Characterization of phenolic constituents inhibiting the formation of sulfur-containing volatiles produced during garlic processing

Article abstract of DOI:10.1021/jf505982f

Garlic (Allium sativum L.), which is a widely distributed plant, is globally used as both spice and food. This study identified five novel phenolic compounds, namely, 8-(3-methyl-(E)-1-butenyl)diosmetin, 8-(3-methyl-(E)-1-butenyl)chrysin, 6-(3-methyl-(E)-1-butenyl)chrysin, and Alliumones A and B, along with nine known compounds 6-14 from the ethanol extract of garlic. The structures of these five novel phenolic compounds were established via extensive 1D- and 2D-nuclear magnetic resonance spectroscopy experiments. The effects of the phenolic compounds isolated from garlic on the enzymatical or nonenzymatical formation of sulfur-containing compounds produced during garlic processing were examined. Compound 12 significantly reduced the thermal decomposition of alliin, whereas compound 4 exhibited the highest percentage of alliinase inhibition activity (36.6%).

Full text of DOI:10.1021/jf505982f

Article
pubs.acs.org/JAFC
Characterization of Phenolic Constituents Inhibiting the Formation
of Sulfur-Containing Volatiles Produced during Garlic Processing
Wen-Qing Li,† Hua Zhou,*,† Mei-Yun Zhou,‡ Xing-Peng Hu,† Shi-Yi Ou,† Ri-An Yan,† Xiao-Jian Liao,‡
Xue-Song Huang,† and Liang Fu†
‡
†Department of Food Science and Engineering, and Department of Chemistry, Jinan University, Guangzhou 510632, Guangdong
China
S
* Supporting Information
ABSTRACT: Garlic (Allium sativum L.), which is a widely distributed plant, is globally used as both spice and food. This study
identified five novel phenolic compounds, namely, 8-(3-methyl-(E)-1-butenyl)diosmetin, 8-(3-methyl-(E)-1-butenyl)chrysin, 6-
(3-methyl-(E)-1-butenyl)chrysin, and Alliumones A and B, along with nine known compounds 6−14 from the ethanol extract of
garlic. The structures of these five novel phenolic compounds were established via extensive 1D- and 2D-nuclear magnetic
resonance spectroscopy experiments. The effects of the phenolic compounds isolated from garlic on the enzymatical or
nonenzymatical formation of sulfur-containing compounds produced during garlic processing were examined. Compound 12
significantly reduced the thermal decomposition of alliin, whereas compound 4 exhibited the highest percentage of alliinase
inhibition activity (36.6%).
KEYWORDS: Allium sativum, phenolic compounds, alliin, alliinase
teristics,23−26 inhibition of ACE activity.27 and binding affinity
INTRODUCTION
■
toward P-glycoprotein.28
Garlic (Allium sativum L.), which is a widely distributed plant,
Besides having various biological activities, phenolic com-
has been used as spice, food, and folk medicine worldwide for
pounds usually play important roles in food processing.29 They
hundreds of years.1,2 Garlic is a leading herbal remedy used by
easily form complexes with other food component such as
alternative medical practitioners. Garlic exhibits a wide range of
protein and lipid via hydrogen bonding interactions, leading to
therapeutic effects, such as hypolipidaemic,3 antiatheroscler-
changes in physicochemical properties of the latter such as
otic,4 antidiabetic,5 antimicrobial,6 anticarcinogen,7 and im-
solubility, thermal stability, and digestibility.30−34 Therefore,
munomodulatory8 activities.
phenolic compounds, as the major metabolites of garlic, could
The characteristic smell and health activities of garlic result
interact with alliin or alliinase during garlic processing. These
from sulfur-containing volatiles;9 these substances are formed
phenolic compounds affect the formation of S-containing
when fresh, raw garlic is chopped or crushed, thereby rupturing
the intracellular compartments that contain the nonprotein
amino acid alliin (S-allyl-L-cysteine sulfoxide), pyridoxal-5-
phosphate-dependent enzyme alliinase (L-(+)-S-alk(en)-
compounds, thereby altering the flavor and health benefits of
garlic. However, little information is available on the systematic
isolation of the phenolic compounds from garlic and the effect
of such compounds on alliin or alliinase.
This research aimed to characterize phenolic compounds
from garlic and study the effects of them on the enzymatical or
nonenzymatical formation of S-containing compounds. Four-
teen phenolic compounds that were isolated from garlic,
ylcysteine sulfoxide lyase, EC 4.4.1.4), and α, β-eliminating
endogenous lyase from Allium spp.10,11 or from alliin, which is
also a product nonenzymatically formed when garlic or alliin is
thermally processed at temperatures >100 °C.9,12−14
Aside from S-containing volatiles, garlic also contains
including five novel flavones, were investigated to determine
phenolic compounds, including the flavonoids15−17 myricetin,
the inhibitory activities of the compounds on the enzymatical
quercetin, and apigenin, as well as phenolic acids,18 such as
or nonenzymatical formation of S-containing compounds. To
gallic, chlorogenic, ferulic, o-coumaric, and cinnamic acids, the
contents of which were affected by the genotype and location of
garlic and the processing condition of “seed” cloves. For
instance, the white and Chinese garlic cultivars contained
our knowledge, this study provided the first evidence of the
functions of phenolic compounds in garlic during alliinase
catalyst reaction and thermal decomposition of alliin.
higher contents of total phenolics and ferulic acid than those of
MATERIALS AND METHODS
General Experimental Procedures. All chemicals, including
alliin (98.5%) and alliinase (EC 4.4.1.4, 1000 U/mg), were purchased
the purple garlic cultivars;18 phenolic compounds were more
■
abundant in the leaves than in the bulbs;19 and low-temperature
conditioning of garlic seed cloves increased the synthesis of
phenolic compounds, resulting in a threefold content increase
compared with those conditioned at room temperature.20 Most
phenolic compounds exhibit interesting biochemical properties,
such as anti-HIV property,21 capability to protect low-density
lipoprotein cholesterol from oxidation,22 antioxidative charac-
Received: September 28, 2014
Revised: January 6, 2015
Accepted: January 11, 2015
Published: January 11, 2015
© 2015 American Chemical Society
787
DOI: 10.1021/jf505982f
J. Agric. Food Chem. 2015, 63, 787−794

Journal of Agricultural and Food Chemistry
Article
from Aladdin Industrial Corporation (China). Distilled water was used
throughout the experiments. All the experiments were performed at
room temperature unless stated otherwise. UV/vis spectra were
obtained using a Spectrumlab 53 UV−visible spectrophotometer
(Shanghai Lengguang Technology Co., Ltd., Shanghai, China).
Nuclear magnetic resonance (NMR) spectra were acquired using a
400 MHz NMR machine (Bruker), and IR spectra were obtained using
a Fourier transform-infrared spectrophotometer (Nicolet Avatar). All
the experiments were performed in triplicate.
