Activity-Guided Identification of in Vitro Antioxidants in Beer

Article abstract of DOI:10.1021/acs.jafc.7b05674

In order to locate the key antioxidants contributing to oxidative stability of beer, activity-guided fractionation in combination with the oxygen radical absorbance capacity (ORAC) assay, hydrogen peroxide scavenging (HPS) assay, and linoleic acid (LA) assay was applied to a pilsner-type beer. LC-MS and 1D/2D NMR experiments led to the identification of a total of 31 antioxidants, among which 3-methoxy-4-hydroxyphenyl-β-d-glucopyranoside (tachioside), 4-(2-formylpyrrol-1-yl)butyric acid, 4-[2-formyl-5-(hydroxymethyl)pyrrol-1-yl]butyric acid, n-multifidol-3-O-β-d-glucoside, quercetin-3-O-(6″-malonyl)-glucoside, 4-feruloylquinic acid, syringaresinol, saponarin, and hordatines A-C have been isolated from beer for the first time. On a molar comparison, the hordatines A-C, saponarin, and quercetin-3-O-β-d-(6″-malonyl)glucoside were evaluated with the highest antioxidant activities of all identified beer constituents, reaching values of 10-17.5 (ORAC), 2.0-4.1 (HPS), and 1.1-6.1 μmol TE/μmol (LA) for hordatines A-C.

Full text of DOI:10.1021/acs.jafc.7b05674

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Article
Activity-Guided Identification of In Vitro-Antioxidants in Beer
Stefan Spreng, and Thomas Hofmann
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05674 • Publication Date (Web): 29 Dec 2017
Downloaded from http://pubs.acs.org on December 30, 2017
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ActivityꢀGuided Identification of In VitroꢀAntioxidants
in Beer
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Stefan Spreng1, and Thomas Hofmann1,2*
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1Chair of Food Chemistry and Molecular and Sensory Science, Technische
Universität München, LiseꢀMeitnerꢀStr. 34, Dꢀ85354 Freising, Germany, and
2Bavarian Center for Biomolecular Mass Spectrometry, GregorꢀMendelꢀStraße 4,
Dꢀ85354 Freising, Germany.
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*
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To whom correspondence should be addressed
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PHONE
FAX
+49ꢀ8161/71ꢀ2902
+49ꢀ8161/71ꢀ2949
EꢀMAIL
thomas.hofmann@tum.de
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ABSTRACT
In order to locate the key antioxidants contributing to oxidative stability of beer,
activityꢀguided fractionation in combination with the oxygen radical absorbance
capacity (ORAC) assay, hydrogen peroxide scavenging (HPS) assay and linoleic
acid (LA) assay was applied to a pilsnerꢀtype beer. LCꢀMS and 1D/2D NMR
experiments led to the identification of a total of 31 antioxidants, amongst which 3ꢀ
methoxyꢀ4ꢀhydroxyphenylꢀβꢀDꢀglucopyranoside (tachioside), 4ꢀ(2ꢀformylpyrrolꢀ1ꢀ
yl)butyric acid, 4ꢀ[2ꢀformylꢀ5ꢀ(hydroxymethyl)pyrrolꢀ1ꢀyl]butyric acid, nꢀmultifidolꢀ3ꢀ
OꢀβꢀDꢀglucoside, quercetinꢀ3ꢀOꢀ(6‘‘ꢀmalonyl)ꢀglucoside, 4ꢀferuloylquinic acid,
syringaresinol, saponarin, and hordatines AꢀC have been isolated from beer for the
first time. On a molar comparison, the hordatines AꢀC, saponarin, and quercetinꢀ3ꢀ
OꢀβꢀDꢀ(6’’ꢀmalonyl)glucoside were evaluated with the highest antioxidant activities
of all identified beer constituents, reaching values 10 – 17.5 (ORAC), 2.0 –
4.1 (HPS), and 1.1 – 6.1 ꢁmol TE/ꢁmol (LA) for hordatines AꢀC.
Keywords:
Antioxidants, beer, hordatines, tachioside
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INTRODUCTION
Freshly brewed beer has been attracting consumers for thousands of years due to
its refreshing character, desirable aroma, and its typical bitter taste profile. Although
microbiological spoilage and haze formation have been limiting factors determining
the shelf life of beer, technological improvements in beer manufacturing and a
better understanding of the chemical transformations occurring during the brewing
process helped to keep these problems largely under control. However, the
instability of the attractive aroma as well as the typical bitter taste of beer became
the main unresolved limiting factor of beer quality.1,2
Strecker aldehydes like phenylacetaldehyde and 2ꢀmethylpropanal were
identified as important staling compounds formed upon beer ageing,3ꢀ6 while
undesirable changes in taste are particularly caused by the degradation of the isoꢀαꢀ
acids, the main contributors to beer’s bitterness which derived from hops.7,8 Two
types of degradation mechanisms have been assigned for the storageꢀinduced isoꢀ
αꢀacids depletion, a transꢀspecific, oxygenꢀindependent and protonꢀcatalyzed
cyclization reaction converting transꢀisoꢀαꢀacids into the less bitter, but harsh and
lingering bitter tricyclohumols and tricyclohumenes9,10 and, by an autoxidative
mechanism, leading to cisꢀ and transꢀconfigured hydroperoxyꢀ and hydroxylꢀalloꢀisoꢀ
αꢀacids.11 While the protonꢀcatalyzed cyclization of isoꢀαꢀacids can be controlled by
keeping the beer’s pH value high and storage temperatures low,10 antioxidants are
believed to counteract the autoxidative mechanisms and to increase the shelf life of
beer.12,13 Therefore, a series of studies has been performed to evaluate the
antioxidant activity of beer components.1 Most previous studies focused on the role
of polyphenols as beer antioxidants by acting as metal ion chelators,14 inhibiting
fatty acid oxidation15 and avoiding formation of unwanted staling aldehyde
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precursors.16,17 Among the polyphenols, hydroxybenzoic acids (1−3, Figure 1) and
hydroxycinnamic acid derivatives (4−7) were considered as antioxidants.18ꢀ24
Originating from both hops and malt,25 they occur in both free and bound form.21
Ferulic acid (6), the most predominant hydroxycinnamic acid in beer,18,19,21 has
been shown to slow down the degradation of isoꢀαꢀacids.26 In addition, Maillard
reaction products, primarily formed during the kilning process,27 were proposed as
antioxidants in beer.21,28ꢀ31 Moreover, sulphur dioxide was found to act as an
antioxidant15 and scavenger for staling aldehydes in beer by forming
monothioacetals.32,33 Whereas all the previous studies focused on the antioxidant
activity of literatureꢀknown compound classes in beer, any activityꢀguided strategy
to locate and identify the key antioxidants in beer is still lacking.
It was therefore the objective of the present study to locate the key
antioxidants in pilsnerꢀtype beer by means of an activityꢀguided fractionation
approach using three in vitro antioxidant assays, namely the oxygen radical
absorbance capacity (ORAC) assay, the hydrogen peroxide scavenging (HPS)
assay, and the linoleic acid (LA) assay. The target antioxidants should then be
isolated, purified and determined in their chemical structure as well as their
antioxidative activity.
