatine contained the same fragment ions as the MS/ € penack et al. (1998) MS spectra presented by von Ro (Supplementary material Figure S2). The mass spectra of glycosides of coumaroylagmatines, shows a typical 162.05 m/z loss resulting from the removal of a hexose unit (Supplementary material Figure S3). Mass spectrum, by MS/MS mode, of feruloylagmatine and feruloylhydroxyagmatine as well as their glycosides are presented as supplementary material Figure S4 and S5. For comparison, MSE and MS/MS spectra of feruloylagmatine and feruloylhydroxyagmatine are presented as supplemental material Figure S6. Although the exact place of hydroxylation cannot be distinguished by the equipment used in current work, the most likely

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J.-M. Pihlava / Journal of Cereal Science 60 (2014) 645e652

Table 1 Hydroxycinnamoylagmatines and their sugar conjugates in barley (Brl) and beer samples. Letter “G” or “H” in the reference (Ref) column indicates that the compound has been reported by Gorzolka et al. (2014) or Heuberger et al. (2014) respectively. EC þ Hþ ¼ elemental composition in positive mode i.e. with additional proton. Agmatines

Abbreviation

EC þ Hþ

calc. [MþH]þ m/z

Found [MþH]þ m/z

Error (ppm)

Rt (min)

Sample

Ref

Coumaroylagmatine hexoside CoumaroyleOHeagmatine hexoside Met-coumaroylagmatine hexoside Feruloylagmatine hexoside FeruloyleOHeagmatine hexoside Met- feruloylagmatine hexoside Sinapoylagmatine hexoside

CouAgm CouAgm-hex CouOHAgm CouOHAgm-hex Met-CouAgm Met-CouAgm-hex FerAgm FerAgm-hex FerOHAgm FerOHAgm-hex Met-FerAgm Met-FerAgm-hex SinAgm SinAgm-hex

C14H21N4O2 C20H31N4O7 C14H21N4O3 C20H31N4O8 C15H23N4O2 C21H33N4O7 C15H23N4O3 C21H33N4O8 C15H23N4O4 C21H33N4O9 C16H25N4O3 C22H35N4O8 C16H25N4O4 C22H35N4O9

277.1666 439.2194 293.1613 455.2141 291.1821 453.2349 307.1771 469.2299 323.1719 485.2247 321.1927 483.2455 337.1876 499.2404

277.1675 439.2192 293.1619 455.2143 291.1817 453.2353 307.1782 469.2308 323.1713 485.2262 321.1921 483.2467 337.1870 499.2416

3.2 0.5 2.0 0.4 1.4 0.9 3.6 1.9 1.9 3.1 1.9 2.5 1.8 2.4

4.63 3.26 3.73 2.61 5.11 3.66 5.24 3.73 4.34 3.08 5.64 4.11 4.78 3.45

Brl, Brl, Brl, Brl, Brl Brl Brl, Brl Brl, Brl Brl Brl Brl, Brl

Beer Beer Beer Beer

G

Beer

G, H

Beer

G

Beer

G

G

Table 2 Hordatines and their sugar, hexose (hex), conjugates in barley (Brl) and beer samples. Numbers distinguished in italic are minor peaks. EC þ Hþ ¼ elemental composition in positive mode i.e. with additional proton. nd ¼ compound not detected, tr ¼ trace amount, but [MþH]þ ion still detectable. For hordatines conjugated with 3 or more hexose units, the value in parenthesis following the [Mþ2H]2þvalue, is the actual [MþH]þ ion. If found, and being less than 1200 m/z, it is listed in parenthesis and if not found it is referred as “nd” in the “Found [MþH]þm/z”column. Letter “G” or “H” in the reference (Ref) column indicates that the compound has been reported by Gorzolka et al. (2014) or Heuberger et al. (2014) respectively. Hordatines

Structure

EC þ Hþ

Calc. [MþH]þ m/z

Found [MþH]þ m/z

Error (ppm)

Rt (min)

