Isolation and structure of whiskey polyphenols produced by oxidation of oak wood ellagitannins

Article abstract of DOI:10.1021/jf8012713

Three new phenolic compounds named whiskey tannins A and B and carboxyl ellagic acid were isolated from commercial Japanese whiskey, along with gallic acid, ellagic acid, brevifolin carboxylic acid, three galloyl glucoses, a galloyl ester of phenolic glucoside, 2,3-(S)-hexahydroxydiphenoylglucose, and castacrenin B. Whiskey tannins A and B were oxidation products of a major oak wood ellagitannin, castalagin, in which the pyrogallol ring at the glucose C-1 position of castalagin was oxidized to a cyclopentenone moiety. These tannins originated from ellagitannins contained in the oak wood used for barrel production; however, the original oak wood ellagitannins were not detected in the whiskey. To examine whether the whiskey tannins were produced during the charring process of barrel production, pyrolysis products of castalagin were investigated. Dehydrocastalagin and a new phenolcarboxylic acid trislactone having an isocoumarin structure were isolated, along with castacrenin F and ellagic acid. However, whiskey tannins were not detected in the products.

Full text of DOI:10.1021/jf8012713

J. Agric. Food Chem. 2008, 56, 7305–7310 7305
Isolation and Structure of Whiskey Polyphenols
Produced by Oxidation of Oak Wood Ellagitannins
MIHO FUJIEDA,† TAKASHI TANAKA,*,† YOSHIHIDE SUWA,‡ SEIICHI KOSHIMIZU,§
†
AND ISAO KOUNO
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852-8521, Japan; Research Center, Suntory Limited, 1-1-1 Wakayamadai Shimamoto-cho,
Mishima-gun, Osaka 616-8503, Japan; and Whisky Blending and Planning Department, Suntory
Limited, 5-2-1 Yamazaki, Wakayamadai Shimamoto-cho, Mishima-gun, Osaka 616-0001, Japan
Three new phenolic compounds named whiskey tannins A and B and carboxyl ellagic acid were
isolated from commercial Japanese whiskey, along with gallic acid, ellagic acid, brevifolin
carboxylic acid, three galloyl glucoses, a galloyl ester of phenolic glucoside, 2,3-(S)-hexahy-
droxydiphenoylglucose, and castacrenin B. Whiskey tannins A and B were oxidation products of
a major oak wood ellagitannin, castalagin, in which the pyrogallol ring at the glucose C-1 position
of castalagin was oxidized to a cyclopentenone moiety. These tannins originated from ellagitannins
contained in the oak wood used for barrel production; however, the original oak wood ellagitannins
were not detected in the whiskey. To examine whether the whiskey tannins were produced during
the charring process of barrel production, pyrolysis products of castalagin were investigated.
Dehydrocastalagin and a new phenolcarboxylic acid trislactone having an isocoumarin structure
were isolated, along with castacrenin F and ellagic acid. However, whiskey tannins were not
detected in the products.
KEYWORDS: Ellagitannin; castalagin; oak; oxidation; whiskey
INTRODUCTION
as a mimic of decomposition during the charring process in
barrel production and the products were characterized.
In whiskey production, distilled spirits are aged for several
years in oak barrels, and the constituents of the wood dissolve
into the spirit to determine its color, flavor, and taste.
Although oak woods contains C-glycosidic ellagitannins, such
as castalagin, vescalagin, grandinin, and roburins A-E (1),
the polyphenols in whiskey are different from the original
oak wood tannins. First, tannins decompose during the
toasting or charring process in barrel production, in which
the inside of the barrel is scorched with fire (2). In addition,
during the aging process, oxygen molecules penetrate into
the spirits through the barrel wood and oxidize the solutes.
Therefore, the polyphenols in whiskey are a mixture of
products generated through a complex chemical process. To
clarify the structures of polyphenols in commercial whiskey,
the present study first compared the polyphenol concentration
of some commercial bottled Japanese whiskey, and then
polyphenols were isolated from the whiskey with the highest
polyphenol content. In addition, pyrolysis of castalagin, the
major C-glycosidic ellagitannin of oak wood, was examined
MATERIALS AND METHODS
Materials. Castalagin was isolated as a white microcrystalline
powder from Japanese chestnut heart wood (Castanea crenata, Fa-
gaceae) (3). Commercial pure malt whiskey was supplied through the
courtesy of Suntory Limited, Osaka, Japan.
General Procedures. Ultraviolet (UV) spectra were obtained with
a JASCO V-560 UV-vis spectrophotometer (JASCO Co., Tokyo,
Japan), and optical rotations were measured with a JASCO DIP-370
digital polarimeter. 1H, 13C NMR, 1H-1H correlation spectroscopy
(COSY), 1H NMR nuclear Overhauser and exchange spectroscopy
(NOESY), heteronuclear single-quantum coherence (HSQC), and
heteronuclear multiple-bond connectivity (HMBC) spectra were re-
corded in acetone-d6, MeOH-d4, DMSO, and a mixture of acetone-d6/
D2O (0.8 mL/2 drops) at 27 °C with a Varian Unity Plus 500
spectrometer (Varian, Inc., Palo Alto, CA) operating at 500 MHz for
1H and at 125 MHz for 13C. Coupling constants are expressed in hertz,
and chemical shifts are given on a δ (parts per million) scale. HMQC
(JCH ) 140 Hz), HMBC (nJCH optimized for 8 Hz), and NOESY (mixing
time ) 0.50 s) experiments were performed using standard Varian pulse
sequences. Mass spectra were recorded on a JEOL JMS-700N
spectrometer (JEOL Ltd., Tokyo, Japan) or an Voyager-DE PRO
(Applied Biosystems, Foster City, CA); glycerol or m-nitrobenzyl
alcohol was used as the matrix for fast atom bombardment mass
spectroscopy (FAB-MS) measurements, and 2,5-dihydroxybenzoic acid
* Author to whom correspondence should be addressed [telephone
81 (0)95 819 2433; fax 81 (0)95 819 2477; e-mail t-tanaka@
nagasaki-u.ac.jp].
† Nagasaki University.
‡ Research Center, Suntory Limited.
§ Whisky Blending and Planning Department, Suntory Limited.
10.1021/jf8012713 CCC: $40.75  2008 American Chemical Society
Published on Web 08/02/2008