MATERIALS AND METHODS
Chemicals. The following compounds were obtained commercially: 2,2′ꢀazoꢀ
bis(2ꢀmethylpropinamidine) (AAPH), fluorescein sodium salt, (±)ꢀ6ꢀhydroxyꢀ2,5,7,8ꢀ
tetramethylchromaneꢀ2ꢀcarboxylic
acid
(Trolox),
2,2′ꢀazinobis(3ꢀ
ethylbenzothiazolineꢀ6ꢀsulfonic acid) diammonium salt (ABTS), acetic acid,
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disodium hydrogen phosphate, ethylenediaminetetraacetic acid hemoglobine,
hydrogen peroxide, iron(II)sulfate heptahydrate, peroxidase from horseradish,
linoleic acid, 2ꢀmethoxyhydroquinone, phenylalanine, pꢀhydroxyphenyllactic acid,
sodium tetraborate, syringic acid, trifluoroacetic acid (99%), triton Xꢀ100,
tryptophan, tween 20, tyrosol (SigmaꢀAldrich, Steinheim, Germany); caffeic acid,
formic acid (98−100%), hydrochloric acid (32%), pꢀhydroxybenzoic acid, potassium
dihydrogen phosphate, potassium hydroxide, sodium hydroxide (Merck, Darmstadt,
Germany); ferulic acid, fluorescein, 2ꢀisopropylmalic acid, pꢀcoumaric acid, sinapic
acid, vanillic acid (Fluka, NeuꢀUlm, Germany); D2O, methanoleꢀd4 (EurisoꢀTop,
Saarbrücken, Germany); sodium hydroxide (RiedelꢀdeꢀHaen, Seelze, Germany);
cyclo (ProꢀTyr) (Bachem, Weil am Rhein, Germany); benzoylleucomethylene blue
(TCI Europe, Zwijndrecht, Belgium). Water for highꢀperformance liquid
chromatography (HPLC) separation was purified by means of a MilliꢀQ water
advantage A 10 water system (Millipore, Molsheim, France). Solvents were of
HPLC grade (J.T. Baker, Deventer, Netherlands) and ethyl acetate was purified by
distillation in vacuum at 40°C. Pilsnerꢀtype beer was purchased from a German
brewery. A flavor hop polyphenol extract was obtained from Simon H. Steiner
GmbH (Mainburg, Germany).
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Solvent Extraction of Beer. Beer (500 mL) was adjusted to pH 2.5 with
traces of formic acid and then extracted with ethyl acetate (3 × 500 mL). The
combined organic extracts (I) and the aqueous layer (II) were separated from
organic solvents in vacuum at 40°C and freezeꢀdried for 48 h. The dried aqueous
phase (38 g) was extracted with acetonitrile/water (70/30, v/v; 3 × 100 mL) for
10 min during ultrasonification. After centrifugation (4000 rpm, 5 min), the combined
supernatants were separated from organic solvents in vacuum at 40°C and
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lyophilised for 48 h to obtain the acetonitrile/water soluble fraction IIA (yield: 40%),
which was kept at −20°C until further used.
Separation of Fractions I and IIA by Medium Pressure Liquid
Chromatography (MPLC). Fraction I (3.9 g) and fraction IIA (16 g), respectively,
was dissolved in 20 mL or 80 mL methanol/water (30/70, v/v), and 20 mL were
injected through a 6ꢀwayꢀinjection valve on a Sepacore system (Büchi, Flawil,
Switzerland) consisting of two Cꢀ605 pumps, a Cꢀ620 control unit, a Cꢀ660 fraction
collector, and a Cꢀ635 UV detector. The separation was performed on a 460 ×
16 mm glass column (Büchi, Flawil, Switzerland) filled with 25−40 ꢁm LiChroprep
RP18 material (Merck KGaA, Darmstadt, Germany). Operating with a flow rate of
30 mL/min, the solvent system consisted of aqueous formic acid (0.1 %, A) and
methanolic formic acid (0.1 %, B) and a gradient was used as follows:
0 min / 5 % B,
20 min / 25 % B,
23 min / 60 % B,
33 min / 68 % B,
40 min / 100 % B, and 55 min / 100 % B. Prior to the next injection, the column was
flushed to 5 % B for 3 min, and kept for 10 min. The effluent was monitored at
λ=254 nm, data recorded by using Sepacore Control Chromatography Software,
version 1.0 (Büchi, Flawil, Switzerland), and a total of 9 MPLC fractions collected,
namely Iꢀ1 to Iꢀ9 (Figure 2). Separation of fraction IIA was performed using the
same conditions, but using another solvent gradient: 0 min / 5 % B,
20 min / 25 % B, 23 min / 60 % B, 33 min / 68 % B, 40 min / 100 % B, and
55 min / 100 % B. The column was flushed to 5 % B for 3 min, and kept for 10 min
prior to next injection. Monitoring the effluent at 254 nm, another 9 MPLC fractions,
namely IIAꢀ1 up to IIAꢀ9, were collected. The subfractions obtained were separated
from solvent in vacuum at 40°C and freezeꢀdried for 48 h, before storing at −20°C
until further used.
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Identification of Key Antioxidants in MPLC Fraction Iꢀ7. Fraction Iꢀ7
(84.5 mg) was dissolved in acetonitrile/water (20/80, v/v; 5 mL) and, after
membrane filtration, 1 mL was injected onto a 250 × 21.2 mm i.d., 5 ꢂm,
HyperClone C18 column (Phenomenex, Aschaffenburg, Germany) using a 2 mL
sample loop. Using a binary gradient of 0.1 % aqueous formic acid as solvent A and
acetonitrile as solvent B, chromatography (flow rate: 21 mL/min) was performed
with the following gradient: 0 min / 10 % B, 5 min / 15 % B, 20 min / 22 % B,
23 min / 100 % B, 27 min / 100 % B, 30 min / 10 % B, and 35 min / 10 % B. The
effluent, monitored λ=228 and 288 nm, was separated into 10 subfractions, namely
Iꢀ7ꢀ1 up to Iꢀ7ꢀ10, which were collected individually in several runs. The
corresponding fractions were combined and separated from solvent in vacuum at
40°C, followed by lyophilization. The subfractions were further purified by reꢀ
chromatography by means of semipreparative RPꢀHPLC using a 250 × 10.0 mm
i.d., 5 ꢂm, Luna PFP column (Phenomenex, Aschaffenburg, Germany), operated
with a flow rate of 5.5 mL/min and using 0.1 % aqueous formic acid in water as
solvent A and methanol as solvent B. For separation of fraction Iꢀ7ꢀ9, the following
solvent gradient was used: 0 min / 35 % B, 10 min / 48 % B, 20 min / 48 % B,
28 min / 55 % B,
33 min / 55 % B,
43 min / 80 % B,
44 min / 100 % B,
47 min / 100 % B, 48,5 min / 35 % B, and 55 min / 35 % B. The effluent, monitored
at λ=280 nm, was collected into 27 subfractions, namely Iꢀ7ꢀ9ꢀ1 up to Iꢀ7ꢀ9ꢀ27. After
reꢀchromatography and verifying the purity of each fraction by means of analytical
HPLC, the target compounds were separated from solvent in vacuum, freezeꢀdried
for 48 h and, then, analyzed by LCꢀTOFꢀMS and 1D/2DꢀNMR spectroscopy for
1
structure determination. Spectroscopic data (UV/Vis, LCꢀTOFꢀMS, HꢀNMR) and
chromatographic data (retention time), followed by coꢀchromatography with
commercially available reference compounds led to the identification of pꢀ
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hydroxybenzoic acid (1), vanillic acid (2), syringic acid (3), pꢀcoumaric acid (4),
caffeic acid (5), ferulic acid (6), sinapic acid (7), pꢀhydroxyphenyllactic acid (8), 2ꢀ
isopropylmalic acid (9), cyclo(ProꢀTyr) (10), tyrosol (11), and cinnamic acid (15,
Table 1). Coꢀ, nꢀ and adꢀmultifidolglucoside (18a−c),34 quercetinꢀ3ꢀOꢀβꢀDꢀglucoside
(19),35
kaempferolꢀ3ꢀOꢀβꢀDꢀglucoside
(20),35
quercetinꢀ3ꢀOꢀβꢀDꢀ(6ꢀOꢀ
malonyl)glucoside (21),36 and kaempferolꢀ3ꢀOꢀβꢀDꢀ(6ꢀOꢀmalonyl)glucoside (22),35
were assigned by comparison of spectroscopic data with literature and by coꢀ
chromatography with the corresponding reference material isolated from a hop
polyphenol extract. In addition, comparison of spectroscopic data with literature led
to the identification of 3ꢀ, 4ꢀ and 5ꢀferuloylquinic acid (12−14),37 Nꢀferuloyltyramine
(16),38 and syringaresinol (17).39
Identification of Key Antioxidants in MPLC Fractions IIAꢀ5, IIAꢀ7, IIAꢀ8
and IIAꢀ9. Identification of Antioxidants in Fraction IIAꢀ5. Fraction IIAꢀ5 was
separated on a 250 × 21.2 mm i.d., 5 ꢂm, Luna PhenylꢀHexyl column
(Phenomenex, Aschaffenburg, Germany) with a flow rate of 21 mL/min. Using a
binary gradient of 1 % formic acid in water (solvent A) and 1 % formic acid in
acetonitrile (solvent B), chromatography was performed with the following gradient
after dissolving fraction IIAꢀ5 (9.4 mg) in 800 ꢂL starting condition solvents:
0 min / 10 % B,
5 min / 15 % B,
20 min / 22 % B,
23 min / 100 % B,
27 min / 100 % B, 30 min / 10 % B, 35 min / 10 % B. Monitoring the effluent at
280 nm, 10 subfractions (IIAꢀ5ꢀ1 to IIAꢀ5ꢀ10) were collected and their antioxidant
activity evaluated by means of the ORACꢀ, HPSꢀ and LAꢀassay, respectively. The
highly active subfraction IIAꢀ5ꢀ9 was purified by reꢀchromatography, separated from
solvent in vacuum, lyophilised for 48 h and, then, used for LCꢀTOFꢀMS, LCꢀMS/MS
as well as 1D/2DꢀNMR spectroscopy to identify phenylalanine (23) and tachioside
(24) by comparison with the corresponding reference compound.
