In vitro activity-guided identification of antioxidants in aged garlic extract

Article abstract of DOI:10.1021/jf305549g

Activity-guided fractionation was applied on an aged garlic extract (AGE), reported to show strong antioxidant activity, in order to locate the key in vitro antioxidant ingredients by means of the hydrogen peroxide scavenging (HPS) assay as well as the OR

Full text of DOI:10.1021/jf305549g

Article
pubs.acs.org/JAFC
In Vitro Activity-Guided Identification of Antioxidants in Aged Garlic
Extract
Toshiaki Matsutomo, Timo D. Stark, and Thomas Hofmann*
Chair of Food Chemistry and Molecular Sensory Science, Technische Universitat Munchen, Lise-Meitner Straße 34, D-85354
̈
̈
Freising, Germany
ABSTRACT: Activity-guided fractionation was applied on an aged garlic extract (AGE), reported to show strong antioxidant
activity, in order to locate the key in vitro antioxidant ingredients by means of the hydrogen peroxide scavenging (HPS) assay as
well as the ORAC assay. Besides the previously reported four tetrahydro-β-carbolines, (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-
tetrahydro-β-carboline-3-carboxylic acid and (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid,
LC-MS/MS, LC-TOF-MS, and 1D/2D-NMR experiments led to the identification of coniferyl alcohol and its dilignols
(−)-(2R,3S)-dihydrodehydrodiconiferyl alcohol, (+)-(2S,3R)-dehydrodiconiferyl alcohol, erythro-guaiacylglycerol-β-O-4′-con-
iferyl ether, and threo-guaiacylglycerol-β-O-4′-coniferyl ether as the major antioxidants in AGE. The purified individual
compounds showed high antioxidant activity, with EC50 values of 9.7−11.8 μM (HPS assay) and 2.60−3.65 μmol TE/μmol
(ORAC assay), respectively.
KEYWORDS: aged garlic extract, antioxidants, dihydrodehydrodiconiferyl alcohol, dehydrodiconiferyl alcohol
INTRODUCTION
■
Reactive oxygen species (ROS) are well known to be generated
as byproducts of the normal cell aerobic respiration that is
essential to life and play a crucial role in cell signaling and
homeostasis.1 However, overproduction of ROS causes
undesirable oxidative stress, which is associated with chronic
and degenerative diseases such as diabetes,2,3 neurodegenera-
tion,4,5 cardiovascular disease,6,7 and cancer8,9 and is involved in
the process of aging.10,11
Although not 100% effective, the human body has developed
a very delicate system to eliminate free radicals from the
body.12,13 Vitamins C and E and antioxidant phytochemicals
such as polyphenols have been thought to be responsible for
most of the antioxidant activity in foods and were examined for
their abilities to attenuate oxidative damage induced by
ROS.14,15
Figure 1. Chemical structures of antioxidants previously identified in
aged garlic extract: Nα-(1-deoxy-D-fructos-1-yl)-L-arginine (1),
(1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (2),
(1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (3),
(1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid
(4), and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-dicarbox-
ylic acid (5).
Moreover, previous studies have provided strong evidence
that aged garlic extract (AGE), manufactured by extracting
fresh garlic with aqueous ethanol and maturing the extract for
more than 10 months, exhibits strong antioxidant activity, e.g.
by increasing the activity of superoxide dismutase, catalase, and
glutathione peroxidase in vascular endothelial cells 16 as well as
by scavenging H2O2 and free radicals.17−19 AGE was also
reported to exhibit cardioprotective,20,21 liver-protective,22,23
and cancer-preventing effects,24,25 although the underlying
chemical components have not yet been elucidated.
Besides some organosulfur compounds such as S-allylcys-
teine (SAC) and S-allylmercaptocysteine (SAMC), originating
from garlic,17 Maillard reaction products such as Nα-(1-deoxy-
D-fructos-1-yl)-L-arginine (1; Figure 1) and the 1-methyl-
1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids 2−5 were
identified in AGE as potent antioxidants.26,27 Even though
these previous studies gave a first insight into antioxidants in
AGE, their antioxidant activities are assumed to be by far
insufficient to show a major contribution to the overall
antioxidative power of AGE.
The objective of the present study was, therefore, to locate
the antioxidants in AGE by means of an activity-guided
fractionation approach using the hydrogen peroxide scavenging
(HPS) assay as well as the oxygen radical absorbance capacity
(ORAC) assay and to identify their chemical structures by
means of LC-MS and NMR experiments.
MATERIALS AND METHODS
■
Chemicals. 2,2′-Azo-bis(2-methylpropinamidine) (AAPH), fluo-
rescein sodium salt (FL), ( )-6-hydroxy-2,5,7,8-tetramethylchromane-
2-carboxylic acid (Trolox), 2,2′-azinobis(3-ethylbenzothiazoline-6-
Received: January 2, 2013
Revised: February 27, 2013
Accepted: February 28, 2013
© XXXX American Chemical Society
A
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Figure 2. RP18-HPLC chromatogram of AGE separation (A) and antioxidant activity of AGE and HPLC fractions (B). Data given for HPS and
ORAC activity are the means of three and four independent repetitions, respectively (error bars represent standard deviation, SD).