Plant Material. In January 2014, garlic (Allium sativum L.) was
collected from Guangzhou, which is located in the Guangdong
province of China. Voucher specimens (Accession No. 201410005)
were deposited at the herbarium of the Jinan University.
Fraction 6B was separated using a silica gel column (30 g, CH2Cl2/
MeOH [50:1]) to obtain compound 13 (10.9 mg).
8-(3-Methyl-(E)-1-butenyl)diosmetin (1). Yellow powder;
C21H20O6; m.p.194−196 °C; UV (MeOH) λmax (log ϵ) 220 (2.24),
267 (4.12) nm; IR (KBr) υmax: 3377, 2949, 1643, 1580, 1399, and 851
cm−1; 1H NMR (CD3OD, 400 MHz) δ 6.58 (1H, s, H-3), 6.24 (1H, s,
H-6), 7.39 (1H, d, J = 2.0 Hz, H-2′), 7.04 (1H, d, J = 8.4 Hz, H-5′),
7.48 (1H, dd, J = 8.4 and 2.0 Hz, H-6′), 3.94 (3H, s, OCH3), 6.54
(1H, d, J = 16.4 Hz, H-1″), 6.48 (1H, dd, J = 16.4 and 6.8 Hz, H-2″),
2.53 (1H, m, H-3″), and 1.18 (6H, d, J = 6.8 Hz, H-4″ and H-5″);
HR−ESI−MS, m/z 369.1331 [M + H]+ (calcd for C21H21O6,
369.1333).
8-(3-Methyl-(E)-1-butenyl)chrysin (2). Yellow powder; C20H18O4;
m.p.184−186 °C; UV (MeOH) λmax (log ϵ) 223 (2.38), 276 (4.49)
Extraction and Isolation. Around 25 kg of air-dried garlic was
peeled and extracted thrice with 95% ethanol (3 × 30 L) at 80 °C. The
extract was evaporated to dryness in a vacuum at 60 °C, and 1500.5 g
of the crude extract was obtained. The crude extract was purified using
an Amberlite A21 column (dimension of column: 10 cm, flow rate: 20
mL/min, eluted with 0%, 25%, 50%, 75%, 90%, and 100% ethanol,
volume of solvent: 6 × 40 L) to afford six fractions, namely, fractions
1−6. Fraction 1 contained high amounts of sugar and amino acids,
which were not further separated. Fraction 2 (10.1 g, 25% ethanol
fraction) was further separated by passing the compounds to a
Sephadex LH-20 column (dimension of column: 4 cm, flow rate: 2
mL/min, solvent: MeOH, volume of solvent: 2000 mL) to afford two
fractions, namely, fractions 2A and 2B. After evaporation of the solvent
at a reduced pressure, fraction 2A (4.2 g) was passed through a 100 g
silica gel column by using MeOH/CH2Cl2 (2:10) as the eluting
solvent to afford compound 12 (300 mg). Fraction 2B (2.4 g) was also
passed through a 50 g silica gel column, developed with PE/EtOAc
(5:0.5, 5:1, and 5:2), and monitored via thin-layer chromatography by
using PE/EtOAc (5:1) to afford compounds 8 (20 mg, Rf = 0.35) and
9 (10 mg, Rf = 0.40). Fraction 3 (14.0 g, 50% ethanol fraction) was
separated using a silica gel column (200 g) and eluted by PE/EtOAc
(5:1). Subsequently, the fraction was separated by a Sephadex LH-20
column (dimension of columns: 1 cm, flow rate: 0.2 mL/min, solvent:
MeOH, volume of solvent: 200 mL) to afford compound 10 (8.8 mg)
and another fraction (subfraction A). Subfraction A was further
purified by HPLC [250 mm × 10 mm YMC-Pack-C18 column, mobile
phase: MeOH/H2O (30:70), detection wavelength: 254 nm, flow rate:
1.0 mL/min] to obtain compound 7 (8.3 mg, Rt = 10.5 min). Fraction
4 (12.1 g, 75% ethanol fraction) was subjected to RP18 gel (200 g)
column by using MeOH−H2O (from 8:2 to 9:1) as the eluting solvent
to obtain two fractions, namely, 4A and 4B. Fraction 4A was separated
using a silica gel column and EtOAc/PE (1:5) as the eluting solvent.
Fraction 4A was then purified via preparative TLC CH2Cl2/PE(1:1) to
afford compound 1 (11 mg, Rf = 0.5). Fraction 4B was subjected to a
Sephadex LH-20 column (dimension of column: 1 cm, flow rate: 0.1
mL/min, solvent: MeOH, volume of solvent: 180 mL) and then
purified via preparative HPLC [250 mm × 10 mm YMC-Pack-C18
column, mobile phase: MeOH/H2O (20:80), detection wavelength:
254 nm, flow rate: 1.0 mL/min] to obtain compounds 4 (10 mg, Rt =
13.5 min) and 5 (11 mg, Rt = 14.8 min). Fraction 5 (25.8 g, 90%
ethanol fraction) was separated using a silica gel column and EtOAc/
PE (1:5) to afford three fractions, including 5A−5C. Fraction 5A was
separated by a Sephadex LH-20 column (dimension of column: 1 cm,
flow rate: 0.2 mL/min, solvent: CH3COCH3, volume of solvent: 170
mL) to obtain compound 14 (14.6 mg), and fraction 5B was
chromatographed using a silica gel column (400 g) with CH2Cl2/
MeOH mixtures of increasing polarity to afford compound 11 (10.4
mg). Fraction 5C was separated using a Sephadex LH-20 column
(dimension of column: 1 cm, flow rate: 0.2 mL/min, solvent: MeOH/
H2O (1:1), volume of solvent: 240 mL) to yield compound 6 (18.8
mg). Meanwhile, fraction 6 (10.2 g, 100% ethanol fraction) was
separated by silica and developed with CH2Cl2/MeOH (100:1, 100:5,
10:1) to obtain two fractions, namely, fractions 6A and 6B. Fraction
6A was purified using a silica gel column [30g, CH2Cl2/CH3COCH3
(100:1)] and further prepared via preparative TLC (EtOAc/PE (5:1)
to obtain compounds 2 (8.1 mg, Rf = 0.4) and 3 (8.4 mg, Rf = 0.34).
1
nm; IR (KBr) υmax: 3401, 2926, 1607, 1578, 1422, and 872 cm−1; H
NMR (CD3OD, 400 MHz) δ 6.61 (1H, s, H-3), 6.17 (1H, s, H-6),
7.88 (2H, d, J = 7.6 Hz, H-2′ and H-6′), 7.52 (3H, m, H-3′, H-4′, and
H-5′), 6.46 (1H, d, J = 16.4 Hz, H-1″), 6.43 (1H, dd, J = 16.4 and 6.4
Hz, H-2″), 2.46 (1H, m, H-3″), and 0.89 (6H, d, J = 6.8 Hz, H-4″ and
H-5″); HR−ESI−MS, m/z 323.1279 [M + H]+ (calcd for C20H19O4,
323.1278).