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Identification of Antioxidants in Fraction IIAꢀ7. Fraction IIAꢀ7 (4.2 mg) was
dissolved in acetonitrile/1 % formic acid (20/80, v/v; 500 ꢂL) and, after membrane
filtration, was injected onto a 250 × 21.2 mm i.d., 5 ꢂm, Luna PhenylꢀHexyl column
(Phenomenex, Aschaffenburg, Germany) with a flow rate of 21 mL/min. Using a
binary gradient of 1 % formic acid in water as solvent A and 1 % formic acid in
acetonitrile as solvent B, the following method was used for separation:
0 min / 0 % B, 5 min / 0 % B, 10 min / 5 % B, 25 min / 5 % B, 27 min / 100 % B,
29 min / 100 % B, 32 min / 0 % B, and 35 min / 0 % B. The effluent, monitored at
λ=280 nm and by means of an ELSD, was separated into 11 subfractions, namely
IIAꢀ7ꢀ1 to IIAꢀ7ꢀ11, and the solvent was removed in vacuum at 40°C, followed by
lyophilisation and analysis by means of the ORACꢀ, HPSꢀ and LAꢀassay,
respectively. Analysis of the most active subfraction IIAꢀ7ꢀ9 by by LCꢀTOFꢀMS, LCꢀ
MS/MS and 1D/2DꢀNMR led to the identification of tryptophan (25).
Identification of Antioxidants in Fraction IIAꢀ8. For further subfractionation,
fraction IIAꢀ8 (6.3 mg) was separated on a 250 × 21.2 mm i.d., 5 ꢂm, Luna Phenylꢀ
Hexyl column (Phenomenex, Aschaffenburg, Germany). Operating with a flow rate
of 21 mL/min, the solvent system consisted of aqueous formic acid (0.1 %, A) and
acetonitrile (B) and a gradient was used as follows: 0 min / 5 % B, 25 min / 25 % B,
32 min / 30 % B,
34 min / 100 % B,
37 min / 100 % B,
40 min / 5 % B,
45 min / 5 % B. Monitoring the effluent at 280 nm, 15 subfractions were collected,
namely IIAꢀ8ꢀ1 to IIAꢀ8ꢀ15. Hordatines (29−31) were purified by an alternative
method using a 250 × 10.0 mm i.d., 5 ꢂm, Luna PFP(2) column (Phenomenex,
Aschaffenburg, Germany) with 0.1 % trifluoroacetic acid in water as solvent A and
0.1 % trifluoroacetic acid in methanol as solvent B. The following gradient was used
for separation at a flow rate of 5.5 mL/min: 0 min / 15 % B, 10 min / 30 % B,
30 min / 48 % B,
32 min / 100 % B,
34 min / 100 % B,
36 min / 15 % B,
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40 min / 15 % B. After separation from solvent in vacuum, the subfractions and
purified compounds were freezeꢀdried and then analyzed by LCꢀTOFꢀMS, LCꢀ
MS/MS, 1D/2DꢀNMR spectroscopy as well as ORACꢀ, HPSꢀ and LAꢀassay,
respectively. In accordance with the spectroscopic data (UV/Vis, LCꢀTOFꢀMS,
1H/13CꢀNMR), the structures of the previously reported 4ꢀ[2ꢀformylꢀ5ꢀ
(hydroxymethyl)pyrrolꢀ1ꢀyl]butyric acid (28)40 and hordatine A (29)41 was confirmed.
The chemical structures of hordatine B (30) and hordatine C (31) have been
determined by means of UV/vis, LCꢀTOFꢀMS, and 1D/2DꢀNMR experiments and, to
the best of our knowledge, no detailed NMRꢀdata have been earlier reported.
Hordatine B, 30, Figure 1. UV/vis (0.1% aqueous formic acid/acetonitrile):
λmax = 228 / 300 / 320 nm; LCꢀTOFꢀMS (ESI)−: m/z (%) 625.3089 (20; measured),
625.3098 (calculated for [C29H40N8O5+HCOOH−H]−) 579.3038 (100; measured),
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579.3043; LCꢀTOFꢀMS (calculated for [C29H40N8O5−H]−); (ESI)+: m/z (%) 581.3198
(5; measured), 581.3200 (calculated for [C29H40N8O5+H]+), 291.1641 (100;
1
measured), 291.1640 (calculated for [C29H40N8O5+2H]2+); H NMR (500 MHz, D2O,
COSY): δ (ppm) 1.29–1.60 [m, 8H, H2ꢀC(2‘‘), H2ꢀC(2‘‘‘), H2ꢀC(3‘‘), H2ꢀC(3‘‘‘)], 2.97–
3.32 [m, 8H, H2ꢀC(1‘‘), H2ꢀC(1‘‘‘), H2ꢀC(4‘‘), H2ꢀC(4‘‘‘)], 3.89 [s, 3H, H3ꢀC(10)], 4.32
[d, 1H, J = 7.4 Hz, HꢀC(8‘)], 5.91 [d, 1H, J = 7.4 Hz, HꢀC(7‘)], 6.06 [d, 1H, J =
12.2 Hz, HꢀC(8)], 6.86 [d, 1H, J = 12.2 Hz, HꢀC(7)], 6.88 [s, 1H, HꢀC(2)], 6.94 [d, 2H,
J = 8.4 Hz, HꢀC(3‘/5‘)], 7.07 [s, 1H, HꢀC(6)], 7.32 [d, 2H, J = 8.4 Hz, HꢀC(2‘/6‘)]; 13C
NMR (125 MHz, D2O, HSQC, HMBC): δ (ppm) 26.1, 26.2, 26.3, 26.6 [C(2‘‘), C(2‘‘‘),
C(3‘‘), C(3‘‘‘)], 39.7 [C(1‘‘‘)], 40.0 [C(1‘‘)], 41.7 [C(4‘‘), C(4‘‘‘)], 57.1 [C(10)], 58.1
[C(8‘)], 89.5 [C(7‘)], 114.5 [C(6)], 116.8 [C(3‘/5‘)], 118.1 [C(2)], 124.1 [C(8)], 127.7
[C(3)], 128.9 [C(2‘/6‘)], 131.1 [C(1)], 131.6 [C(1‘)], 137.2 [C(7)], 144.7 [C(5)], 148.4
[C(4)], 157.3 [C(4‘)], 157.7 [C(5‘‘), C(5‘‘‘)], 171.6 [C(9)], 173.9 [C(9‘)].