Sample

Ref

Hordatine A

CouAgmeCouAgm

C28H39N8O4

551.3094 713.3623 875.4151 519.2379 (1037.4679) 600.2643 (1199.5207) 681.2907 (1361.5736) 762.3171 (1523.6264) 843.3435 (1685.6792) 924.3699 (1847.7320) 1005.3963 (2009.7849) 581.3200

hex

C35H51N8O10

743.3728

2*hex 3*hex 4*hex 5*hex 6*hex 7*hex 8*hex Hordatine C

FerAgmeFerAgm

C41H61N8O15 C47H71N8O20 C53H81N8O25 C59H91N8O30 C65H101N8O35 C71H111N8O40 C77H121N8O45 C30H43N8O6

905.4256 534.2432 615.2696 696.2960 777.3224 858.3488 939.3752 611.3306

CouOHAgm-CouAgm

C36H53N8O11 C42H63N8O16 C48H73N8O21 C54H83N8O26 C28H39N8O5

773.3834 935.4362 549.2484 (1097.4890) 630.2748 (1259.5418) 567.3043

CouOHAgm-FerAgm

C34H49N8O10 C40H59N8O15 C46H69N8O20 C52H79N8O25 C29H41N8O6

729.3572 891.4099 527.2353 (1053.2627) 608.2617 (1215.5155) 597.3146

4.4 2.0 6.4 1.8 1.4 1.7 1.0 1.4 4.6 3.9 4.5 2.9 3.8 3.6 0.2 6.2 2.4 3.2 1.0 2.3 0.1 4.6 0.7 3.9 1.8 2.0 1.6 2.0 5.1 0.3 1.9 1.4 3.7 7.6 - 0.7 0.7 1.8 2.7 6.4 0.3 1.8 2.2 4.4 2.1 3.3 2.5 2.9 3.5

5.70 6.20 4.83 5.06 4.68/4.91 4.54/5.00 4.39/4.98 4.28/4.95 4.28/4.97 4.25/4.94 4.16/4.92 5.48 6.17 6.31 6.57 4.81 5.16 4.91 4.68/4.88 4.56/4.94 4.43/4.93 4.36/4.91 4.32/4.91 4.30/4.89 6.51 6.60 5.10 4.78/5.03 4.89/5.09 4.74 5.58 5.89 4.60 4.68 4.59 4.60 5.57 5.82 6.04 4.36 4.53 4.61 4.60 5.98 6.19 4.63 4.82 4.88

Brl, Beer Brl, Beer Brl, Beer Brl, Beer Beer Beer Beer Beer Beer Beer Beer Brl Brl, Beer Brl, Beer Brl, Beer Brl, Beer Brl Brl, Beer Brl, Beer Beer Beer Beer Beer Beer Brl, Beer Brl, Beer Brl Brl/Beer Brl, Beer Beer Brl, Beer Brl, Beer Brl, Beer Brl/Beer Beer Beer Brl, Beer Brl, Beer Brl Brl Brl, Beer Brl, Beer Brl, Beer Brl/Beer Brl/Beer Brl Brl Brl

H G, H G G

CouAgm-FerAgm

C34H49N8O9 C40H59N8O14 C46H69N8O19 C52H79N8O24 C58H89N8O29 C64H99N8O34 C70H109N8O39 C76H119N8O44 C82H129N8O49 C29H41N8O5

551.3118 551.3105 713.3669 875.4135 519.2386 (1037.4653) 600.2653 (nd) 681.2900 762.3160 843.3474 924.3663 1005.4009 581.3217 581.3222 581.3221 581.3199 743.3774 743.3746 905.4285 534.2437 (1067.491) 615.271 696.2959 777.326 858.3494 939.3715 611.3317 611.3318 773.3846 935.4381/tr 549.2512 (1097.4843) 630.2750 567.3054 567.3051 729.3599 891.4089 527.2349 (nd) 608.2621 597.3157 597.3162 597.3184 759.3681 759.3693 921.4226 542.243 (1083.4980) 627.3268/tr 627.3276 789.3803 789.3806 951.4344