6-(3-Methyl-(E)-1-butenyl)chrysin (3). Yellow powder; C20H18O4;
m.p.188−189 °C; UV (MeOH) λmax (log ϵ) 218 (2.36), 275 (3.11)
1
nm; IR (KBr) υmax: 3363, 2924, 1644, 1580, 1379, and 850 cm−1; H
NMR (CD3OD, 400 MHz) δ 6.67 (1H, s, H-3), 6.43 (1H, s, H-8),
7.54 (2H, d, J = 6.8 Hz, H-2′ and H-6′), 7.95 (2H, dd, J = 6.8 and 2.0
Hz, H-3′ and H-5′), 7.56 (1H, d, J = 6.8 Hz, H-4′), 6.63 (1H, d, J =
16.4 Hz, H-1″), 6.68 (1H, dd, J = 16.4 and 6.4 Hz, H-2″), 2.44 (1H, m,
H-3″), 1.07 (6H, d, J = 6.8 Hz, H-4″ and H-5″); HR−ESI−MS, m/z
323.1271 [M + H]+ (calcd for C20H19O4, 323.1278).
Alliumone A (4). Yellow powder; C35H28O8; m.p.312−314 °C; UV
(MeOH) λmax (log ϵ) 216 (2.44), 277 (5.19), nm; IR (KBr) υmax
:
3469, 2924, 1647, 1580, 1397, and 845 cm−1; 1H NMR (CD3OD, 400
MHz) δ 6.61 (2H, s, H-3), 6.16 (2H, s, H-6), 7.14 (4H, dd, J = 8.0 and
7.8 Hz, H-2′ and H-6′), 7.62 (4H, d, J = 7.8 Hz, H-3′ and H-5′), 7.22
(2H, dd, J = 8.0 and 7.8 Hz, H-4′), 5.54 (1H, t, J = 8.0 Hz, H-1″), 2.12
(2H, t, J = 8.0 Hz, H-2″), 1.44 (1H, m, H-3″), 0.84 (6H, d, J = 6.4 Hz,
H-4″ and H-5″); HR−ESI−MS, m/z 577.1840 [M + H]+ (calcd for
C35H29O8, 577.1857).
Alliumone B (5). Yellow powder; C35H28O8; m.p.331−333 °C;
D
[α]20 +10.04 (c 0.001, methanol); UV (MeOH) λmax (log ϵ) 216
(2.78), 277 (5.79), and 345 (2.45) nm; IR (KBr) υmax: 3433, 2953,
1656, 1590, 1416, and 878 cm−1; 1H NMR (CD3OD, 400 MHz) unit
I: δ 6.72 (1H, s, H-3), 6.34 (1H, s, H-8), 7.59 (2H, m, H-2′ and H-6′),
8.34 (2H, dd, J = 7.8 and 1.2 Hz, H-3′ and H-5′), 7.51 (1H, m, H-4′),
5.74 (1H, t, J = 8.4 Hz, H-1″), 2.28 (1H, m, H-2″α), 2.21 (1H, m, H-
2″β), 1.44 (1H, m, H-3″), 0.92 (3H, m, H-4″), 0.93 (3H, m, H-5″);
unit II: δ 6.60 (1H, s, H-3), 6.12 (1H, s, H-6), 7.50 (2H, m, H-2′ and
H-6′), 7.94 (2H, d, J = 7.0 Hz, H-3′ and H-5′), 7.57 (1H, m, H-4′);
HR−ESI−MS, m/z 577.1857 [M + H]+ (calcd for C35H29O8,
577.1857).
Alliin and Allicin Analysis. Quantitative analyses of alliin and
allicin were performed using an Agilent 1260 infinity quaternary liquid
chromatography (Hewlett-Packard, Wilmington, NC) equipped with a
250 cm × 4.6 mm ODS-A-C18 (5 nm particle size) column. The
HPLC conditions were as follows: alliin and allicin, as mobile phase of
MeOH/H2O (20:80) and (15:85), detection wavelength of 220 and
254 nm, and flow rate of 0.8 and 1.0 mL/min, respectively.
Allicin Synthesis. Alliin was synthesized as previously described
with slight modifications.35 Diallyl disulfide was placed under vacuum
at 0 °C to remove traces of allyl disulfide. A solution of diallyl disulfide
(1.00 g, 7.0 mmol) in chloroform (30 mL) and m-chloroperbenzoic
acid (77%, 1.79 g, 8.0 mmol) in chloroform (5 mL) was added
dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 1 h.
Anhydrous sodium carbonate (8 g) was added in small portions with
vigorous stirring. The reaction mixture was stirred for an additional 0.5
h at 0 °C and then filtered through a pad of Celite and magnesium
sulfate. The filtrate was concentrated under reduced pressure to yield
0.90 g (85% yields) of crude allicin, which was purified by column
chromatography (PE: EtOAc, 80:20). 1HNMR (400 MHz,
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Figure 1. Structures of compounds 1−14.
CD3CO2D): δ 3.72−3.92 (m, 4H), 5.20−5.56 (m, 4H), 5.88−5.99 (m,
2H); 13CNMR (100 MHz, CD3CO2D): δ 35.0, 59.9, 119.1, 124.0,
125.8, 132.9 ppm.
Procedure for Allicin Decomposition. Allicin decomposition
was estimated by determining the content of allicin in the samples
according to a previously described method.35 Exactly 0.5 mg of
phenolic compound (control: no phenolic compound added) was
added into 0.5 mL of 10 mmol L−1 allicin aqueous solution at room
temperature. The aliquots were withdrawn from the reaction mixture
at a regular time interval (24 h). The resulting solution was
immediately analyzed for allicin content, after which the decom-
position percentage of allicin was calculated. Each value is the average
obtained from three runs of chromatography performed on the
sample. Allicin decomposition (%) = [(initial content of allicin −
content of allicin)/initial content of allicin] × 100%.
Thermal Decomposition of Alliin. The thermal decomposition
of alliin was estimated by determining its content in the samples
according to a previously described method.12,36 Exactly 5 mg of alliin,
1.0 mg of phenolic compounds isolated from garlic (control: no
phenolic compound added), and water were placed in a 5 mL glass
tube. The tube was then sealed and equilibrated for 24 h. After
equilibration, the tube was heated in an oven at 120 °C for 2 h, cooled
in a freezer to −18 °C, and crushed into water to a total volume of 5
mL. The resulting solution was immediately analyzed for alliin
content.10,21 The inhibition percentage was calculated as follows:
inhibition percent = [(content of alliin − content of control)/content
of control] × 100%. Triplicate measurements were performed.