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Hordatine C, 31, Figure 1. UV/vis (0.1% aqueous formic acid/acetonitrile):
λmax = 228 / 300 / 320 nm; LCꢀTOFꢀMS (ESI)−: m/z (%) 655.3221 (20; measured),
655.3204 (calculated for [C30H42N8O6+HCOOH−H]−), 609.3147 (100; measured),
609.3149 (calculated for [C30H42N8O6−H]−); LCꢀTOFꢀMS (ESI)+: m/z (%) 611.3289
(5; measured), 611.3306 (calculated for [C30H42N8O6+H]+), 306.1691 (100;
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measured), 306.1691 (calculated for [C30H42N8O6+2H]2+); H NMR (500 MHz, D2O,
COSY): δ (ppm) 1.31–1.62 [m, 8H, H2ꢀC(2‘‘), H2ꢀC(2‘‘‘), H2ꢀC(3‘‘), H2ꢀC(3‘‘‘)], 2.98–
3.32 [m, 8H, H2ꢀC(1‘‘), H2ꢀC(1‘‘‘), H2ꢀC(4‘‘), H2ꢀC(4‘‘‘)], 3.87 [s, 3H, H3ꢀC(10‘)], 3.92
[s, 3H, H3ꢀC(10)], 4.35 [d, 1H, J = 7.4 Hz, HꢀC(8‘)], 5.93 [d, 1H, J = 7.4 Hz, HꢀC(7‘)],
6.07 [d, 1H, J = 12.2 Hz, HꢀC(8)], 6.88 [d, 1H, J = 12.2 Hz, HꢀC(7)], 6.90 [s, 1H, Hꢀ
C(2)], 6.96 [s, 1H, HꢀC(2‘)], 6.97 [d, 1H, J = 7.4 Hz, HꢀC(5‘)], 7.08 [d, 1H, J = 7.4 Hz,
HꢀC(6‘)], 7.10 [s, 1H, HꢀC(6)]; 13C NMR (125 MHz, D2O, HSQC, HMBC): δ (ppm)
26.2, 26.3, 26.6 [C(2‘‘), C(2‘‘‘), C(3‘‘), C(3‘‘‘)], 39.8 [C(1‘‘‘)], 40.0 [C(1‘‘)], 41.7 [C(4‘‘),
C(4‘‘‘)], 57.1 [C(10‘)], 57.1 [C(10)], 58.2 [C(8‘)], 89.4 [C(7‘)], 111.2 [C(6‘)], 114.6
[C(6)], 116.7 [C(5‘)], 118.0 [C(2)], 120.2 [C(2‘)], 124.0 [C(8)], 127.8 [C(3)], 131.3
[C(1)], 132.3 [C(1‘)], 137.1 [C(7)], 144.6 [C(5)], 146.7 [C(4‘)], 148.5 [C(4)], 148.7
[C(3‘)], 157.6 [C(5‘‘), C(5‘‘‘)], 171.6 [C(9)], 173.9 [C(9‘)].
Identification of Antioxidants in Fraction IIAꢀ9. Fraction IIAꢀ9 (11.3 mg) was
dissolved in acetonitrile/1% formic acid (40/60, v/v; 1 mL) and, after membrane
filtration, injected onto a 250 × 21.2 mm i.d., 4 ꢂm, Synergi HydroꢀRP column
(Phenomenex, Aschaffenburg, Germany). Chromatography (flow rate: 21 mL/min)
was performed using a binary gradient of 1% formic acid in water (A) and 1%
formic acid in acetonitrile (B), the following chromatographic method was used:
0 min / 5 % B,
12 min / 12 % B,
32 min / 32 % B,
40 min / 50 % B,
42 min / 100 % B, 44.5 min / 100 % B, 47 min / 5 % B, and 50 min / 5 % B.
Monitoring the effluent at λ=280 nm, 20 subfractions were collected, namely IIAꢀ9ꢀ1
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to IIAꢀ9ꢀ20, solvent separated in vacuum, lyophilised for 48 h, and their antioxidant
activity analysed by means of the ORACꢀ, HPSꢀ and LAꢀassay, respectively. After
reꢀchromatography, comparison of spectroscopic data (UV/Vis, LCꢀTOFꢀMS,
1H/13CꢀNMR) with literature data revealed saponarin (26)42 and 4ꢀ(2ꢀformylpyrrolꢀ1ꢀ
yl)butyric acid (27)40 as the key antioxidants.
Isolation of Antioxidants from Hop Polyphenol Extract. A commercial
flavor hop polyphenol extract (20 g) was suspended with water (200 mL), the pH
adjusted to 2.5 by adding traces of formic acid and, then, extracted with ethyl
acetate (3 × 200 mL). The combined organic extracts were separated from organic
solvents in vacuum at 40°C, dissolved in methanol/water (70/30, v/v) and, after
membrane filtration, injected onto a 250 × 21.2 mm i.d., 5 ꢂm, Luna PhenylꢀHexyl
column (Phenomenex, Aschaffenburg, Germany) using a 2 mL sample loop.
Chromatography (flow rate: 21 mL/min) was performed using 0.1% aqueous formic
acid as solvent A and acetonitrile as solvent B, and the following gradient:
0 min / 20 % B,
20 min / 20 % B,
24 min / 25 % B,
26 min / 25 % B,
29 min / 100 % B, 32 min / 100 % B, 35 min / 20 % B and 40 min / 20 % B.
Monitoring the effluent at λ=264 nm, a total of 7 subfractions was collected, namely
IIIꢀ1 to IIIꢀ7, separated from solvent in vacuum at 40°C, followed by lyophilization.
Comparison of spectroscopic data (UV/vis, LCꢀTOFꢀMS, 1H/13CꢀNMR) with literature
confirmed the structure of coꢀ, nꢀ and adꢀmultifidolglucoside (18a−c),34 quercetinꢀ3ꢀ
OꢀβꢀDꢀglucoside (19),35 kaempferolꢀ3ꢀOꢀβꢀDꢀglucoside (20),35 quercetinꢀ3ꢀOꢀβꢀDꢀ(6’’ꢀ
Oꢀmalonylglucoside (21),36 and kaempferolꢀ3ꢀOꢀβꢀDꢀ(6’’ꢀOꢀmalonylglucoside (22)35
as key antioxidants.
Chemical Synthesis of Tachioside (24) and Isotachioside (24a).
Following a literature protocol with some modifications,43 2ꢀmethoxyhydroquinone
(4 mmol) was dissolved in acetone (10 mL), acetobromoꢀαꢀDꢀglucose (3 mmol) and
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sodium hydroxide (2 mmol/L in water; 1.5 mL) were added, followed by stirring for
24 h at room temperature in the dark. After filtration, the solvent was removed in
vacuum, sodium hydroxide (1 mmol/L in methanol/water, 50/50, v/v; 10 mL) was
added and, after incubation for 5 min to achieve deꢀacetylation, the pH value was
adjusted to 5.5 with hydrochloric acid, followed by purification by means of MPLC
using a 150 × 40 mm cartridge (Büchi, Flawil, Switzerland) filled with 25−40 ꢁm
LiChroprep RP18 material (Merck KGaA, Darmstadt, Germany). Chromatography
(flow rate: 40 mL/min) was performed using a binary gradient of 0.1% aqueous
formic acid (solvent A) and methanol (solvent B): 0 min / 0% B, 5 min / 0% B,
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30 min / 30% B,
37 min / 100% B,
45 min / 100% B,
50 min / 0% B,
and
60 min / 0% B. After solvent separation, the target compounds were reꢀ
chromatographed by HPLC on a 250 × 21.2 mm i.d., 5 ꢂm, Luna PhenylꢀHexyl
column (Phenomenex, Aschaffenburg, Germany) operated at a flow rate of
21 mL/min. Monitoring the effluent at 280 nm, the target molecules were eluted with
3% acetonitrile in 0.1 % aqueous formic acid, separated from solvent in vacuum
and freezeꢀdried for 48 h to afford tachioside (24, 12 mg) und isotachioside (24a, 20
mg).