sulfonic acid) diammonium salt (ABTS), peroxidase from horseradish,
quercetin, (−)-epicatechin, ascorbic acid, and coniferyl alcohol were
purchased form Sigma-Aldrich (Steinheim, Germany), and hydrogen
peroxide was from Merck (Hohenbrunn, Germany). Water for
chromatographic separations was purified with a Milli-Q Gradient
A10 system (Millipore, Schwalbach, Germany), and solvents used
were of HPLC-grade (Merck, Darmstadt, Germany). Aged garlic
extract was produced by Wakunaga Pharmaceutical Co. Ltd. by
hydroalcoholic extraction of sliced, fresh garlic cloves in a stainless
steel tank for 20 months at room temperature. The AGE samples used
contained the well-known organosulfur compound S-allylcysteine in
the concentration of 1.6−2.4 mg/g dry weight.19 Nα-(1-Deoxy-D-
fructos-1-yl)-L-arginine (1) was synthesized as reported in the
literature.26 Chemical synthesis of (1R,3S)-1-methyl-1,2,3,4-tetrahy-
dro-β-carboline-3-carboxylic acid (2), (1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-β-carboline-3-carboxylic acid (3), (1R,3S)-1-methyl-1,2,3,4-
tetrahydro-β-carboline-1,3-dicarboxylic acid (4), and (1S,3S)-1-meth-
yl-1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid (5) was per-
formed following a literature procedure.27
Hydrogen Peroxide Scavenging Assay. In accordance with a
literature protocol,26 sample solutions of AGE fractions and purified
compounds were prepared in phosphate buffer (100 mM, pH 6.0)
using the test materials in the concentration ratios naturally occurring
in AGE. An aliquot (100 μL) of the sample solution or phosphate
buffer (100 mM, pH 6.0) used for control was placed in each well of a
96-well clear microplate (VWR, Ismaning, Germany). Then,
phosphate buffer (100 mM, pH 6.0; 30 μL) and aqueous hydrogen
peroxide solution (500 μM in water; 10 μL) or phosphate buffer used
for blank was added in each well. Afterward a solution of peroxidase
(150 U/mL in water; 40 μL) and a solution of ABTS (0.1% in water;
40 μL) were added. After incubation for 15 min at 37 °C, the
absorbance of each well was measured at 414 nm by means of a
FLUOstar OPTIMA equipment (BMG LABTECH, Offenburg,
Germany). Using the measured absorbance of the sample solution
(AS), the sample-blank solution (ASB, without hydrogen peroxide),
the control solution (AC, without sample solution), and control-blank
solution (ACB, without sample solution and hydrogen peroxide), the
scavenging effect (E) was calculated using the following formula, and
EC50 was calculated by the probit method: E = [(AC − ACB) − (AS
− ASB)]/(AC − ACB) × 100.
Oxygen Radical Absorbance Capacity Assay. Following a
literature protocol28 with some modification, the peroxyl radical
scavenging efficacy was measured using the ORAC assay. Sample
solutions were prepared in the same way as in the hydrogen peroxide
scavenging assay using phosphate buffer (10 mM, pH 7.4). A series of
Trolox solutions (200, 100, 50, 25, 12.5 μM) was prepared by diluting
an ethanolic solution of Trolox (2 mM) with phosphate buffer (10
mM, pH 7.4). Sample solution, Trolox dilution (25 μL), or phosphate
buffer (10 mM, pH 7.4) used as blank was placed in wells of a 96-well
black microplate (VWR). Then fluorescein sodium salt (150 μL in
phosphate buffer, 10 nM) was added to each well, and the microplate
was incubated at 37 °C for 30 min. Afterward, the decay of
fluorescence was measured every 90 s at the excitation wavelength of
485 nm and the emission wavelength of 520 nm using a FLUOstar
OPTIMA plate reader. The first three cycles were taken to determine
the background signal. After 3 cycles, AAPH (25 μL in phosphate
buffer, 240 mM) was added, and then the measurement was resumed
and continued up to 90 min (60 cycles in total). The ORAC values
were calculated according to the method of Cao et al.29 Briefly, a
standard curve was obtained from the area under the fluorescence
versus time curve (AUC) for Trolox dilutions minus the area-under-
the-curve (AUC) for blank. Then the AUC for the sample solution
minus the AUC for the blank was calculated and compared to the
standard curve. ORAC values were expressed as Trolox equivalents
(μmol TE/μmol).
Fractionation of AGE. An aliquot of AGE (80 g, dry weight) was
fractionated by using a preparative HPLC system (PrepStar, Varian,
Darmstadt, Germany) consisting of two HPLC pumps (model SD-1),
a two-wavelength UV detector (Prostar 325), and a fraction collector
(model 701), equipped with a 50 × 200 mm, 8 μm, C-18 column
(Microsorb, Varian, Darmstadt, Germany) packed by using a load-and-
lock packing station (Varian, Darmstadt, Germany). Monitoring the
effluent (100 mL/min) at 220 nm, chromatography was performed
starting with aqueous formic acid (0.1% in water, pH 2.5) for 10 min,
then increasing the methanol content up to 100% within 3 min, and
finally held for 7 min at 100% methanol. Four fractions, namely,
fractions 1 (28.3 g), 2 (0.3 g), 3 (6.7 g), and 4 (43.5 g), were collected
(Figure 2), concentrated under reduced pressure at 40 °C, and then
further separated on a semipreparative HPLC system (Jasco, Groß-
Umstadt, Germany) equipped with a 250 × 21.2 mm, 5 μm, C-18
B
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Figure 3. RP18-HPLC chromatogram of the separation of fraction 4 (A) and antioxidant activity of HPLC fractions (B). Data given for HPS and
ORAC activity are the means of three and four independent repetitions, respectively (error bars represent SD).