Assay of Alliinase Activity. Alliinase activity was assayed by
determining the production of pyruvate from alliin according to a
previously described method with slight modifications.10 Briefly, the
alliinase activity was assayed by determining the production of allicin
from alliin. Around 0.5 mL of a 890 U mL−1 alliinase sample was
added to 1 mL of the standard reaction mixture containing 60 mmol
L−1 sodium phosphate buffer (pH = 6.5), 25 μmol L−1 pyridoxal
phosphate, 1 mmol L−1 phenolic compounds isolated from garlic
(control: no addition of phenolic compound), and 20 mmol L−1 pure
alliin as substrate. The enzymatic reaction was incubated at 25 °C for 5
min, and the reaction was terminated by adding 2 mL of 0.1 g L−1
trichloroacetic acid. The solution was centrifuged to remove the
precipitated protein, and the supernatant was assayed for pyruvate
concentration via colorimetric analysis. Around 0.5 mL of 1.0 g kg−1
2,4-dinitrophenylhydrazine was added to the supernatant and
incubated at 25 °C for 5 min. Exactly 5 mL of 0.5 mol L−1 NaOH
was then added to the solution and incubated at 25 °C for 10 min. The
absorbance at 520 nm was determined using a Spectrumlab 53 UV−
visible spectrophotometer (Shanghai Lengguang Technology Co.,
Ltd., Shanghai, China). The pyruvate concentration was calculated
according to the pyruvate standard curve, and the inhibition
percentage was calculated as follows: alliinase inhibition (%) =
[(content of control − content of pyruvate)/content of control] ×
100%. Triplicate measurements were performed.
RESULTS AND DISCUSSION
■
Identification of Compounds 1−14. Exactly 95%
aqueous ethanol extract of garlic was separated successively
by partitioning the extract using Amberlite A21 column (eluted
with 0%, 25%, 50%, 75%, 90%, and 100% ethanol) and
repeated Sephadex LH-20 columns. Gel column chromatog-
raphy afforded five new phenolic compounds 1−5 (Figure 1).
Nine known compounds, namely, diosmetin (6),37 2-(3,4-
dimethoxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (7),38 5-
hydroxy-3,7-dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-
one (8),39 5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-4H-
chromen-4-one (9),40 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-
methoxy-4H-chromen-4-one (10),41 chrysin (11),42 quercetin
(12),4,43 (E)-methyl 3-(4-hydroxy-3,5-dimethoxyphenyl)-
acrylate (13),44 and methyl-4-hydroxybenzoate (14),45 were
also obtained and identified based on their spectral data and
comparison with literature reports.
Compound 1 exhibited the molecular structure C21H20O6.
This result was inferred from the HR-EIMS, 13C NMR, and
DEPT data, which indicated 12 degrees of unsaturation. The IR
spectrum indicated the presence of hydroxyl groups (3377
cm−1) and a carbonyl group (1643 cm−1), which was
1
conjugated with an aromatic ring. The H NMR spectrum
provided signals of most functional groups, including five
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Journal of Agricultural and Food Chemistry
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aromatic protons [δH 6.58 (1H, s), 6.24 (1H, s), 7.39 (1H, d, J
= 2.0) 7.04 (1H, d, J = 8.4 Hz), and 7.48 (1H, dd, J = 8.4 and
2.0 Hz)] and a 3-methyl-1-butenyl group [6.54 (1H, d, J = 16.4
Hz, H-1″), 6.48 (1H, dd, J = 16.4 and 6.8 Hz, H-2″), 2.53 (1H,
m, H-3″), and 1.18 (6H, d, J = 6.8 Hz, H-4″ and H-5″)]. The
13C NMR spectrum (Table 1) also exhibited signals for one
compound 2. The 13C NMR spectra of 2 displayed signals for
one carbonyl group, seven aromatic methane groups, including
two symmetrical aromatic methane groups, as well as seven
aromatic quaternary C, including three oxygenated C signals at
δC 165.3, 161.1, and 165.5. Analysis of the 13C NMR of
compound 2 showed that an aromatic methoxy and two
oxygenated aromatic quaternary C signals were eliminated, and
two aromatic methane signals were added compared with those
of compound 1, which indicated that the C-3′ and C-4′
positions cannot be substituted by the methoxy and hydroxyl
groups. The results revealed that compound 2 contained a
chrysin skeleton.42 Compound 2 was deduced as an isomer of
8-prenychrysin.47 By contrast, the 13C NMR and DEPT data of
2 are similar to those of 8-prenylchrysin, except that a
downfield quaternary carbon signal and a methylene signal in
8-prenylchrysin were replaced by two methane signals in 2,
which indicated that 2 contained 3-methyl-1-butenyl group
instead of prenyl group in 8-prenylchrysin.44 In the HMBC
NMR experiments, proton δH 6.46 (H-1″) showed correlations
with C-7 (δC 165.5), C-8 (δC 106.8), and C-9 (δC 155.9),
thereby confirming that the 3-methyl-1-butenyl group was
attached at C-8. Accordingly, compound 2 was identified as
(E)-5,7-dihydroxy-8-(3-methylbut-1-enyl)-2-phenyl-4H-chro-
men-4-one, which was named as 8-(3-methyl-(E)-1-butenyl)-
chrysin.
Table 1. 13C NMR Chemical Shift Assignments (δ) of 1−5
(in CD3OD, 100 MHz)
4
5
positions
1
2
3
unit I, II
unit I
unit II
2
166.1
104.3
184.2
161.1
100.0
164.1
106.6
155.9
105.3
125.3
114.2
148.3
152.6
112.4
120.1
116.9
142.7
34.2
165.3
105.7
184.0
161.1
100.5
165.5
106.8
155.9
105.1
132.7
130.1
127.5
132.9
127.5
130.1
117.3
142.3
34.1
164.7
105.6
183.4
160.3
111.5
168.6
95.7
165.5
106.2
183.6
161.2
103.5
172.1
110.4
157.3
104.0
132.2
129.8
127.3
132.2
127.3
129.8
30.1
165.3
105.3
183.1
161.0
116.7
171.4
97.7
164.5
105.0
184.0
160.8
103.3
172.8
112.0
157.2
103.4
132.9
129.9
127.2
132.6
127.2
129.9
3
4
5
6
7
8
9
157.7
104.3
132.6
130.2
127.3
132.6
127.3
130.2
118.2
141.9
34.5
158.3
104.1
133.1
130.1
128.0
132.7
128.0
130.1
28.0
10
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
MeO
Compound 3 exhibited the same molecular formula
(C20H18O4) according to the HR−ESI−MS ([M + H]
323.1271; calcd for C20H19O4, 323.1278). The IR bands
showed the presence of OH groups (3363 cm−1). A
comparison of the NMR spectra of compounds 2 and 3
(Table 1) showed the similarity of the two compounds. The
major difference indicated that one aromatic methane signal at
δC 100.5 and an aromatic quaternary C signal at δC 106.8 in
compound 2 were replaced by that at δC 95.7 and δC 111.5 in
compound 3. This finding implied that the 3-methyl-1-butenyl
group, which substituted the position in compound 3, was
different from that in compound 2. The correlations between a
methane doublet at δH 6.63 (H-1″) and C-7 (δC 168.6), C-8
(δC 95.7), and C-9 (δC 157.7) in the HMBC spectrum
suggested that the 3-methyl-1-butenyl group was linked to a C-
6 carbon. Thus, the structure was characterized as (E)-5,7-
dihydroxy-6-(3-methylbut-1-enyl)-2-phenyl-4H-chromen-4-
one, which was named as 6-(3-methyl-(E)-1-butenyl)chrysin.