3ꢀMethoxyꢀ4ꢀhydroxyphenylꢀβꢀDꢀglucopyranoside (tachioside), 24, Figure 1.
UV/vis (0.1% aqueous formic acid/acetonitrile): λmax = 224 / 284 nm; LCꢀTOFꢀMS
(ESI)−: m/z (%) 301.0919 (100; measured), 301.0923 (calculated for [C13H18O8−H]−),
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139.0399 (40; measured), 139.0401 (calculated for aglycone [C7H8O3−H]−); LCꢀ
TOFꢀMS (ESI)+: m/z (%) 325.0894 (100; measured), 325.0894 (calculated for
1
[C13H18O8+Na]+); H NMR (500 MHz, D2O, COSY): δ (ppm) 3.42–3.63 [m, 4H, Hꢀ
C(4‘), HꢀC(3‘), HꢀC(2‘), HꢀC(5‘)], 3.73 [dd, 1H, J = 5.9, 12.5 Hz, HaꢀC(6‘)], 3.85 [s,
3H, H3ꢀC(1‘‘)], 3.92 [dd, H, J = 2.0, 12.5 Hz, HbꢀC(6‘)], 4.99 [d, H, J = 7.6 Hz, Hꢀ
C(1‘)], 6.66 [dd, 1H, J = 2.7, 8.7 Hz, HꢀC(6)], 6.84 [d, 1H, J = 2.7 Hz, HꢀC(2)], 6.87
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[d, 1H, J = 8.7 Hz, HꢀC(5)]; 13C NMR (125 MHz, D2O, HSQC, HMBC): δ (ppm) 56.9
[C(1‘‘)], 61.6 [C(6‘)], 70.1 [C(4‘)], 74.0 [C(2‘)], 76.6 [C(3‘)], 77.1 [C(5‘)], 102.3 [C(1‘)],
103.9 [C(2)], 109.8 [C(6)], 116.6 [C(5)], 141.6 [C(4)], 149.0 [C(3)], 151.8 [C(1)].
2ꢀMethoxyꢀ4ꢀhydroxyphenylꢀβꢀDꢀglucopyranoside
(Isotachioside),
24a,
Figure 1. UV/vis (0.1% aqueous formic acid/acetonitrile): λmax = 224 / 284 nm; LCꢀ
TOFꢀMS (ESI)−: m/z (%) 301.0922 (100; measured), 301.0923 (calculated for
[C13H18O8−H]−), 139.0406 (45; measured), 139.0401 (calculated for aglycone
[C7H8O3−H]−); LCꢀTOFꢀMS (ESI)+: m/z (%) 325.0891 (100; measured), 325.0894
1
(calculated for [C13H18O8+Na]+); H NMR (500 MHz, D2O, COSY): δ (ppm) 3.40–
3.57 [m, 4H, HꢀC(4‘), HꢀC(3‘), HꢀC(2‘), HꢀC(5‘)], 3.74 [dd, 1H, J = 4.6, 12.3 Hz, Haꢀ
C(6‘)], 3.84 [s, 3H, H3ꢀC(1‘‘)], 3.88 [dd, H, J = 1.6, 12.3 Hz, HbꢀC(6‘)], 4.85 [d, 1H,
J = 7.5 Hz, HꢀC(1‘)], 6.41 [dd, 1H, J = 2.6, 8.7 Hz, HꢀC(5)], 6.57 [d, 1H, J = 2.6 Hz,
HꢀC(3)], 7.05 [d, 1H, J = 8.7 Hz, HꢀC(6)]; 13C NMR (125 MHz, D2O, HSQC, HMBC):
δ (ppm) 58.6 [C(1‘‘)], 63.7 [C(6‘)], 72.5 [C(4‘)], 76.2 [C(2‘)], 78.8 [C(3‘)], 79.3 [C(5‘)],
103.7 [C(6)], 105.1 [C(1‘)], 109.6 [C(5)], 121.3 [C(3)], 142.5 [C(1)], 153.1 [C(2)],
155.5 [C(4)].
Oxygen Radical Absorbance Capacity (ORAC) Assay. The antioxidant
capability of extracts, fractions and purified compounds was measured by using the
ORACꢀAssay following a literature protocol.44 Sample solutions were prepared
using phosphate buffer (10 mmol/L, pH 7.4) and aliquots in four replicates (each 25
ꢁL) were pipetted into the wells of a black 96ꢀwellꢀplate (Greiner BioꢀOne,
Frickenhausen, Germany). After addition of fluorescein (10 nmol/L in phosphate
buffer, 150 ꢂL) to each well and incubation of the microplate (30 min, 37°C) the
decline of fluorescence was recorded every 90 s at an excitation wavelength of 485
nm and an emission wavelength of 520 nm using a plate reader (FLUOstar
OPTIMA, BMG Labtech, Offenburg, Germany). After three cycles, AAPH
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(240 mmol/L in phosphate buffer, 25 ꢁL) was added to each well before performing
another 57 cycles (90 min in total). For calibration, a serial dilution of trolox (200,
100, 50, 25, 12.5 ꢂmol/L) from a stock solution (4 mmol/L in ethanol) was treated in
the same way as well as a blank of phosphate buffer (25 ꢁL) and a C1ꢀwell (50 ꢁL
of phosphate buffer instead of sample and AAPH). Calculation was carried out as
described in literature45 with slight modifications. After normalising to C1ꢀwell (C1ꢀ
value = 1) and determination of the area under the fluorescence versus time curve
(AUC) the AUC of the blank was subtracted. With the obtained netAUC the
antioxidant capacity was expressed as trolox equivalents (TE) using the calibration
curve of trolox standards.
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Hydrogen Peroxide Scavenging (HPS) Assay. Following a literature
protocol with slight modifications,46 stock solutions of the samples in water were
diluted stepwise (1+1, v+v; 8 dilution steps) using a phosphate buffer (100 mmol/L,
pH 6.0) and afterwards, three replicates (each 20 ꢁL) of each dilution step were
pipetted into the wells of a clear 96ꢀwellꢀplate (Greiner BioꢀOne, Frickenhausen,
Germany). Phosphate buffer (100 mmol/L, pH 6.0, 100 ꢁL) was added to each well
as well as aqueous solutions of hydrogen peroxide (250 ꢁmol/L, 20 ꢁL), horseradish
peroxidase (150 U/ml, 40 ꢁL) and ABTS (0.1%, 40 ꢁL). After incubation at 37°C,
the absorption at λ=414 nm was recorded four times within 15 min using a
FLUOstar OPTIMA plate reader (BMG Labtech, Offenburg, Germany). Considering
the different dilutions of a sample, the EC50ꢀvalue of the gained absorption versus
concentration curve was calculated on the base of a logistic doseꢀresponseꢀmodel
with Origin 8G as software. The antioxidant activity was expressed as trolox
equivalents (TE) dividing the EC50ꢀvalue of trolox by the EC50ꢀvalue of the sample.