hex 2*hex 3*hex 4*hex 5*hex 6*hex 7*hex 8*hex 9*hex Hordatine B

hex 2*hex 3*hex 4*hex Hordatine A1 hex 2*hex 3*hex 4*hex Hordatine B1

(1067.4706) (1229.5234) (1391.5762) (1553.6291) (1715.6819) (1877.7347)

hex

C35H51N8O11

759.3679

2*hex 3*hex Hordatine C1

C41H61N8O16 C47H71N8O21 C30H43N8O7

921.4206 542.2406 (1083.4734) 627.3255

hex

C36H53N8O12

789.3783

2*hex

C42H63N8O17

951.4311

FerOHAgm-FerAgm

H G, H

G G

G G G

G G G

G

G, H

G G G

J.-M. Pihlava / Journal of Cereal Science 60 (2014) 645e652

649

Table 2 (continued ) Hordatines 3*hex Hordatine A2 hex 2*hex 3*hex Hordatine B2 hex 2*hex 3*hex 4*hex Hordatine hex 2*hex 3*hex 4*hex Hordatine hex Hordatine hex 2*hex Hordatine hex 2*hex

C2

Structure

EC þ Hþ

Calc. [MþH]þ m/z

Found [MþH]þ m/z

Error (ppm)

Rt (min)

Sample

CouOHAgmeCouOHAgm

C48H73N8O22 C28H39N8O6

557.2459 (1113.4839) 583.2993

CouOHAgm-FerOHAgm

C34H49N8O11 C40H59N8O16 C46H69N8O21 C29H41N8O7

745.3521 907.4049 535.2328 (1069.4577) 613.3098

C35H51N8O12 C41H61N8O17 C47H71N8O22 C53H81N8O27 C30H43N8O8 C36H53N8O13 C42H63N8O18 C48H73N8O23 C54H83N8O28 C29H41N8O4 C35H51N8O9 C30H43N8O5 C36H53N8O10 C42H63N8O15 C31H45N8O6 C37H55N8O11 C43H65N8O16

775.3626 937.4155 550.2378 631.2645 643.3207 805.3734 967.4258 565.2433 646.2698 565.3251 727.3779 595.3356 757.3885 460.2249 625.3462 787.3994 949.4519

557.2524 (1113.5050) 583.301 tr/583.2985 745.3600/tr 907.4088 535.2323 (nd) 613.3123 613.3098 775.3644 937.4193 550.2401 (nd) 631.2708 643.3217 805.3742 967.4278 565.2427 (nd) 646.2675 565.3276 727.3805 595.3377/tr 757.3902 460.2256 (919.4538) 625.3476/tr (as fragment of 2*glc) 949.4545

11.8 2.9 1.4 10.6 4.3 0.9 4.1 0.0 2.3 4.1 3.7 10.0 1.6 1.0 2.1 1.1 3.5 4.4 3.6 3.5 2.2 2.3 2.2

4.82 5.23 5.31 4.11 4.09 3.91 5.16 5.43 4.04 4.15 4.16 4.04 5.53 4.28 4.36 4.09 4.13 6.52 5.14 6.43 5.08 5.16 6.81 (5.37) 5.37

Brl, Beer Brl, Beer Brl/Beer Brl/Beer Beer Beer Brl, Beer Brl, Beer Brl, Beer Brl, Beer Brl, Beer Beer Brl Brl Brl Beer Beer Brl, Beer Brl Brl/Beer Brl Brl Brl/Beer (Brl) Brl

FerOHAgmeFerOHAgm

A-Met

Met(CouAgm-FerAgm)

B-Met

Met(CouAgm-FerAgm)

C-Met

Met(FerAgmeFerAgm)

(1099.4683) (1261.5211)

(1129.4789) (1291.5317)

(919.4413)