column (Microsorb, Varian, Darmstadt, Germany) as the stationary
phase consisting of two PU-2087 Plus pumps, a DG-2080-53 degasser,
an LG-2080-02 gradient unit, and a 2010 Plus multiwavelength
detector. Using a flow rate of 21.0 mL/min, an aliquot (10 g) of
fraction 4 was fractionated using the following gradient of 0.1%
aqueous formic acid (solvent A) and methanol (solvent B): 0−3 min
(0% B), 3−33 min (0 → 90% B), 33−34 min (90 → 100% B), 34−39
min (100% B). Monitoring the effluent at 220 nm, a total of 16
subfractions were collected, separated from solvent under vacuum, and
then freeze-dried to obtain fractions 4-1 (286 mg), 4-2 (47 mg), 4-3
(86 mg), 4-4 (455 mg), 4-5 (1542 mg), 4-6 (3129 mg), 4-7 (153 mg),
4-8 (46 mg), 4-9 (115 mg), 4-10 (9 mg), 4-11 (11 mg), 4-12 (25 mg),
4-13 (21 mg), 4-14 (6 mg), 4-15 (39 mg), and 4-16 (90 mg) with the
yields given in parentheses (Figure 3). The corresponding fractions
collected from four separations were combined. Fraction 4-14 was
further separated by means of semipreparative HPLC (HPLC pump
system PU 2087, high-pressure gradient unit, and PU-2075 UV
detector, Jasco, Groß-Umstadt, Germany) equipped with a Luna
PhenylHexyl 10 × 250 mm, 5 μm column (Phenomenex) as the
stationary phase using the following gradient of 0.1% aqueous formic
acid (solvent A) and acetonitrile (solvent B) at a flow rate of 4.2 mL/
min: 0−35 min (22% B), 35−38 min (22 → 100% B), 38−42 min
Figure 4. Chemical structures of coniferyl alcohol (6), (−)-(2R,3S)-
dihydrodehydrodiconiferyl alcohol (7), (+)-(2S,3R)-dehydrodiconifer-
yl alcohol (8), erythro-guaiacylglycerol-β-O-4′-coniferyl ether (9), and
threo-guaiacylglycerol-β-O-4′-coniferyl ether (10).
(100% B). Monitoring the HPLC effluent at 220 nm showed two main
compounds, which were isolated, freed from solvent under vacuum,
and freeze-dried to afford compounds 7 and 8 as white, amorphous
powders in yields of 14.7 and 11.7 mg (Figure 4). LC-TOF-MS, 1D/
2D-NMR, and circular dichroism (CD) spectroscopic experiments led
to the identification of these compounds as (−)-(2R,3S)-dihydrodehy-
drodiconiferyl alcohol (7) and (+)-(2S,3R)-dehydrodiconiferyl alcohol
(8), respectively.
= 6.4 Hz, H−C(10)], 3.51 [m, 1H, J = 6.4, 6.6 Hz, H−C(3)], 2.62 [t,
2H, J = 7.7 Hz, H−C(8)], 1.79 [m, 2H, J = 6.4, 7.7 Hz, H−C(9)]. 13C
NMR (100 MHz, acetone-d6, HSQC, HMBC): δ 148.35 [C(3′)],
147.35 [C(7a)], 147.19 [C(4′)], 144.89 [C(7)], 136.30 [C(5)],
134.70 [C(1′)], 130.00 [C(3a)], 119.54 [C(6′)], 117.57 [C(4)],
115.64 [C(5′)], 113.89 [C(6)], 110.45 [C(2′)], 88.18 [C(2)], 64.78
[C(11)], 61.84 [C(10)], 56.42 [C(7-OMe)], 56.27 [C(3′-OMe)],
55.10 [C(3)], 36.00 [C(9)], 32.70 [C(8)].
(−)-(2R,3S)-Dihydrodehydrodiconiferyl alcohol, 7, Figure 4.
UV/vis (MeOH/H2O, 5:5, v/v): λmax = 232, 281 nm. LC-TOF-MS
(ESI−): m/z 359.1497 ([M − H]−, measured; m/z 359.1495,
calculated for [C20H24O6−H]−). CD (MeOH, 0.28 mmol/L): λmax
1
(Δε) = 294 (−1.1), 242 (−2.8), 224 (+1.4). H NMR (400 MHz,
acetone-d6, COSY): δ 7.04 [d, 1H, J = 1.9 Hz, H−C(2′)], 6.89 [dd,
1H, J = 1.9, 8.2 Hz, H−C(6′)], 6.81 [d, 1H, J = 8.2 Hz, H−C(5′)],
6.75 [d, 1H, J = 1.6 Hz, H−C(4)], 6.73 [d, 1H, J = 1.6 Hz, H−C(6)],
5.52 [d, 1H, J = 6.6 Hz, H−C(2)], 3.83 [s, 3H, H−C(7-OMe)], 3.82
[s, 3H, H−C(3′-OMe)], 3.76−3.92 [m, 2H, H−C(11)], 3.57 [t, 2H, J
(+)-(2S,3R)-Dehydrodiconiferyl alcohol, 8, Figure 4. UV/vis
(MeOH/H2O, 5:5, v/v): λmax = 225, 275 nm. LC-TOF-MS(ESI−):
m/z 357.1350 ([M − H]−, measured; m/z 357.1338, calculated for
[C20H22O6−H]−); CD (MeOH, 0.28 mmol/L): λmax(Δε) = 286
1
(+1.4), 231 (−1.2). H NMR (400 MHz, acetone-d6, COSY): δ 7.04
C
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[d, 1H, J = 1.9 Hz, H−C(2′)], 6.98 [d, 1H, J = 1.6 Hz, H−C(4)], 6.95
[d, 1H, J = 1.6 Hz, H−C(6)], 6.89 [dd, 1H, J = 1.9, 8.1 Hz, H−C(6′)],
6.81 [d, 1H, J = 8.2 Hz, H−C(5′)], 6.53 [d, 1H, J = 15.9 Hz, H−
C(8)], 6.25 [dt, 1H, J = 5.3, 15.9 Hz, H−C(9)], 5.57 [d, 1H, J = 6.5
Hz, H−C(2)], 4.20 [d, 2H, J = 5.3 Hz, H−C(10)], 3.87 [s, 3H, H−
C(7-OMe)], 3.82 [s, 3H, H−C(3′-OMe)], 3.93−3.79 [m, 2H, H−
C(11)], 3.54 [dd, 1H, J = 6.3, 6.5 Hz, H−C(3)]. 13C NMR (100 MHz,
acetone-d6, HSQC, HMBC): δ 151.77 [C(3′)], 149.27 [C(4′)],
148.84 [C(3)], 147.20 [C(4)], 133.79 [C(1)], 133.17 [C(1′)], 131.42
[C(7′)], 128.63 [C(8′)], 120.81 [C(6′)], 120.75 [C(6)], 118.87
[C(5′)], 115.85 [C(5)], 111.76 [C(2)], 111.30 [C(2′)], 87.14 [C(8)],
74.04 [C(7)], 63.75 [C(9′)], 61.92 [C(9)], 56.55 [C(3′-OMe)], 56.35
[C(3-OMe)].