Linoleic Acid (LA) Assay. Following a literature protocol,47,48 the samples
were dissolved and diluted stepwise (1+1, v+v, 8 dilution steps) using an
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ethanol/water mixture (1+1, v+v). Aliquots of three replicates (each 25 ꢁL) were
pipetted into the wells of a clear 96ꢀwellꢀplate (Greiner BioꢀOne, Frickenhausen,
Germany) before addition of oxygenꢀsaturated phosphate buffer (200 mmol/L,
pH 6.75, 60 ꢁL), an aqueous hydrogen peroxide solution (16 mmol/L, 20 ꢁL) and an
aqueous iron(II)sulfate/EDTA solution (16 mmol/L/ 15 mmol/L, 20 ꢁL) to each well.
After mixing linoleic acid (25 ꢁL), tween 20 (25 ꢁL), sodium hydroxide (1 mol/L,
300 ꢁL) and water (5 mL) and filling up to 10 mL with borate buffer (50 mmol/L,
pH 9.0), this linoleic acid substrate (20 ꢁL) was added to each well and the
degradation of linoleic acid was performed for exactly 10 min at room temperature.
To stop this process, 80 ꢁL of a colorant solution, consisting of haemoglobin
(28 mg), benzoylleucomethylene blue (25.5 mg) and triton Xꢀ100 (7 mL), which
were dissolved in 250 mL phosphate buffer (400 mmol/L, pH 5.0), were added to
each well. After a further incubation for 30 min at room temperature, the absorption
at λ=666 nm was recorded using an infinite M200 spectrometer (Tecan, Männedorf,
Switzerland) with iꢀcontrol 1.10 as software. Out of the sigmoidal absorption versus
concentration curve of the serial dilution, the EC50ꢀvalue of each sample was
calculated on the base of a logistic doseꢀresponseꢀmodel with Origin 8G as
software. The antioxidant activity was expressed as trolox equivalents (TE) dividing
the EC50ꢀvalue of trolox by the EC50ꢀvalue of the sample.
High Performance Liquid Chromatography (HPLC). The HPLC system
(Jasco, GroßꢀUmstadt, Germany) consisted of two PUꢀ2087 Plus pumps, a DGꢀ
2080ꢀ53 degaser, respectively, and a MDꢀ2010 Plus diode array detector monitoring
the effluent in a range between 220 and 500 nm using Chrompass 1.8.6.1 (Jasco,
GroßꢀUmstadt, Germany) as software. For sample injection, an ASꢀ2055 Plus
autosampler was used in analytical mode and a 7725i type Rheodyne injection
valve (Rheodyne, Bensheim, Germany) in preparative and semipreparative mode.
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For detection with ELSD a Sedex LTꢀELSD Model 80 (Sedere, Alfortville,
Frankreich) was used and the split ratio was set to 1.0 mL/min for the ELSD.
UPLC/TimeꢀofꢀFlight Mass Spectrometry (UPLCꢀTOFꢀMS). Aliquots (2 ꢂL)
of all antioxidants were injected into an Acquity UPLC core system (Waters,
Manchester, UK), consisting of a binary solvent manager, a sample manager and a
column oven. The chromatographic separation was performed on a 150 × 2 mm
i.d., 1.7 ꢁm, BEH C18 column (Waters, Manchester, UK) at a flow rate of
0.3 mL/min and a temperature of 40°C. Aqueous formic acid (0.1%, A) and
acetonitrile (B) were used as solvents for the following gradient: 0 min / 5% B,
3 min / 100% B, and 4 min / 100% B. Highꢀresolution mass spectra were recorded
on a Synapt G2ꢀS HDMS (Waters, Manchester, UK) in negative and positive ESI
resolution mode using −3.0 and +2.5 kV capillary voltage, respectively, 30 kV
sampling cone, 4.0 kV extraction cone, 150°C source temperature, 450°C
desolvation temperature, 30 L/h cone gas and 850 L/h desolvation gas. The
instrument was calibrated (m/z 50−1200) using a solution of sodium formate
(0.5 mmol/L) dissolved in 2ꢀpropanol/water (9/1, v/v). All data were lock mass
corrected using leucine enkephaline as reference (m/z 554.2615, [M−H]− and m/z
556.2771, [M+H]+). Data acquisition and interpretation were performed using
MassLynx (version 4.1) and the tool “elemental composition” as software.
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Nuclear Magnetic Resonance Spectroscopy (NMR). 1Dꢀ and 2DꢀNMR
spectra were recorded on a 400 MHz ultrashield Avance III spectrometer with a
Broadband Observe BBFOplus probehead and a 500 MHz ultrashield plus Avance
III spectrometer with a Triple Resonance Cryo Probe TCI probehead (Bruker,
Rheinstetten, Germany), respectively. Using methanolꢀd4 and D2O as solvents, the
chemical shifts were quoted in parts per million relative to the solvent signal. The
pulse sequences for recording 2D NMR experiments (i.e. COSY, HSQC, and
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HMBC) were taken from the Bruker software library. Data processing was
performed by using XWinꢀNMR version 3.5 (Bruker, Rheinstetten, Germany) and
MestreꢀNova 8 (Mestrelab Research, Santiago de Compostela, Spain) as software.
RESULTS AND DISCUSSION
Using several tests as recommended for antioxidant screening,49,50 a Pilsnerꢀtype
beer was iteratively fractionated and each fraction analysed by three different in
vitroꢀassays, namely the oxygen radical absorbance capacity (ORAC) assay, the
hydrogen peroxide scavenging (HPS) assay, and the linoleic acid (LA) assay,
respectively.
Antioxidant AssayꢀGuided Fractionation of Beer. First, beer was
extracted with ethyl acetate to afford the ethyl acetate soluble fraction I and the
aqueous fraction II (Figure 2). Without considering the yields of these fractions,
fraction I showed with 210 (ORAC assay), 79 (HPS assay), and 38 ꢁmol TE/g (LA
assay) by far high antioxidant activity when compared to the aqueous fraction II with
97 (ORAC assay), 23 (HPS assay), and 16 ꢁmol TE/g (LA assay), thus indicating
hydrophobic antioxidants to be more active than the waterꢀsoluble compounds.
However, considering the high yield of 90% of fraction II when compared to fraction
I (10%), the aqueous fraction II seems to have a higher contribution to the
antioxidant activity of beer, e.g. fraction II showed antioxidant activity with
3600 (ORAC), 840 (HPS), and 580 ꢁmol TE/L beer (LA), whereas fraction I showed
lower activity with 800 (ORAC), 310 (HPS), and 150 ꢁmol TE/L beer (LA). As
consideration of the natural ratios of the fractions is crucial to evaluate the
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contribution to the antioxidant activity of the beer, the following activity
measurements were done by taking the different yields of fractions into account.
Identification of Key Antioxidants in Fraction I. To achieve the isolation of
individual antioxidants, fraction I was separated by means of RP18ꢀMPLC to yield 9
fractions, namely Iꢀ1 to Iꢀ9 (Figure 2), which were then subjected to antioxidant
activity measurement. Recombination of aliquots of these fractions revealed an
antioxidant activity of the mixture with values of 670 (ORAC), 270 (HPS), and 130
ꢁmol TE/L (LA) which is comparable to the activity found for the entire fraction I,
thus confirming the stability of the key antioxidants during separation. Among the
individual fractions, fraction Iꢀ7 was found to exhibit by far the highest activity with
530 (ORAC), 220 (HPS), and 58 ꢁmol TE/L (LA), respectively, and was therefore
further subꢀfractionated by means of preparative HPLC. A total of 10 subfractions (Iꢀ
7ꢀ1 to Iꢀ7ꢀ10) was collected and analysed using the three antioxidant assays.
Except for fraction Iꢀ7ꢀ10, all fractions showed some relevant antioxidant activity. In
particular, fraction Iꢀ7ꢀ2 with 93 (ORAC), 87 (HPS), and 7 ꢁmol TE/L (LA), and
fraction Iꢀ7ꢀ9 with 32 (ORAC), 36 (HPS), and 24 ꢁmol TE/L (LA) showed the highest
activities, followed by fractions Iꢀ7ꢀ1, Iꢀ7ꢀ4, and Iꢀ7ꢀ7, respectively.