€penack place is the g(3)-position of the agmatine structure (von Ro et al., 1998). The other possibility could have been the hypothetical agmatine analog of Nu -hydroxy-L-arginine (NOHA). But since Nuhydroxy-agmatine structure has not been reported, while Hamana and Matsuzaki (1993) reported g(3)-hydroxyagmatine in the seeds of leguminous plants, the most likely structure would also in this case be g(3)-hydroxyagmatine. As novel compounds, N6-methylated agmatines of coumaric and ferulic acids and their glycosides were found from barley. The assumption that the hydroxygroup in position 4 of the p-coumaroyl- or feruloylagmatine structure is not methylated, was confirmed by the presence of the hexose conjugate of these agmatines. Since the only methylated agmatine structure reported in the literature is the N6-methylagmatine in the seeds of some leguminous plants (Matsuzaki et al., 1990), the same methylation site is presumably in question also in this case. However, it is not directly possible to confirm the exact methylation site by the mass spectrometric analysis used. The major differences, besides the [MþH]þ ions, in the MS/MS spectrum of feruloyl-N6-methylagmatine and feruloylagmatine, was the methylagmatine (C6H14N3 128.119 m/z) fragment, which was absent from the spectrum of feruloylagmatine, and the relative abundance of the agmatine fragment (C5H12N3 114.103 m/z), which was lower in the spectrum of feruloyl-N6-methylagmatine (Supplemental material Figures S4eS6). For coumaroyl-N6-methylagmatine and its hexoside, no MS/MS data is available and so their identification is based solely on the MS and MSE data. It was impossible to obtain a clear MSE spectrum of coumaroyl-N6-methylagmatine due to the coeluting hordatine C hexoside, so the identification relies on the exact mass in MS mode and the good match by Elemental Composition (not shown). In case of coumaroyl-N6-methylagmatine hexoside, the MSE spectrum showed methylagmatine fragment (128.117 m/z) and a number of the same fragments as coumaroylagmatine hexoside (Supplementary material Figure S3). Sinapoylagmatine, which was first reported by Gorzolka et al. (2014), was also found in barley and beer samples together with its hexose conjugate. However, the elution times of these compounds in this study were somewhat conflicting, i.e. sinapoylagmatine eluting before feruloylagmatine (Table 2). In MSE mode, the co-eluting compounds made the identification challenging and

2.7

Ref

H

H

even in the mass spectrum by MS/MS, the fragmentation of sinapoylagmatine seemed not to produce, at least at the same abundance, the specific agmatine residues like coumaroyl- and feruloylagmatine did (Supplementary material Figure S7). This difference is especially well seen on the hexoside spectra (Supplementary material Figure S8). Sinapoylhydroxyagmatine, as reported also for the first time by Gorzolka et al. (2014), was not found. Mass spectra of the hydroxycinnamoyl agmatines produced in the MSE mode, were generally in good agreement with the ones produced by the more selective MSeMS mode (Supplemental material Figures S2 and S6). However, in some cases, additional fragments originating from the co-eluting compounds, were present in the MSE spectra. 3.2. Hordatines The aglycons, hordatine A (CouAgmeCouAgm), hordatine B (CouAgm-FerAgm) and hordatine C (FerAgmeFerAgm) were found in barley and beer samples. All of these three compounds had analogues, containing either one or two hydroxyagmatine (OHAgm) residue. These hordatines were named as follows: hordatine A1 (CouOHAgm-CouAgm), hordatine B1 (CouOHAgm-FerAgm), hordatine C1 (FerOHAgm-FerAgm), hordatine A2 (CouOHAgmeCouOHAgm), hordatine B2 (CouOHAgm-FerOHAgm) and hordatine C2 (FerOHAgmeFerOHAgm) (Fig. 2). Hordatines A, C, A1, C1, A2 and B2 had one minor peak, hordatine B three and hordatine B1 two minor peaks besides the major peak in their [MþH]þ ion chromatograms (minor peaks are indicated in italic in the Table 2). These would be cis- and trans-isomers of hordatines (Nomura et al., 2007). In case of hordatine B, two of the minor peaks could be the regioisomer FerAgm-CouAgm of CouAgm-FerAgm and its cis/trans eisomer. Lower concentration of hordatine B1 could explain why one minor peak from the ion chromatogram was not detected. Hordatine A partly co-eluted with hordatine B, so that it was not possible to get a spectrum of hordatine A without ions originating from hordatine B (see [MþH]þ ion chromatograms in Figure S9 in supplemental material). The mass spectrum in MS mode of all hordatine aglycons showed a double charged base peak [Mþ2H]2þ, while the second biggest