Synthesis of erythro-Guaiacylglycerol-β-O-4′-coniferyl Ether
(9) and threo-Guaiacylglycerol-β-O-4′-coniferyl Ether (10).
Following a literature protocol30 with some modifications, coniferyl
alcohol (0.56 mmol) was dissolved in phosphate buffer (85 mL; 100
mM, pH 6.0), and a solution (5 mL) of horseradish peroxidase (3.4
μg/mL) in phosphate buffer and an aqueous solution (85 mL) of
hydrogen peroxide (0.01% in water) were added dropwise within 30
min while stirring at room temperature. After 5 h of continued stirring,
the reaction was stopped by the addition of hydrogen chloride (2 mL,
1 M), the reaction mixture was extracted with ethyl acetate (2 × 200
mL), the combined organic layers were separated from solvent under
vacuum, the residue was taken up in methanol/water (75/25, v/v; 15
mL), and then the target compounds 9 and 10 were isolated by means
of preparative HPLC (Jasco) equipped with a Luna PhenylHexyl 21.2
× 250 mm, 5 μm column (Phenomenex). Using a flow rate of 21 mL/
min, chromatography was performed with the following gradient using
0.1% aqueous formic acid (solvent A) and methanol (solvent B): 0−12
min (10 → 20% B), 12−32 min (20 → 60% B), and 32−34 min (60
→ 100% B). Monitoring the effluent at 220 nm, the effluent of the
peaks detected at 16.2 and 16.8 min, respectively, were collected
individually and freed from solvent under vacuum to give compounds
9 (4.0 μmol) and 10 (7.2 μmol) as white, amorphous powders after
freeze-drying.
Ultra-Performance Liquid Chromatography/Mass Spec-
trometry (UPLC-MS/MS). Mass spectral analyses were performed
on a Waters Xevo TQ-S mass spectrometer (Waters, Manchester, UK)
coupled to an Acquity UPLC i-class core system (Waters, Milford,
MA, USA) equipped with a 2 × 150 mm, 1.7 μm, BEH Phenyl column
(Waters, Manchester, UK) in a column oven. Using the negative
electrospray ionization mode (ESI−), capillary voltage (−2.0 kV),
sampling cone voltage (50 V), source offset voltage (30 V), source
temperature (150 °C), desolvation temperature (500 °C), cone gas
(150 L/h), desolvation gas (1000 L/h), collision gas (0.25 mL/min),
and nebulizer gas (6.5 bar) were set as given in parentheses.
Calibration of the mass spectrometer in the range m/z 40−1963 was
performed using a solution of phosphoric acid (0.1% in acetonitrile).
The UPLC-MS/MS system was operated with MassLynx software
(Waters, Manchester). Data processing and analysis were performed
using TargetLynx 4.1 SCN 813 software (Waters, Manchester).
UPLC-MS/MS Detection of Compounds 7−10 in AGE. After
1:10 dilution with 20% methanol, aliquots (1 μL) of AGE were
injected into the UPLC-MS/MS system equipped with a 2 × 150 mm,
1.7 μm, BEH Phenyl column (Waters, Manchester, UK). Operating
with a flow rate of 0.4 mL/min and a temperature of 45 °C,
chromatography was performed using the following gradient of 0.1%
aqueous formic acid in water (solvent A) and 0.1% formic acid in
acetonitrile (solvent B): 0.0−9.0 min (12 → 40% B), 9.0−9.1 min (40
→ 99% B), 9.1−9.6 min (99% B), 9.6−9.7 min (99 → 12% B), 9.7−
10.2 min (12% B). ESI− mass and product ion spectra were acquired
for compounds 7−10 with direct flow infusion using IntelliStart. The
MS/MS parameters were tuned for each individual compound,
detecting the fragmentation of the [M − H]− molecular ions into
specific product ions after collision with argon. By means of the
multiple reaction monitoring (MRM) mode, compounds 7 (m/z 359.3
→ 341.1/329.1), 8 (m/z 357.3 → 339.3/327.2), 9 (m/z 375.2 →
327.2/149.2), and 10 (m/z 375.2 → 327.2/195.1) were analyzed using
the mass transitions given in parentheses (20 ms duration).