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UV/Vis, LCꢀTOFꢀMS, and NMR analysis and comparison with data obtained
from the corresponding reference substances revealed tyrosol (11) in fraction Iꢀ7ꢀ2,
pꢀhydroxybenzoic acid (1) in fraction Iꢀ7ꢀ3, vanillic acid (2), caffeic acid (5) and
syringic acid (3) in fraction Iꢀ7ꢀ4, pꢀcoumaric acid (4) in fraction Iꢀ7ꢀ6, ferulic acid (6)
and sinapic acid (7) in fraction Iꢀ7ꢀ7, and cinnamic acid (15) in fraction Iꢀ7ꢀ8 as most
active antioxidants (Table 1), thus confirming literature data.18ꢀ23,51ꢀ53 In addition, pꢀ
hydroxyphenyllactic acid (8) and 2ꢀisopropylmalic acid (9), both wellꢀknown
metabolites of Saccharomyces cerevisiae,54,55 and the diketopiperazine cyclo(Proꢀ
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Tyr) (10) were identified in fraction Iꢀ7ꢀ1 and, to the best of our knowledge, have not
yet been reported in beer.
Next to 3ꢀferuloylquinic acid (14), known from barley and beer,56,57 also 4ꢀ
and 5ꢀferuloylquinic acid (13, 14) could be unequivocally identified in fraction Iꢀ7ꢀ5.
Nꢀferuloyltyramine (16), recently reported to be present in hops and beer,58 was
found in fraction Iꢀ7ꢀ9ꢀ15, while fraction Iꢀ7ꢀ9ꢀ21 contained syringaresinol (17),
which has been reported earlier in barley and brewer’s spent grain,59,60 but not yet
in beer. Comparison of spectroscopic chromatographic data with those of reference
substances isolated and purified from a hop polyphenol extract, followed by coꢀ
chromatography, led to the identification of coꢀmultifidolglucoside (18a) and
quercetinꢀ3ꢀOꢀβꢀDꢀglucoside (19) in fraction Iꢀ7ꢀ8, quercetinꢀ3ꢀOꢀβꢀDꢀ(6’’ꢀOꢀ
malonyl)glucoside (21) in fraction Iꢀ7ꢀ9ꢀ12, adꢀmultifidolglucoside in fraction Iꢀ7ꢀ9ꢀ17
(18c), nꢀmultifidolglucoside (18b) in fraction Iꢀ7ꢀ9ꢀ19, and kaempferolꢀ3ꢀOꢀβꢀDꢀ
glucoside (20) and kaempferolꢀ3ꢀOꢀβꢀDꢀ(6’’ꢀOꢀmalonylglucoside (22) in fraction Iꢀ7ꢀ
9ꢀ20. Apart from compounds 18a, 18c, 19, 21 and 22, which have already been
reported as constituents in beer,34,62 nꢀmultifidolglucoside (18b) and quercetinꢀ3ꢀOꢀ
βꢀDꢀ(6’’ꢀOꢀmalonyl)glucoside (20) were just known from hops.58,61
Identification of Key Antioxidants in the Fraction II. Due to its complex
composition, the lyophilized fraction II was extracted with an acetonitrile/water
(70/30, v/v) to separate less polar compounds from highly polar carbohydrates and
peptides. As the nonꢀextractable residue exhibited only a negligible antioxidant
activity compared to the highly active extract fraction IIA with 3300 (ORAC), 770
(HPS), and 460 ꢁmol TE/L (LA), fraction IIA was further separated by means of
RP18ꢀMPLC to give subfractions IIAꢀ1 to IIAꢀ9, which were again assayed for
antioxidant activity. The highest ORACꢀactivities were found for fraction IIAꢀ9
(1700 ꢁmol TE/L), IIAꢀ8 (1200 ꢁmol TE/L), followed by IIAꢀ7 (800 ꢁmol TE/L). The
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HPSꢀassay identified fractions IIAꢀ9 (580 ꢁmol TE/L), IIAꢀ8 (470 ꢁmol TE/L) and IIAꢀ
5 (340 ꢁmol TE/L) as the most active ones, while fractions IIAꢀ5 (300 ꢁmol TE/L),
IIAꢀ8 (160 ꢁmol TE/L) and IIAꢀ9 (160 ꢁmol TE/L) exhibited the highest impact in the
LAꢀassay. Taking all these data together, fractions IIAꢀ5, IIAꢀ7, IIAꢀ8, and IIAꢀ9 were
most active and, therefore, further separated by means of preparative HPLC to give
the subfractions IIAꢀ5ꢀ1 to IIAꢀ5ꢀ10, IIAꢀ7ꢀ1 to IIAꢀ7ꢀ11, IIAꢀ8ꢀ1 to IIAꢀ8ꢀ15, and IIAꢀ
9ꢀ1 to IIAꢀ9ꢀ15.
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
Within fraction IIAꢀ5, subfraction IIAꢀ5ꢀ9 showed by far the highest antioxidant
activity (200 ꢁmol TE/L in ORAC, 91 ꢁmol TE/L in HPS, 140 ꢁmol TE/L in LA;
Figure 2). Subfraction IIAꢀ7ꢀ9 (230 ꢁmol TE/L in ORAC, 13 ꢁmol TE/L in HPS,
13 ꢁmol TE/L in LA) contained the most important antioxidants of fraction IIAꢀ7. In
comparison, the antioxidant activity in the subfractions of IIAꢀ8 and IIAꢀ9 was rather
spread over the fractions.
UV/vis, LCꢀTOFꢀMS and 1D/2DꢀNMR analysis of the compounds isolated
from fraction IIAꢀ5ꢀ9 revealed, besides the amino acid Lꢀphenylalanine (23), a
phenolic compound exhibiting two UVꢀmaxima at 224 and 284 nm. LCꢀTOFꢀMS
showed m/z 301.0919 ([C13H18O8–H]−) as the pseudomolecular ion ([M−H]−) and a
1
cleavage of 162 Da, thus indicating the presence of a hexose moiety. H NMR
spectroscopy revealed proton signals at 6.66 (HꢀC(6)), 6.84 (HꢀC(2)), und 6.87 ppm
(HꢀC(5)), thus indicating an threeꢀfold substituted aromatic system. Moreover, a βꢀDꢀ
glucopyranoside moiety was assigned with the anomeric proton (HꢀC(1‘)) resonating
at 4.99 ppm and showing a coupling constant of 7.6 Hz. Heteronuclear correlation
spectra (HMBC) revealed the connection between HꢀC(1‘) and C(1) at 151.8 ppm
and between H3ꢀC(1’’) at 3.85 ppm and C(3) at 149.0 ppm, thus indicating the
presence of tachioside (24) or isotachioside (24a) as outlined in Figure 3.
Independent chemical synthesis and comparison of chromatographic and
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531
532
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535
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547
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549
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551
552
553
554
555
556
spectroscopic data, followed by coꢀchromatography, revealed tachioside (24;
Figure 1) as the key antioxidant in fraction IIAꢀ5.