Compound 4 was isolated as a yellow powder. The molecular
formula of compound 4 was C35H28O8 according to the results
of 13C (DEPT), NMR spectra, and HR−ESI−MS ([M + H]
42.0
41.0
26.9
27.7
23.2
23.0
23.3
23.0
23.1
23.2
23.0
23.3
23.0
23.3
56.4
carbonyl group, seven aromatic methane groups, and seven
aromatic quaternary C, including at least four oxygenated C
signals at δC 155.9, 161.1, 164.1, and 166.1. This result revealed
that compound 1 contained a flavone skeleton. Compound 1
was deduced as 3-methyl-1-butenyl group-substituted diosme-
tin22 based on 2D NMR spectrum. In the HMBC spectrum, the
following long-range cross peaks (2J and 3J) were observed in a
ring: singlet at δH 6.24 (H-6) with C-5 (δC 161.1), C-7 (δC
164.1), C-8 (δC 106.6), and C-10 (δC 105.3); as well as
methane doublet at δH 6.54 (H-1″) correlated with C-7 (δC
164.1), C-8 (δC 106.6), and C-9 (δC 155.9), which indicated
that the 3-methyl-1-butenyl group was attached at C-8. An
aromatic methoxy group at δH 3.94 correlated with C-4′ (δC
152.6), and another proton at δH 6.48 (H-2′) showed cross
peaks with C-6′ (δC 120.1), C-3′ (δC 148.3), and C-4′ (δC
152.6). These results indicated the presence of a 3′-hydroxyl-4′-
methoxybenzoyl moiety. The stereochemistry at C-2″ was E
because the coupling constant between H-1″ and H-2″ was
16.4 Hz.46 Thus, the structure of compound 1 was (E)-5,7-
dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-8-(3-methylbut-1-
enyl)-4H-chromen-4-one, which was named as 8-(3-methyl-
(E)-1-butenyl) diosmetin.
1
577.1840; calcd for C35H29O8, 577.1857). The H and 13C
NMR spectral data of compound 4 were analogous to those of
compound 2. The 13C NMR spectrum showed that two signals,
which were ascribed to methane C atoms (δC = 30.1 ppm) at
C-1″ and C-2″ (δC = 42.0 ppm) in compound 4, were
observed in place of the two olefinic C atoms (δC = 117.3 and
142.3 ppm, respectively) in compound 2. The characteristic
proton signal at δH = 5.54 ppm, which was attributed to C-1″
(δC = 30.1 ppm), suggested that C-1″ was linked with two
symmetric flavone moieties that combined the molecular
formula with 13C NMR spectral data and was confirmed by
the significant HMBC cross peaks from the methane proton at
δH 5.54 to C-8 (δC = 110.4 ppm) and C-7 (δC = 172.1 ppm).
Thus, compound 4 was determined as 8,8′-(3-methylbutane-
1,1-diyl)bis(5,7-dihydroxy-2-phenyl-4H-chromen-4-one),
which is a novel natural compound named Alliumone A.
Compound 2 was obtained as a yellow amorphous powder.
The EIMS exhibited a molecular ion peak at m/z 323 [M + 1].
The elemental formula of the compound was C20H18O4, as
confirmed by the results of HR−ESI−MS ([M + H] 323.1279;
calcd for C20H19O4, 323.1278). The IR and UV data of
compound 2 were compared with those of compound 1, and
the same basic skeleton as compound 1 was observed in
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Figure 2. Proposed biosynthesis of 4 and 5.
Compound 5 was obtained as a yellow amorphous powder.
The molecular formula of compound 5 was C35H28O8
according to the 13C (DEPT), NMR spectra, and HR−ESI−
MS ([M + H] 577.1857; calcd for C35H28O8, 577.1857). These
results were the same as those for compound 4. The NMR
signals of compound 5 appeared in pairs at the same region as
those of compound 4, which suggested that compound 5 was
the nonsymmetrical isomer of compound 4. The presence of a
correlation peak from the characteristic proton signal at δH =
5.74 ppm, which was ascribed to C-1″, C-8, and C-6, in the
HMBC experiment (δC = 28.0 ppm), demonstrated that C-1″
was attached to the C-8 of unit I and C-6 of unit II. A detailed
analysis of the HMBC, H-2″(I) to C-4″(I) and C-1″(I), H-8(I)
to C-10(I) and C-7(I), H-6(II) to C-10(II) and C-8(II), H-3(I
II) to C-4(I II), C-10(I II), and C-1′(I II), as well as H-4′(I II)
to C-3′/5′(I II) and C-2′/6′(I II), facilitated the assignment of
all the protons and Cs. An S configuration of a chiral center was
Figure 3. Effect of phenolic compounds from garlic on thermal
degradation of alliin.
D
tentatively assigned according to the optical rotation [(α)20
+10.04 (c 0.001, methanol)] of compound 5 with that of (S)-1-
ferrocenyl-4-(1-cyclopentylethyl)benzene.48 In conclusion, the
structure of compound 5 was determined as (S)-8-(1-(5,7-
dihydroxy-4-oxo-2-phenyl-4H- chromen-6-yl)-3-methylbutyl)-
5,7-dihydroxy-2-phenyl-4H-chromen-4-one, which is a novel
natural compound named Alliumone B.
A plausible biogenetic pathway to 4 and 5 was proposed
(Figure 2). Compound 11 generated 8-prenylchrysin though
prenylation reaction. The prenyl group of the latter was
oxidized and then basified, leading to form 8-(3-hydroxy-3-
methyl-but-1-enyl)chrysin, which was similar to the last step of
the formation of hyperualones B.49 The resulting compound
was reduced to generate 2, which further produced the
electrophilic addition reaction at the C(1″)= C(2″) bond
with C-8 of 11 via a or C-6 of 11 via b (Figure 2), thereby
forming 4 and 5, respectively.