Comparison of retention times with the reference compounds,
followed by cochromatography, led to the unequivocal identification
of compounds 7−10 in AGE.
LC/Time-of-Flight Mass Spectrometry (LC-TOF-MS). Aliquots
(1−5 μL) of the analytes dissolved in methanol/water (8:2, v/v; 1
mL) were injected into an Acquity UPLC core system (Waters,
Milford, MA, USA) connected to a SYNAPT G2-S HDMS
spectrometer (Waters, Manchester, UK) operating in the electrospray
(ESI) modus with the following parameters: capillary voltage +2.5 or
−3.0 kV, sampling cone 30, extraction cone 4.0, source temperature
150 °C, desolvation temperature 450 °C, cone gas 30 L/h, and
desolvation gas 850 L/h. The instrument was calibrated over a mass
range from m/z 50 to 1200 using a solution of sodium formate (0.5
mmol/L) in 2-propanol/water (9:1, v/v). All data were lock mass
corrected using leucine enkephaline as the reference (m/z 556.2771
for [M + H]+; m/z 554.2615 for [M − H]−). Data acquisition and
analysis was performed by using the MassLynx software (version 4.1;
Waters).
erythro-Guaiacylglycerol-β-O-4′-coniferyl Ether, 9, Figure 4. UV/
vis (MeOH/H2O, 5:5, v/v): λmax = 265 nm; LC-TOF-MS (ESI−): m/
z 375.1446 ([M − H]−, measured; m/z 375.1444, calculated for
1
[C20H24O7−H]−). H NMR (500 MHz, methanol-d4, COSY): δ 7.02
[d, 1H, J = 1.9 Hz, H−C(2)], 7.00 [s, 1H, H−C(2′)], 6.87 [brs, 2H,
H−C(5′, 6′)], 6.84 [dd, 1H, J = 1.8, 8.1 Hz, H−C(6)], 6.73 [d, 1H, J
= 8.1 Hz, H−C(5)], 6.51 [dt, 1H, J = 1.4, 15.9 Hz, H−C(7′)], 6.24
[dt, 1H, J = 5.8, 15.9 Hz, H−C(8′)], 4.82 [overlapped, 1H, H−C(7)],
4.35 [m, 1H, H−C(8)], 4.19 [dd, 2H, J = 1.4, 5.8 Hz, H−C(9′)], 3.80
[m, 2H, H−C(9)], 3.80 [s, 3H, H−C(3′-OMe)], 3.80 [s, 3H, H−C(3-
OMe)]. 13C NMR (125 MHz, methanol-d4, HSQC, HMBC): δ
151.93 [C(3′)], 148.96 [C(4′)], 148.72 [C(3)], 147.04 [C(4)],
134.10 [C(1)], 133.07 [C(1′)], 131.46 [C(7′)], 128.51 [C(8′)],
121.04 [C(6)], 120.66 [C(6′)], 118.92 [C(5′)], 115.66 [C(5)],
111.90 [C(2)], 111.40 [C(2′)], 86.22 [C(8)], 74.12 [C(7)], 63.76
[C(9′)], 62.23 [C(9)], 56.52 [C(3-OMe)], 56.34 [C(3′-OMe)].
threo-Guaiacylglycerol-β-O-4′-coniferyl Ether, 10, Figure 4. UV/
vis (MeOH/H2O, 5/5, v/v): λmax = 264 nm. LC-TOF-MS (ESI−): m/
z 375.1447 ([M − H]−, measured; m/z 375.1444, calculated for
Circular Dichroism Spectroscopy. CD spectra were acquired by
means of a Jasco J810 spectropolarimeter (Jasco, Tokyo, Japan).
1
Nuclear Magnetic Resonance Spectroscopy. H, 13C, COSY,
1
[C20H24O7−H]−). H NMR (500 MHz, methanol-d4, COSY): δ 7.09
HSQC, and HMBC experiments were performed on an Avance III 400
MHz spectrometer with a BBO probe and an Avance-III-500
spectrometer, respectively, the latter of which was equipped with a
Cryo-CTCI probe (Bruker, Rheinstetten, Germany). Methanol-d4 and
acetone-d6 were used as solvents, and trimethylsilane (TMS) was used
as the internal standard. Data processing was performed by using
Topspin software (version 2.1; Bruker) as well as Mestre-C software
(version 4.8.6; Mestrelab Research, Santiago de Compostella, Spain).
[d, 1H, J = 1.9 Hz, H−C(2′)], 7.06 [d, 1H, J = 1.9 Hz, H−C(2)], 7.03
[d, 1H, J = 8.3 Hz, H−C(5′)], 6.95 [dd, 1H, J = 1.9, 8.3 Hz, H−
C(6′)], 6.90 [dd, 1H, J = 1.9, 8.1 Hz, H−C(6)], 6.79 [d, 1H, J = 8.1
Hz, H−C(5)], 6.57 [d, 1H, J = 15.9 Hz, H−C(7′)], 6.30 [dt, 1H, J =
5.7, 15.9 Hz, H−C(8′)], 4.92 [d, 1H, J = 5.8 Hz, H−C(7)], 4.33 [m,
1H, H−C(8)], 4.24 [dd, 2H, J = 1.4, 5.7 Hz, H−C(9′)], 3.91 [s, 3H,
H−C(3′-OMe)], 3.86 [s, 3H, H−C(3-OMe)], 3.77 [dd, 1H, J = 4.0,
11.9 Hz, H−C(9a)], 3.51 [dd, 2H, J = 5.3, 11.9 Hz, H−C(9b)]. 13C
NMR (125 MHz, methanol-d4, HSQC, HMBC): δ 151.77 [C(3′)],
149.27 [C(4′)], 148.84 [C(3)], 147.20 [C(4)], 133.79 [C(1)], 133.17
[C(1′)], 131.42 [C(7′)], 128.63 [C(8′)], 120.81 [C(6′)], 120.75
[C(6)], 118.87 [C(5′)], 115.85 [C(5)], 111.76 [C(2)], 111.30
[C(2′)], 87.14 [C(8)], 74.04 [C(7)], 63.75 [C(9′)], 61.92 [C(9)],
56.55 [C(3′-OMe)], 56.35 [C(3-OMe)].