In addition, tryptophan (25) was identified in fraction IIAꢀ7ꢀ9 by comparison
with the reference substance, and saponarin (26), previously reported in barley and
the PVPP residue of beer,63,64 was identified in fraction IIAꢀ9ꢀ11 by means of LCꢀMS
and NMR spectroscopic experiments. Moreover, LCꢀTOFꢀMS analysis of fraction
IIAꢀ9ꢀ11 revealed a major compound (27) with m/z 180.0668 as the [M−H]− ion,
fitting well with the molecular formula of C9H11NO3, and m/z 94.0300 as the
1
fragment ion ([C5H5NO–H]−), thus indicating a butyric acid side chain. The H NMR
spectrum exhibited an aldehyde proton (HꢀC(1)) at 9.46 ppm and the typical proton
coupling pattern of a pyrrol system with three aromatic double doublets resonating
at 6.26 (HꢀC(4)), 7.04 (HꢀC(3)), and 7.17 ppm (HꢀC(5)) and exhibiting characteristic
coupling constants of 1.7, 2.5, and 4.1 Hz, respectively. The HMBC spectrum of 27
revealed connectivity of the signal of C(2) at 132.7 ppm with the protons at
4.37 ppm (H2ꢀC(4‘)) as well as the aldehyde proton (HꢀC(1)) and the aromatic
proton signals HꢀC(3), HꢀC(4), and HꢀC(5), thus indicating the butyric acid side
chain to be linked to the pyrrol via the nitrogen atom. Taking all spectroscopic data
into consideration, 4ꢀ(2ꢀformylpyrrolꢀ1ꢀyl)butyric acid (27) was identified as the key
compound in fraction IIAꢀ9ꢀ7.
LCꢀTOFꢀMS analysis of the major compound (28) in fraction IIAꢀ8ꢀ10 showed
a similar fragmentation pattern as observed for compound 27, although a mass shift
of 30 Da was observed, e.g. m/z 210.0770 was found as the pseudomolecular ion
([C10H13NO4–H]−) and m/z 124.0404 ([C6H7NO2–H]−) as the main fragment ion
(Figure 4). In comparison to 27, the 1H NMR spectrum of compound 28 showed an
additional signal of a hydroxymethyl group (H2ꢀC(6)) at 4.64 ppm, while the pyrrol
proton at C(5) was lacking. Although isolated earlier as a Maillard reaction product
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from carbohydrate/aminobutyric acid model reactions,40,65 4ꢀ[2ꢀformylꢀ5ꢀ
(hydroxymethyl)pyrrolꢀ1ꢀyl]butyric acid (28), isolated from fraction IIAꢀ8ꢀ10, has not
been previously been reported as a constituent of beer (Figure 1).
558
559
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582
HPLCꢀUV/vis analysis of fractions IIAꢀ8ꢀ6, IIAꢀ8ꢀ8, and IIAꢀ8ꢀ11, respectively,
showed typical local absorption maxima at 228 nm, 300 nm and 320 nm as
expected for hydroxycinnamic acid derivatives. LCꢀTOFꢀMS of the main compounds
29−31 revealed m/z 551.3091, 581.3194, and 611.3296 as pseudomolecular ions
([M+H]+), fitting well with the molecular formula of C28H38N8O4, C29H40N8O5, and
C30H42N8O6, respectively, and indicated the presence of hordatines (29−31), which
has been reported earlier.66 Full spectroscopic signal assignment has been
achieved by 1D/2DꢀNMR experiments (Table 2). The 3J proton coupling constant for
the protons HꢀC(7) and HꢀC(8) of the double bond of 12.2 Hz indicated a cisꢀ
configuration, that seemed to be caused by a trans to cis isomerisation during the
3
isolation procedure.67 Moreover, the J proton coupling constant of 7.4 Hz between
the protons HꢀC(7’) and HꢀC(8’) demonstrated a transꢀconfiguration of the amide
side chain and the hydroxyphenyl side chain. HMBC spectroscopy confirmed the
position of the methoxy groups by the 3J coupling of H3ꢀC(10) at 3.89 ppm and C(5)
3
at 144.7 ppm of hordatines B (30) and C (31) and the J coupling of H3ꢀC(10’) at
3.87 ppm and C(3’) at 148.7 ppm of hordatine C (31). This is the first time, that
1
detailed H/13C NMR data are available for hordatine B (30; Figure 1), although it
has been suggested earlier as a natural product from barley.68 Since its first
suggestion in barley,66 the chemical structure of hordatine C (31, Figure 1) has, to
the best of our knowledge, not been confirmed earlier.
Antioxidant Activity of Identified Compounds. Prior to the analysis of their
antioxidant activity by means of the ORACꢀ, HPSꢀ, and LAꢀassay (Table 3),
respectively, a purity of more than 98% was confirmed for each target compound by
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583
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608
HPLCꢀTOFꢀMS and H NMR spectroscopy. The highest antioxidant activity of 10 –
17.5 (ORAC), 2.0 – 4.1 (HPS), and 1.1 – 6.1 ꢁmol TE/ꢁmol (LA) was found for the
hordatines (29−31). In the ORACꢀassay, only saponarin (26) reached also a high
activity of 13 ꢁmol TE/ꢁmol, while the LAꢀassay indicated high antioxidant activity
for compounds exhibiting an aromatic orthoꢀdihydroxy moiety, such as, e.g. caffeic
acid
(5;
2.8 ꢁmol TE/ꢁmol),
and
quercetinꢀ3ꢀOꢀβꢀDꢀglucopyranoside
(19;
(21;
1.8 ꢁmol TE/ꢁmol),
quercetinꢀ3ꢀOꢀβꢀDꢀ(6ꢀmalonyl)glucoside
3.0 ꢁmol TE/ꢁmol). While monoꢀphenolic compounds, such as, e.g. pꢀ
hydroxybenzoic acid (1), pꢀcoumaric acid (4), pꢀhydroxyphenyllactic acid (8), cyclo
(ProꢀTyr) (10), and tyrosol (11) showed almost negligible activities
(< 0.2 ꢁmol TE/ꢁmol) in the TE assay, pꢀcoumaric acid (4) showed high antioxidant
activity of 8 ꢁmol TE/ꢁmol in the ORACꢀassay and 1.0 ꢁmol TE/ꢁmol in the HPSꢀ
assay, thus indicating the different specificities of the test systems used. In
comparison,
4ꢀ(2ꢀformylpyrrolꢀ1ꢀyl)butyric
acid
(27)
and
4ꢀ[2ꢀformylꢀ5ꢀ
(hydroxymethyl)pyrrolꢀ1ꢀyl]butyric acid (28) showed only low activity of 0.13 and
0.12 ꢁmol TE/ꢁmol in the LAꢀassay, and 2ꢀisopropylmalic acid (9), cinnamic acid
(15) and phenylalanine (23) did not show any significant antioxidant activity
(< 0.01 ꢁmol TE/ꢁmol).
For the first time, an activityꢀguided fractionation, followed by unequivocal
structure determination by means of LCꢀTOFꢀMS, LCꢀMS/MS, and NMR
spectroscopy led to the identification of 31 antioxidant compounds in beer. Whereas
previous studies focused on the antioxidant activity of literatureꢀknown compound
classes in beer, such as, e.g. chalcones, flavanones, procyanidins, hydroxybenzoic
acids, and hydroxycinnamic acids,14,18ꢀ24 the activityꢀguided strategy applied in the
present study demonstrate for the first time hordatines (29−31), saponarin (26), and
quercetinꢀ3ꢀOꢀβꢀDꢀ(6’’ꢀOꢀmalonyl)glucoside (21) to exhibit with the highest
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antioxidant activities. To answer the question as to which of the compounds
identified contribute most to the antioxidant activity of beer, quantitative and doseꢀ
activity studies are currently under investigation and will be published separately.
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619
620
621
622
623
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625
ACKNOWLEDGEMENT
This IGF Project of the FEI is supported via AiF within the programme for promoting
the Industrial Collective Research (IGF) of the German Ministry of Economic Affairs
and Energy (BMWi), based on a resolution of the German Parliament. We are also
grateful to the industry partners of this project for providing us with authentic
samples.
SUPPORTING INFORMATION AVAILABLE
MPLC and HPLC chromatograms and antioxidant activity of the activityꢀguided
fractionation. This material is available free of charge via the Internet at
http://pubs.acs.org.
626 Literature Cited
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