Effect of Phenolic Compounds on Thermal Degradation
of Alliin. Previous research reported that alliin is self-degraded
or thermally decomposed to generate volatile S-containing
compounds.12−14 These compounds are generated from alliin
via hydrolysis or free-radical rearrangement.14 Most phenolic
compounds are strong radical scavengers; thus, we investigated
the effects of phenolic compounds isolated from garlic on the
thermal decomposition of alliin (Figure 3). The phenolic
compounds positively affected the inhibition of the thermal
degradation of alliin, in which addition of flavones, such as
compounds 11 and 12, led to stronger inhibition than that of
other phenolic compounds, including 13 and 14. Compound
12 exhibited a maximum inhibition of about 51% compared
with compound 14, which exhibited 5.2% inhibition.
The hydroxyl group in the flavones affected the inhibition
ability of the compounds. Compound 11 exhibited more
potential inhibition ability than those of compounds 9 and 10
because the latter compounds lacked a hydroxyl substituent at
the 7-position of the A ring. This result suggested that the
hydroxyl group substituent at the 7-position of the A ring can
increase the inhibition ability of the compound. The 3-methyl-
1-butenyl group substituent on the flavone skeleton also
increased the inhibition capability of the flavone, particularly
when the substituent was located at the 8-position of the A ring.
This result was exhibited by compounds 2, 3, and 11.
Compounds 2 and 3 exhibited one 3-methyl-1-butenyl group
substituent at the 8- and 6-positions, respectively, whereas
compound 11 lacked a 3-methyl-1-butenyl group substituent in
the skeleton. Thermal degradation assay results showed that
compounds 2 and 3 exhibited stronger inhibition ability than
that of compound 11. Compound 2 also exhibited better
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Journal of Agricultural and Food Chemistry
Article
inhibition activity than compound 3, which suggested that the
presence of a 3-methyl-1-butenyl group at the 8-position in the
skeleton caused stronger inhibition ability relative to that with a
3-methyl-1-butenyl group at the 6-position.
The above results indicated that most flavones clearly
exhibited strong inhibition during the alliin degradation. This
finding was expected because the flavones behaved as strong
radical scavengers, which prevented free radical reaction during
the thermal process. The 3-methyl-1-butenyl group substituent,
which behaved as an electron-donating substituent at the ring,
also decreased the vertical ionization potentials, thereby
resulting in better antioxidant activity.50 This behavior can
strongly inhibit the thermal degradation of alliin.
Effect of Phenolic Compounds on Alliinase Activity. The
alliinase catalyst reaction was affected by the temperature, pH,
concentration, solvent, and presence of additives.8,9,51,52
However, the functions of the phenolic compounds, which
are major secondary metabolites of garlic in the alliinase
activity, remain unknown. Figure 4 shows the effects of 14
Figure 5. Decomposition percentage of allicin with phenolic
compounds 4 and 12. Each value is the average obtained from three
runs of chromatography performed on the sample.
In the present study, five novel flavones, namely, 8-(3-
methyl-(E)-1-butenyl)diosmetin, 8-(3-methyl-(E)-1-butenyl)-
chrysin, 6-(3-methyl-(E)-1-butenyl)chrysin, and Alliumones A
and B, together with nine known compounds 6−14, were
identified from the ethanol extract of garlic. To our knowledge,
this research is the first systematic report on phenolic
compounds from garlic and thus provides the first evidence
of the effects of phenolic compounds in garlic on the
enzymatical or nonenzymatical formation of S-containing
compounds. This study demonstrated that compounds 12
exhibited the strongest inhibition ability for the thermal
degradation of alliin (51%), and biflavone 4 showed the
highest percentage of alliinase inhibition activity (36.6%). The
results may serve as important references for garlic processing.
Figure 4. Alliinase inhibition activity of phenolic compounds isolated
from garlic.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, 13C (DEPT) NMR, HSQC, HMBC, and H−1H
1
phenolic compounds isolated from garlic on the alliinase
activity. The results indicated that flavones with more hydroxyls
(e.g., compound 12) exhibited higher inhibitory activity than
that with more methoxyls (e.g., compounds 8 and 9). Biflavone
4 also showed the highest percentage of alliinase inhibition
activity at 36.6%. Moreover, compound 5 exhibited 32.7%
alliinase inhibition activity. The alliinase inhibition activities of
compounds 4 and 5 were correspondingly 3.6 and 3.2 times
more potent than that of compound 11. These results implied
that the efficiency of flavones as inhibitors of alliinase depended
largely on their chemical structures, relative orientation, and
number of hydroxyl groups attached to the aromatic ring.
In addition to interactions with alliinase and alliin, phenolics
may also interact with allicin as the reactive species formed
upon the alliinase cleavage of alliin. Allicin is very unstable and
thus easily decomposes into S-containing volatiles. Phenolics
could affect allicin stability. Therefore, two phenolics 4 and 12,
which respectively showed strong interaction with alliinase and
alliin, were selected to evaluate their effects on allicin stability
(Figure 5). After 144 h of the test, the addition of phenolics 4
and 12 caused the decomposition percentage of allicin to reach
47% and 48%, respectively, compared with the control at 52%.
This finding is consistent with that obtained by Fujisawa.53 This
result implies that the addition of phenolics slightly promoted
allicin stability at room temperature.
COSY spectra and HR−ESI−MS data of compounds 1−5 and
1H and 13C NMR data of compounds 6−14. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +8620-85226630. Fax: +8620-85226630. E-mail:
zhouhua5460@jnu.edu.cn.
■
Funding
This work was financially supported by the NSFC of China
(Nos. 31101323 and 31000816) and the high-performance
computing platform of Jinan University.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
PE, petroleum ether; EtOAc, ethyl acetate; Rf, retention factor;
Rt, retention time; HIV, human immunodeficiency virus; ACE,
angiotensin 1 converting enzyme; TLC, thin-layer chromatog-
raphy
REFERENCES
■
(1) Eric, B. Garlic and Other Alliums: The Lore and the Science; Royal
Society of Chemistry: Cambridge, U.K., 2010.
792
DOI: 10.1021/jf505982f
J. Agric. Food Chem. 2015, 63, 787−794

Journal of Agricultural and Food Chemistry
Article
(2) Koch, H. P.; Lawson, L. D. Garlic. The Science and Therapeutic
Application of Allium sativum L. and Related Species, 2nd ed.; Williams
& Wilkens: Baltimore, MD, 1996.
(3) Sher, A.; Fakhar-ul-Mahmood, M.; Shah Syed Nisar, H.; Bukhsh,
S.; Murtaza, G. Effect of garlic extract on blood glucose level and lipid
profile in normal and alloxan diabetic rabbits. Adv. Clin. Exp. Med.