RESULTS AND DISCUSSION
■
Since no single antioxidant assay alone is able to give a
comprehensive picture of the antioxidant capacity of the total
aged garlic extract and fractions isolated thereof, the following
study was performed with two different antioxidant assays as
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Figure 5. TOF-MS (ESI−) spectra of (A) (−)-(2R,3S)-dihydrodehydrodiconiferyl alcohol (7) and (B) (+)-(2S,3R)-dehydrodiconiferyl alcohol (8).
recommended in the literature.31 The HPS assay, based on a
single electron transfer mechanism, measures the radical
reduction ability of an antioxidant by transferring one electron,
whereas the ORAC assay, based on a hydrogen atom transfer
mechanism, determines the radical-quenching ability of an
antioxidant by hydrogen donation.
In Vitro Activity-Guided Fractionation of AGE. Aliquots
of the aged garlic extract were separated by means of
preparative RP18-HPLC to give four fractions (Figure 2A),
which were used in their natural concentration ratios for the
determination of their antioxidant activity using the HPS and
the ORAC assay, respectively. Fraction 4 was identified with by
far the highest antioxidant activity, accounting for 80% and 50%
of the HPS and ORAC activity, respectively, found for the total
AGE (Figure 2B). In comparison, fraction 1 showed only
marginal activity in the HPS assay and did not exhibit any
activity in the ORAC assay, whereas the opposite was observed
for fraction 3. Fraction 2 did not show any activity in the assays
used. UPLC-MS/MS screening of AGE for the known
antioxidant Nα-(1-deoxy-D-fructos-1-yl)-L-arginine (1; Figure
1),26 followed by cochromatography with the synthetic
reference substance, revealed the Amadori compound 1 to be
present in fraction 1 (data not shown). Nα-(1-deoxy-D-fructos-
1-yl)-L-arginine was absent in fraction 4, and the following
studies were focused on previously unknown antioxidants in
fraction 4.
In order to identify the key antioxidants in AGE, the most
active fraction (4) was further separated by means of RP-HPLC
to give a total of 16 subfractions, namely, fraction 4-1 to 4-16,
which were freed from solvent under vacuum and then used for
the determination of their antioxidant activities using the HPS
and the ORAC assay, respectively (Figure 3). The highest
antioxidant activity in both assays was found for subfractions 4-
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Figure 6. HMBC spectrum (400 MHz, acetone-d6) of (−)-(2R,3S)-dihydrodehydrodiconiferyl alcohol (7).
9 and 4-15, followed by subfractions 4-12 to 4-14, and 4-16,
respectively.
(−18 Da) and formaldehyde (−30 Da), respectively. In
comparison, the MS spectrum of 8 (Figure 5B) showed the
pseudomolecular m/z 357.1350 and the fragment ions m/z
339.1240 and 327.1240, which are well in alignment with the
cleavage of one molecule of water and formaldehyde,
respectively. As literature studies reported on losses of 18 Da
(water) and 30 Da (formaldehyde) in the MS spectra of
dilignols,32 compounds 7 and 8 were suggested to exhibit a
dilignol structure.
Rechromatography of fractions 4-9 and 4-15, followed by
HPLC-MS/MS analysis, revealed the presence of the
tetrahydro-β-carbolines 2−5, reported recently as antioxidants
in AGE,27 besides a series of minor constituents, which could
not be isolated in the purity and amounts needed for an
unequivocal structure determination by NMR spectroscopy.
HPLC-MS/MS analysis, followed by cochromatography with
the corresponding reference compound, led to the identi-
fication of (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-
dicarboxylic acid (4) and (1R,3S)-1-methyl-1,2,3,4-tetrahydro-
β-carboline-3-carboxylic acid (2) as active antioxidant in the
most potent fractions 4-9 and 4-15, respectively. In addition,
fractions 4-11 and 4-12 were found to contain (1S,3S)-1-
methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (3) as
the primary antioxidant, and (1S,3S)-1-methyl-1,2,3,4-tetrahy-
dro-β-carboline-1,3-dicarboxylic acid (5) was detected in
fraction 4-11. Rechromatography of the active subfraction 4-
14 indicated the presence of two unknown antioxidants (7 and
8), showing UV absorption maxima at 232 and 281 nm.
Structure Determination of Antioxidants 7 and 8.