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degrees of glycosylation, as in the case of hordatine A, with up to 9 hexose units, and B, with 8 hexose units, were found in beer samples (Table 2 and Supplemental material figure S16 and S17). Because of the double charge, the mass difference between adjacent hexosides was 81.0 m/z. It must be kept in mind that the tentative identifications are based only on the [Mþ2H]2þ masses found, although the errors (in ppm) between calculated and measured masses are low (Table 2). Because of the experimental set-up (i.e. extraction using 80% methanol), it is not possible to rule out or to confirm whether these kinds of conjugates of high glycosylation would also be present in the dormant barley grain. As novel compounds, monomethylated forms of hordatines A, B and C were found in barley samples (Supplemental material Figure S9, S18, and S19). As in the case of methylated agmatines, since the hexose conjugates of these compounds were also found, methylation could not be in the 4-hydroxy group of the aromatic ring. Ions 145.145 (C6H17N4), 171.125 (C7H15N4O) and a specific 128.119 (C6H14N3) m/z indicating methylagmatine structure, were found in the MSE spectra of these hordatines, as well as ions from the agmatine residue following the loss of a methyl group in N6position (Supplemental material Figure S1, S18 and S19). No dimethylated hordatines were found. Structures of methylated hordatines B and C, were further confirmed by the MSeMS experiment. Because the abundance of the precursor ions of these compounds was low, MSE spectra suffered from additional “noise” of coeor close-eluting compounds (Supplemental material S20). Hordatine D, consisting of feruloyl- and sinapoylagmatine, as reported by Gorzolka et al. (2014), was not found from the samples of this study.

Fig. 2. Structures of hordatines found in barley and beer. In hordatine aglycons R3 ¼ H and in glycosidic forms R3 ¼ 1-9*hexose unit. Numbering of hordatine structure is adopted from Kageyama et al., 2011.

peak was [MþH]þ as shown as an example with hordatine C in Fig. 3a. The double charged ions were absent from the MSE spectra of hordatine aglycons (Fig. 3d). The MSE spectra of hordatine A, B and C aglycons contained ions 114.103 (C5H12N3), 131.130 (C5H15N4) and 157.109 (C6H13N4O) m/z derived from the agmatine residues (Fig. 3 and Supplemental material Figures S10 and S12). The same fragments are well seen in the spectra of glycosides (Supplemental material Figure S11). Hydroxyagmatine residues in the structures of hordatines A1-C1 and A2-C2, were verified by the presence of ions 129.114 (C5H15N4), 147.125 (C5H15N4O) and 173.104 (C6H13N4O2) m/z in the MSE spectra (Supplemental material Figures S12 and S14). These fragments were especially visible in the spectra of glycosides (Fig. 3 Supplemental material Figures S13 and S15). Glycosides with one hexose unit were present of almost all of the hordatines in barley and beer samples (Table 2). Their MS and MSE spectra contained a double charged base peak [Mþ2H]2þ and a double charged [Mþ2H-hex]2þ ion originating from the removal of hexose unit (Fig. 3b and e). One minor peak was seen on the [MþH]þ ion chromatograms of hordatine B, B1 and C1 hexosides (marked as italic in Table 2). Dihexosides of hordatines were also found, except for methylated hordatine A (Table 2). Although the intensity of the [MþH]þ ion was relatively poor in the mass spectra of hordatine dihexosides, the presence of higher intensity [Mþ2H]2þ ion allowed their confident identification (Fig. 3c and f). To check whether hordatines with higher degree of glycosylation were present, the MS data was filtered using the [Mþ2H]2þ m/ zevalues of hordatine with 3e10 hexose units. In this way, a number of trihexosides were found first. Surprisingly, even higher