2013, 21, 705−711.
(4) Ide, N.; Lau, B. H. S. Garlic compounds minimize intracellular
oxidative stress and inhibit nuclear factor-κB activation. J. Nutr. 2001,
131, 1020S−1026S.
(5) Padiya, R.; Banerjee, S. K. Garlic as an anti-diabetic agent: Recent
progress and patent reviews. Recent Pat. Food, Nutr. Agric. 2013, 5,
105−127.
(6) Marques, A.; Encarnacao, S.; Pedro, S.; Nunes, M. L. In vitro
antimicrobial activity of garlic, oregano and chitosan against
Salmonella enterica. World J. Microbiol. Biotechnol. 2008, 24, 2357−
2360.
(7) Modem, S.; Di Carlo, S. E.; Reddy, T. R. Fresh garlic extract
induces growth arrest and morphological differentiation of MCF7
breast cancer cells. Genes Cancer 2014, 3, 177−186.
(8) Schafer, G.; Kaschula, C. H. The immunomodulation and anti-
inflammatory effects of garlic organosulfur compounds in cancer
chemoprevention. Anti-Cancer Agents Med. Chem. 2014, 14, 233−240.
(9) Zhang, M.; Lei, N.; Zhu, T.; Zhang, Z. Thermal processing effects
on the chemical constituent and antioxidant activity of S-alk(en)-
ylcysteine S-oxides (alliin) extract. LWTFood Sci. Technol. 2012, 51,
309−313.
(10) Wang, J.; Cao, Y.; Wang, C.; Sun, B. Low-frequency and low-
intensity ultrasound accelerates alliinase-catalyzed synthesis of allicin in
freshly crushed garlic. J. Sci. Food Agric. 2011, 91, 1766−1772.
(11) Wang, J.; Cao, Y.; Sun, B.; Wang, C.; Mo, Y. Effect of ultrasound
on the activity of alliinase from fresh garlic. Ultrason. Sonochem. 2010,
18, 534−540.
(21) Murata, R. M.; Pasetto, S.; Pardi, V. The anti-HIV activity of
flavonoids: Quercetin and Pinocembrin. Planta Med. 2014, 80, 811−
811.
(22) Gong, M. K.; Garige, M.; Varatharajalu, R.; Marmillot, P.;
Gottipatti, C.; Leckey, L. C.; Lakshman, R. M. Quercetin up-regulates
paraoxonase 1 gene expression with concomitant protection against
LDL oxidation. Biochem. Biophys. Res. Commum. 2009, 379, 1001−
1004.
(23) Lu, X.; Ross, C. F.; Powers, J. R.; Aston, D. E.; Rasco, B. A.
Determination of total phenolic content and antioxidant activity of
garlic (Allium sativum) and elephant garlic by attenuated total
reflectance-fourier transformed infrared spectroscopy. J. Agric. Food
Chem. 2012, 59, 5215−5221.
(24) Chen, S. X.; Shen, X. Q.; Cheng, S. Q.; Li, P. P.; Du, J. N.;
Chang, Y. X.; Meng, H. W. Evaluation of garlic cultivars for
polyphenolic content and antioxidant properties. PLoS One 2013, 8,
1−12.
(25) Bozin, B.; Mimica-Dukic, N.; Samojlik, I.; Goran, A.; Igic, R.
Phenolics as antioxidants in garlic (Allium sativum L., Alliaceae). Food
Chem. 2008, 111, 925−929.
(26) Du, J.; Chen, S.; Cheng, Z.; Chang, Y.; Zhou, J. Response
surface methodology for optimization of extraction conditions of total
phenolic compounds from garlic and determination of antioxidant
activity. Food Sci. 2012, 33, 72−77.
(27) Oboh, G.; Akinyemi, A. J.; Ademiluyi, A. O. Inhibitory effect of
phenolic extract from garlic on angiotensin-1 converting enzyme and
cisplatin induced lipid peroxidation-in vitro. Int. J. Biomed. Sci. 2013, 9,
98−106.
(28) Comte, G.; Daskiewicz, J. B.; Bayet, C.; Conseil, G.; Viornery-
Vanier, A.; Dumontet, C.; Di Pietro, A.; Barron, D. C-isoprenylation of
flavonoids enhances binding affinity toward P-glycoprotein and
modulation of cancer cell chemoresistance. J. Med. Chem. 2001, 44,
763−768.
(29) Ozdal, T.; Esra, C.; Altay, F. A review on protein−phenolic
interactions and associated changes. Food Res. Int. 2013, 51, 954−970.
(30) Jung, D. M.; Ropp, J. S. D.; Ebeler, S. E. Study of interactions
between food phenolics and aromatic flavors using one- and two-
(12) Kubec, R.; Velisek, J.; Dolezal, M.; Kubelka, V. Sulfur-containing
volatiles arising by thermal degradation of alliin and deoxyalliin. J.
Agric. Food Chem. 1997, 45, 3580−3585.
(13) Yu, T. H.; Wu, C. M.; Ho, C. T. Meat-like flavor generated from
thermal interactions of glucose and alliin or deoxyalliin. J. Agric. Food
Chem. 1994, 42, 1005−1009.
(14) Yu, T. H.; Wu, C. M.; Rosen, R. T.; Hartman, T. G.; Ho, C. T.
Volatile compounds generated from thermal degradation of alliin and
deoxyalliin in an aqueous solution. J. Agric. Food Chem. 1994, 42, 146−
153.
1
dimensional H NMR spectroscopy. J. Agric. Food Chem. 2000, 48,
407−412.
(31) Viljanen, K.; Kivikari, R.; Heinonen, M. Lipid interactions
during liposome oxidation with added anthocyanin and other phenolic
compound. J. Agric. Food Chem. 2004, 52, 1104−1111.
(32) Tramer, Z.; Shugar, D. Studies on phenolic hydroxyl binding in
proteins. Acta Biochim. Polym. 1959, 6, 235−251.
(33) Hassan, Z. M. R.; Fakhr El Din, H. M.; Ali, A. A.; Mehanna, N.
S.; El-Messery, T. M. Interaction of some low molecular weight
phenolics with milk proteins. World Appl. Sci. J. 2013, 23, 182−187.
(34) Alu’datt, M. H.; Rababah, T.; Ereifej, K.; Brewer, S.; Alli, I.
Phenolic−protein interactions in oilseed protein isolates. Food Res. Int.
2013, 52, 178−184.
(35) Vaidya, V.; Ungold, K. U.; Pratt, D. A. Garlic: Source of the
ultimate antioxidants−sulfenic acids. Angew. Chem., Int. Ed. 2009, 48,
157−160.
(36) Yu, T. H.; Shu, C. K.; Ho, C. T. Thermal decomposition of
alliin, the major flavor component of garlic, in an aqueous solution.