UPLC-TOF-MS analysis of compound 7 showed the three ions
m/z 359.1497, 341.1391, and 329.1391 (Figure 5A), which
matched the empirical formula of the pseudomolecular ion
[C20H24O6−H]− (calcd m/z 359.1495) and the fragment ions
[C20H22O5−H]− (calcd m/z 341.1389) and [C19H22O5−H]−
(calcd m/z 329.1389), most likely formed by cleavage of water
1
Besides two methoxy groups, the H NMR spectrum of 7
showed aromatic proton signals for H−C(2′), H−C(6′), and
H−C(5′) resonating at 7.04, 6.89, and 6.81 ppm and indicated
the presence of a 1,3,4-trisubstituted benzene ring. In addition,
the meta-coupled aromatic protons H−C(4) and H−C(6),
showing a coupling constant of J = 1.6 Hz, resonated at 6.75
and 6.73 ppm and indicated the presence of a 1,2,4,5-
tetrasubstituted benzene ring. Moreover, three aliphatic protons
were observed at 5.52 ppm [d, 1H, J = 6.6 Hz, H−C(2)], 3.51
ppm [dd, 1H, J = 6.4, 6.6 Hz, H−C(3)], and 3.76−3.92 ppm
[m, 2H, H−C(11)], which coupled to each other, and another
three aliphatic protons coupling with each other were observed
at 3.57 ppm [t, 2H, J = 6.4 Hz, H−C(10)], 2.62 ppm [t, 2H, J =
7.7 Hz, H−C(8)], and 1.79 ppm [m, 2H, J = 6.4, 7.7 Hz, H−
C(9)].
Well in agreement with the molecular formula C20H25O6
proposed by TOF-MS, the 13C NMR spectrum showed 20
carbon resonances. Comparison of 13C NMR data with those
obtained by means of a DEPT-135 experiment revealed nine
F
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primary or tertiary carbons, four secondary carbons, and seven
quaternary carbon atoms in compound 7. Signal alignment by
means of a heteronuclear multiple-bond correlation spectros-
copy (HMBC) experiment revealed that compound 7 is
composed of two phenylpropanoids. The HMBC experiment
exhibits correlations of H−C(2) with C(3a) and C(7a),
correlations of H−C(3) with C(4) as well as C(3a) and
C(7a), and correlations of H−C(11) with only C(3a), thus
implying that C(2) and C(3) are connected to an aromatic ring
of another phenylpropanoid through an O-linkage and a C-
linkage, respectively (Figure 6). Taking all spectroscopic data
into consideration, compound 7 was identified as dihydrodehy-
drodiconiferyl alcohol (Figure 4). Although this compound was
earlier reported in Cleistopholis glauca and Lawsonia alba,33,34
this is the first report on the presence of 7 as an antioxidant in
garlic.
identification of antioxidant 8 as (+)-(2S,3R)-dehydrodiconi-
feryl alcohol.
Preparation of erythro-Guaiacylglycerol-β-O-4′-con-
iferyl Ether (9) and threo-Guaiacylglycerol-β-O-4′-con-
iferyl Ether (10) and Identification in AGE. Dihydrodehy-
drodiconiferyl alcohol (7) and dehydrodiconiferyl alcohol (8)
are reported as dimeric dehydrogenation products of coniferyl
alcohol (6) formed by an enzymatic radical coupling of a β-
radical of one coniferyl alcohol unit with a 5-radical of another
coniferyl alcohol unit.40 As coniferyl alcohol (6) is reported to
dimerize preferentially to form β-O-4 intermolecular linkages,41
the question arose as to whether dimers with a β-O-4 linkage
do exist also in AGE. Therefore, reference material of the β-O-4
linked dilignols 9 and 10 (Figure 4) was synthesized by
oxidative coupling of coniferyl alcohol with hydrogen peroxide
in the presence of horseradish peroxidase. After chromato-
graphic purification, the structures of 9 and 10 were confirmed
by means of LC-MS, TOF-MS, and NMR spectroscopy and
were in alignment with literature data.42,43
In order to investigate the presence of these dilignols in
AGE, the MS/MS parameters were tuned for each individual
compound, and then the AGE fractions were screened for 9
and 10 by means of UPLC-ESI-MS/MS. This MS screening,
followed by cochromatography with the corresponding
reference compounds, led to the unequivocal identification of
erythro-guaiacylglycerol-β-O-4′-coniferyl ether (9) and threo-
guaiacylglycerol-β-O-4′-coniferyl ether (10) in fractions 4-12
and 4-13, respectively. Moreover, their precursor molecule
coniferyl alcohol (6) was identified in AGE fraction 4 by means
of UPLC-MS/MS.
The 1H NMR signal pattern recorded for compound 8
showed major similarities to that observed for 7, except for the
aliphatic proton signals H−C(8) and H−C(9) at 6.53 and 6.25
ppm, respectively, showing a large coupling constant of J = 15.9
Hz and indicating a trans-configured double bond between
C(8) and C(9) instead of a single bond. Careful signal
assignment by means of heteronuclear correlation experiments
(HSQC, HMBC) led to the unequivocal identification of
compound 8 as dehydrodiconiferyl alcohol (Figure 4).
Although published earlier as a phytochemical in Rosa
multiflora,35 this is the first report on compound 8 as an
antioxidant in garlic.