3.3. Other phenolamides: hydroxycinnamoylputrescine and -spermidines In addition to agmatine conjugates, a number of possible hydroxycinnamic acid-polyamide conjugates containing putrescine, spermidine or spermine, were searched from the mass spectral data. Out of these, only mono- and dihydroxycinnamic acid conjugates of spermidine were found in barley and/or beer samples. Feruloylputrescine was found only in beer samples (Table 3). Most of the phenolamides listed in Table 3 have not been reported from beer before. Savolainen et al. (2014) reported dicoumaroylspermidine, dicaffeoylspermidine, coumaroylcaffeoylspermi dine, coumaroylferuloylspermidine and caffeoylferuloylspermidine in fermented wheat bran, and Moheb et al. (2011) in wheat leaf, and these were also found in this study from barley and beer samples. Coumaroylcaffeoyl- and caffeoylferuloylspermidine were present as two isomers as in tobacco plants reported by Onkokesung et al. (2012). The isomeric forms had similar fragmentation ions produced in the MSE mode, but there were differences in the intensities of the fragments (Supplementary material Figure S21 and S22). The mass spectrum of coumaroylferuloylspermidine by MSE (Supplementary material Figure S23) was in good agreement to the MS/MS spectrum presented by Savolainen et al. (2014), although with different relative intensities of the fragments. 4. Conclusions This work shows that the number of various forms of hordatines, as well as their hydroxycinnamic acid agmatine precursors, in barley is much larger than previously thought. The presence of these compounds as such, as well as their glycosylated forms in the dormant barley seeds, indicates their role as a preformed reservoir of defense compounds for the early stages of germination and development of a new plant.

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Fig. 3. Mass spectrum in MS mode of hordatine C aglycon (a.), hexoside (b.) and dihexoside (c.). Mass spectra d., e. and f. present the MSE mass spectrum of the same compounds respectively. Thick black arrows in MSE spectrum of hordatine C dihexoside (f.) indicate the fragments typical to agmatine residue.

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Table 3 Other phenolamides found in barley and beer samples. Compound

Abbreviation

EC þ Hþ

calc. [MþH]þ m/z

Found [MþH]þ m/z

Error (ppm)

Rt (min)

Sample

Putrescines Feruloyl putrescine Spermidines Caffeoyl spermidine Coumaroyl spermidine Feruloyl spermidine Di-caffeoyl spermidine Di-coumaroyl spermidine Di-feruloyl spermidine Caffeoylferuloyl spermidine

FerPtr

C14H21N2O3

265.1552

265.1551

0.4

4.15

Beer

CafSpd CouSpd FerSpd CafSpdCaf CouSpdCou FerSpdFer CafSpdFer

C16H26N3O3 C16H26N3O2 C17H28N3O3 C25H31N3O6 C25H31N3O4 C27H35N3O6 C26H33N3O6

308.1974 292.2027 322.2132 470.2291 438.2393 498.2604 484.2448

Coumaroylcaffeoyl spermidine

CouSpdCaf

C25H31N3O5

454.2342

Coumaroylferuloyl spermidine

CouSpdFer

C26H33N3O5

468.2498

308.1967 292,2027/tr 322,2134/tr 470.2303 438.2383 498.2619 484.2463 484.2462 454.2367 454.2360 468.2516

2.3 0.0 0.6 2.6 2.3 3.0 3.1 2.9 5.5 4.0 3.8

1.92 2.43 3.01 6.45 7.87 8.45 7.40 7.50 7.10 7.20 8.16

Brl Brl/Beer Brl/Beer Brl, Beer Brl, Beer Brl, Beer Brl, Beer Brl, Beer Brl, Beer Brl, Beer Brl, Beer

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