ACS Symp. Ser. 1994, 546, 144−152.
(37) Park, Y.; Moon, B. H.; Yang, H.; Lee, Y.; Lee, E.; Lim, Y.
Spectral assignments and reference data complete assignments of
NMR data of 13 hydroxymethoxy-flavones. Magn. Reson. Chem. 2007,
45, 1072−1075.
(38) Seijas, J. A.; Vazquez-Tato, M. P.; Carballido-Reboredo, R.
Solvent-free synthesis of functionalized flavones under microwave
irradiation. J. Org. Chem. 2005, 70, 2855−2858.
(39) Lu, K.; Chu, J.; Wang, H.; Fu, X.; Quan, D.; Ding, H.; Yao, Q.;
Yu, P. Regioselective iodination of flavonoids by N-iodosuccinimide
under neutral conditions. Tetrahedron Lett. 2013, 54, 6345−6348.
(40) Kim, J.; Park, K.-S.; Lee, C.; Chong, Y. Synthesis of a complete
series of O-methyl analogues of naringenin and apigenin. Bull. Korean
Chem. Soc. 2007, 28, 2527−2530.
(15) Lanzotti, V. The analysis of onion and garlic. J. Chromatogr., A
2006, 1112, 3−22.
(16) Kim, J. S.; Kang, O. J.; Gweon, O. C. Comparison of phenolic
acids and flavonoids in black garlic at different thermal processing
steps. J. Funct. Foods 2012, 5, 80−86.
(17) Isabel Alarcon-Flores, M.; Romero-Gonzalez, R.; Martinez
Vidal, J. L.; Garrido Frenich, A. Determination of phenolic compounds
in artichoke, garlic and spinach by ultra-high- performance liquid
chromatography coupled to tandem mass spectrometry. Food Anal.
Method 2014, 7, 2095−2106.
(18) Manuel Beato, V.; Orgaz, F.; Mansilla, F.; Montano, A. Changes
in phenolic compounds in garlic (Allium sativum l.) owing to the
cultivar and location of growth. Plant Foods Hum. Nutr. 2011, 66,
218−223.
(19) Tuan, P. A.; Li, X.; Park, N. I.; Lee, S. Y.; Kim, H. H.; Park, S. U.
Molecular cloning of 4-coumarate: CoA ligase and total phenolic
content in garlic (Allium sativum). Plant Omics 2011, 4, 25−28.
(20) Dufoo-Hurtado, M. D.; Zavala-Gutierrez, K. G.; Cao, C. M.;
Cisneros-Zevallos, L.; Guevara-Gonzalez, R. G.; Torres-Pacheco, I.;
Vazquez-Barrios, M. E.; Rivera-Pastrana, D. M.; Mercado-Silva, E. M.
Low-temperature conditioning of “seed” cloves enhances the
expression of phenolic metabolism related genes and anthocyanin
content in “coreano” garlic (Allium sativum) during plant develop-
ment. J. Agric. Food Chem. 2013, 61, 10439−10446.
793
DOI: 10.1021/jf505982f
J. Agric. Food Chem. 2015, 63, 787−794

Journal of Agricultural and Food Chemistry
Article
(41) Nguyen, T. K. P.; Nguyen, K. P. P.; Kamounah, F. S.; Zhang,
W.; Hansen, P. E. NMR of a series of novel hydroxyflavothiones.
Magn. Reson. Chem. 2009, 47, 1043−1054.
(42) Pandurangan, N. A new synthesis for acacetin, chrysoeriol,
diosmetin, tricin and other hydroxylated flavones by modified baker-
venkataraman transformation. Lett. Org. Chem. 2014, 11, 225−229.
(43) Lopes, R. M.; Agostini-Costa, T. d. S.; Gimenes, M. A.; Silveira,
D. Chemical composition and biological activities of Arachis species. J.
Agric. Food Chem. 2011, 59, 4321−4330.
(44) Kim, H. J.; Chen, F.; Choi, J. H.; Wang, X.; Jiang, Y.
Identification and evaluation of antioxidant phenolic compounds in
parsley (Petroselinum crispum neapolitanum) and radish (Raphanus
sativus L.) sprout. ACS Symp. Ser. 2008, 993, 165−180.
(45) Deleon, S.; Hope, P. H.; Macias, C. Synthesis of p-
hydroxybenzoic acid and its ester. Rev. Soc. Quim. Mex. 1964, 8,
13−18.
(46) Zheng, Z. P.; Xu, Y.; Qin, Q.; Zhang, S.; Gu, X. H.; Lin, Y. Y.;
Xie, G..B.; Wang, M. F.; Chen, J. Characterization of antiproliferative
activity constituents from Artocarpus heterophyllus. J. Agric. Food Chem.
2014, 62, 5519−5527.
(47) Daskiewicz, J. B.; Bayet, C.; Barron, D. Regioselective syntheses
of 6-(1,1-dimethylallyl)chrysin and 8-(3,3-dimethylallyl)chrysin. Tetra-
hedron 2002, 58, 3589−3595.
(48) Binder, J. T.; Cordier, C. J.; Fu, G. C. Catalytic enantioselective
cross-couplings of secondary alkyl electrophiles with secondary
alkylmetal nucleophiles: Negishi reactions of racemic benzylic
bromides with achiral alkylzinc reagents. J. Am. Chem. Soc. 2012,
134, 17003−17006.
(49) Zhang, J. J.; Yang, J.; Liao, Y.; Yang, X. W.; Ma, J. Z.; Xiao, Q. L.;
Yang, L. X.; Xu, G. Hyperuralones A and B, new acylphloroglucinol
derivatives with intricately caged cores from Hypericum uralum. Org.
Lett. 2014, 16, 4912−4915.
(50) Reis, M.; Lobato, B.; Lameira, J.; Santos, A. S.; Alves, C. N. A
theoretical study of phenolic compounds with antioxidant properties.
Eur. J. Med. Chem. 2007, 42, 440−446.
(51) Kim, K. W.; Kim, Y. T.; Kim, M.; Noh, B. S.; Choi, W. S. Effect
of high hydrostatic pressure (HHP) treatment on flavor, phys-
icochemical properties, and biological functionalities of garlic. LWT
Food Sci. Technol. 2013, 55, 347−354.
(52) Anifantaki, E.; Touloupakis, E.; Ghanotakis, D. F. Alliinase
immobilization in calcium alginate beads and layered double
hydroxides matrices. J. Food Biochem. 2012, 36, 12−20.
(53) Fujisawa, H.; Suma, K.; Origuchi, K.; Kumagai, H.; Seki, T.;
Ariga, T. Biological and chemical stability of garlic-derived allicin. J.
Agric. Food Chem. 2008, 56, 4229−4235.
794
DOI: 10.1021/jf505982f
J. Agric. Food Chem. 2015, 63, 787−794