7306 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Fujieda et al.
Figure 1. Structures of compounds 1-7.
was used as the matrix for matrix-assisted laser desorption ionization
MS (MALDI-TOF-MS).
EtOAc-soluble fraction (2.7 g) was suspended in 50% MeOH, and
insoluble precipitates of 2 (156 mg) were collected by filtration; the
filtrate was applied to an MCI gel CHP-20P column (35 cm × 3.5 cm
i.d., 0-100% MeOH) and a Chromatorex ODS column (20 cm × 1.5
cm i.d., 0-40% MeOH) to give lyoniresinol (6) (42 mg). The water-
soluble fraction was subjected to MCI gel CHP20P column chroma-
tography (25 cm × 3.5 cm i.d.) with H2O, which contained increasing
proportions of MeOH (10% stepwise elution from 0 to 100%, each
200 mL), to give nine fractions. Fraction 1, mainly composed of sugars,
was further fractionated by Sephadex LH-20 column chromatography
(20 cm × 3 cm i.d.) with H2O/MeOH (0-40%, 10% stepwise, and
then 40-100%, 20% stepwise, each 200 mL) to give fraction 1-1
(sugars and related compounds), gallic acid (53 mg), and fraction 1-2
(851 mg). Fraction 1-2 was applied to a Chromatorex ODS column
(25 cm × 3 cm i.d.) with 0-40% MeOH (5% stepwise elution, each
200 mL) to give 6-O-galloyl glucose (7) (122 mg) and a mixture of
3-O-galloyl glucose and 2,3-(S)-hexahydroxydiphenoyl (HHDP)-D-
glucose, which was purified by chromatography on a Toyopearl HW40F
column (17 cm × 1 cm i.d.) with 0-5% MeOH (5% stepwise elution,
each 100 mL) to give 3-O-galloyl glucose (8) (33 mg) and 2,3-(S)-
HHDP-D-glucose (9) (6 mg). Fraction 3 (663 mg) was separated by
Sephadex LH-20 chromatography (20 cm × 2.5 cm i.d.) with 0-100%
MeOH (0-40%, 5% stepwise, and then 40-100%, 10% stepwise each
100 mL) to give 2,3-di-O-galloyl glucose (10) (7.6 mg) and a fraction
containing whiskey tannin A (3), which was finally purified by
Chromatorex ODS chromatography (18 cm × 2 cm i.d.) with 0-30%
MeOH (5% stepwise, each 100 mL) to give 3 (21.8 mg). Fraction 4
was successively subjected to Sephadex LH-20 (0-100% MeOH),
Avicel cellulose (2% AcOH), and MCI gel CHP-20P (0-20% MeOH)
column chromatography to give brevifolin carboxylic acid (5.0 mg)
(11). Fraction 5 (961 mg) was separated by Sephadex LH-20 (22 cm
× 2.5 cm i.d., with 0-100% MeOH), Avicel cellulose (12 cm × 1.5
cm i.d., 2% AcOH), MCI gel CHP-20P (15 cm × 1.0 cm i.d., 0-100%
MeOH), and Toyopearl HW40F (25 cm × 3.0 cm i.d., 0-100% MeOH)
chromatography to give brevifolin carboxylic acid (1.6 mg), 3-methoxy-
4-hydroxyphenol 1-O-(6′-O-galloyl)-ꢀ-D-glucopyranoside (12) (4.0 mg),
carboxyl ellagic acid (1) (5.9 mg), whiskey tannin B (4) (7.1 mg), and
castacrenin B (7) (3) (4.8 mg). Fraction 8 was separated by Sephadex
LH-20 column chromatography (23 cm × 3.0 cm i.d., 0-100% MeOH)
Column chromatography was performed using a Diaion HP20SS,