Using the 1H NMR signal and coupling constant of H−C(2)
as diagnostic markers to differentiate between cis- and trans-
diastereoisomers of dilignols,36 the doublet signal of H−C(2) at
5.52 ppm (J = 6.6 Hz) of 7 and at 5.57 ppm (J = 6.5 Hz) of 8
clearly indicated a trans-configuration in both antioxidants. To
confirm the absolute configuration of carbon atoms C(2) and
C(3) in 7 and 8, both compounds were analyzed by means of
circular dichroism spectroscopy. As depicted in Figure 7, the
Antioxidant Activities of Purified Compounds from
AGE. After checking the purity of each compound by means of
1
HPLC-ELSD, UPLC-TOF-MS, and H NMR spectroscopy,
coniferyl alcohol (6), the dilignols 7−10, and the compounds
1−5 (Figure 1) previously identified as antioxidants in
AGE26,27 were studied for their antioxidant activity using the
HPS as well as the ORAC assay (Table 1). Analysis of the
reference compounds ascorbic acid, quercetin, and (−)-epi-
catechin, well known for their high antioxidant activities,
showed comparable activity of these compounds with that
reported in the literature.28,44,45
As expected from the activity-guided fractionation, com-
pounds 6−10 showed high antioxidant activities; for example,
EC50 values of 9.7−11.8 μM were found in the HPS assay and
high activities of 2.60−3.65 μmol TE/μmol were found when
using the ORAC assay. Compared to the low activities found
for Nα-(1-deoxy-D-fructos-1-yl)-L-arginine (1) in the HPS
(EC50: 139.2 μM) and the ORAC assay (0.01 μmol TE/
μmol), the phenols 6−10 showed ∼13 and ∼330 times higher
antioxidant activities, respectively. Coniferyl alcohol (6) and its
dimers 7−10 showed comparable activities with the tetrahydro-
β-carbolines 2−5; for example, the HPS and the ORAC
activities of (−)-(2R,3S)-dihydrodehydrodiconiferyl alcohol (7)
and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-dicar-
boxylic acid (5) were in the same range. These data confirm
previous studies reporting on the in vitro antioxidant activity of
dilignols and their cellular activity against high glucose-
stimulated ROS production.46−49
Figure 7. CD spectra of (−)-(2R,3S)-dihydrodehydrodiconiferyl
alcohol (7) and (+)-(2S,3R)-dehydrodiconiferyl alcohol (8).
CD spectrum of 7 showed a positive Cotton effect at 224 nm
and negative Cotton effects at 242 and 294 nm, being well in
line with the data published for the (−)-(2R,3S)-configumer.37
Therefore, the structure of antioxidant 7 could be deduced as
(−)-(2R,3S)-dihydrodehydrodiconiferyl alcohol. On the other
hand, comparison of the CD spectrum of 8, exhibiting a
negative Cotton effect at 231 nm and a positive Cotton effect at
286 nm (Figure 7), with literature data38,39 enabled the
In conclusion, activity-guided fractionation using both the
HPS and the ORAC assay enabled the identification of the
tetrahydro-β-carbolines 2−5, coniferyl alcohol (6),
(−)-(2R,3S)-dihydrodehydrodiconiferyl alcohol (7),
(+)-(2S,3R)-dehydrodiconiferyl alcohol (8), erythro-guaiacyl-
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Table 1. Antioxidant Activity of Compounds 1−10 and Reference Compounds
c,d
ORAC assay
(μmol TE/μmol)
ab
,
test compound
H2O2 assay (EC50 in μM)
Nα-(1-deoxy-D-fructos-1-yl)-L-arginine (1)
(1R,3S)-/(1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (mixture of 2 and 3)
(1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid (4)
(1S,3S)- 1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid (5)
coniferyl alcohol (6)
139.2
168.2
36.7
14.3
9.7
(104.1−225.8)
(137.7−209.8)
(29.0−49.7)
(12.2−16.9)
(8.6−11.1)
(9.2−12.8)
(10.2−13.9)
(9.0−11.8)
(9.6−12.5)
(15.0−18.3)
(5.3−7.1)
0.01
( 0.00)
( 0.06)
( 0.08)
( 0.09)
( 0.18)
( 0.20)
( 0.37)
( 0.14)
( 0.09)
( 0.10)
( 0.07)
( 0.53)
1.48
1.68
1.73
3.29
2.60
3.65
3.27
3.53
0.34
5.61
9.65
(−)-(2R,3S)-dihydrodehydrodiconiferyl alcohol (7)
(+)-(2S,3R)-dehydrodiconiferyl alcohol (8)
erythro-guaiacylglycerol-β-O-4′-coniferyl ether (9)
threo-guaiacylglycerol-β-O-4′-coniferyl ether (10)
ascorbic acid
10.8
11.8
10.3
11.0
16.5
6.1
quercetin
(−)-epicatechin
4.1
(3.7−4.6)
a
b
The HPS assay was performed in three independent repetitions for each sample. The range in parentheses represents 95% confidence interval.
The ORAC assay was performed in four independent repetitions for each sample. The numerical value in parentheses represents the SD.
c
d
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glycerol-β-O-4′-coniferyl ether (9), and threo-guaiacylglycerol-
β-O-4′-coniferyl ether (10) as potent in vitro antioxidants in
aged garlic extract. Quantitative studies, followed by recon-
stitution and omission experiments, are currently in progress in
order to answer the question as to what extent the individual
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AUTHOR INFORMATION
■
Corresponding Author
(13) Davies, K. J. A. Oxidative stress, antioxidant defenses, and
damage removal, repair, and replacement systems. IUBMB Life 2000,
50, 279−289.
*Phone: +49-8161-71-2902. Fax: +49-8161-71-2949. E-mail:
thomas.hofmann@tum.de.
(14) Esterbauer, H.; Dieber-Rotheneder, M.; Striegl, G.; Waeg, G.
Role of vitamin E in preventing the oxidation of low-density
lipoprotein. Am. J. Clin. Nutr. 1991, 53, 314s−321s.
Funding
We are grateful to Wakunaga Pharmaceutical Co., Ltd., for
financial support.
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ascorbate inhibit the oxidative modification of low-density lipoprotein.
Atherosclerosis 1990, 82, 185−191.
Notes
The authors declare no competing financial interest.
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augments antioxidant enzyme activity in vascular endothelial cells.
Nutr. Res. (N.Y.) 1998, 18, 61−70.
ACKNOWLEDGMENTS
■
We thank Sofie Losch and Evelyn Eggenstein for their skillful
support.
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