MCI gel CHP20P (75-150 µm) (Mitsubishi Chemical Co., Tokyo,
Japan), Sephadex LH-20 (25-100 µm) (GE Healthcare Bio-Sciences,
Uppsala, Sweden), TSK gel Toyopearl HW-40F (TOSOH Co. Tokyo,
Japan), Funacel microcrystalline cellulose (Funakoshi, Co., Tokyo,
Japan), and Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai,
Japan). Thin-layer chromatography (TLC) was performed on 0.2 mm
precoated Kiesel gel 60 F254 plates (Merck Ltd. Japan, Tokyo, Japan)
with toluene/ethyl formate/formic acid (1:7:1, v/v) or chloroform/
methanol/water (14:6:1, v/v). TLC was also performed on a 0.1 mm
precoated Cellulose F plate (Merck) with 2% acetic acid. Spots were
detected by UV illumination and by spraying with 2% ethanolic FeCl3
or 10% sulfuric acid reagent, followed by heating. Analytical HPLC
was performed on a 250 × 4.6 mm i.d. Cosmosil 5C18-AR II column
(Nacalai Tesque, Inc. Kyoto, Japan) with gradient elution of 4-30%
(39 min) and 30-75% (15 min) of CH3CN in 50 mM H3PO4 (flow
rate, 0.8 mL/min; detection, JASCO photodiode array detector MD-
910).
Measurement of Total Polyphenol. Total polyphenol concentrations
of four commercial whiskeys were compared by the Folin-Ciocalteu
method (4). The whiskeys were diluted 5, 10, 25, and 50 times, and
each sample solution (0.5 mL) was mixed with water (1.5 mL) and
phenol reagent (0.5 mL) (Kanto Chemical Co., Tokyo, Japan) and kept
at room temperature for 3 min. To the mixture was added 10% Na2CO3
(0.5 mL), and after 1 h, absorption at 700 nm was measured. A
calibration curve was made using recrystallized gallic acid as
standard.
Fractionation of Whiskey. Commercial whiskey (12 L), a pure malt
whiskey produced by Suntory Limited, Japan, was directly applied to
a Diaion HP20SS column (40 cm × 5 cm i.d.). Most of the phenolic
compounds passed through the column, and nonpolar compounds
adsorbed on the gel were eluted with MeOH to give a solid (3.1 g).
The first fraction that contained phenolic compounds was concentrated
by a rotary evaporator to give a brown syrup (33.6 g), which was
successively partitioned with Et2O and EtOAc to give an Et2O fraction
(1.35 g), an EtOAc fraction (2.7 g), and a water-soluble fraction (26.0
g). The Et2O fraction gave precipitates when dissolved in MeOH, which
was collected by filtration (47.3 mg) and identified as ellagic acid (2)
(Figure 1) by co-HPLC and comparison of UV absorption (5). The

Structure of Whiskey Polyphenols
J. Agric. Food Chem., Vol. 56, No. 16, 2008 7307
1
Table 1. 13C (125 MHz) and H (500 MHz) NMR Data for 3 and 4a
3
4
position
1H
13C
1H
13C
glucose
1
2
3
4
5
6
5.35 (dd, 3.0, 6.0)
5.22 (br d, 6.0)
5.85 (br d, 7.1)
4.29 (dd, 7.1, 9.2)
4.95 (ddd, 3.2, 4.6, 9.2)
3.89 (dd, 4.6, 12.4)
3.80 (dd, 3.2, 12.4)
63.1
77.0
69.4
68.4
75.1
62.2
5.45 (dd, 3.2, 6.4)
4.99 (d, 6.4)
5.97 (d, 7.8)
62.8
76.5
66.4
69.6
70.6
64.8
5.42 (t, 7.8)
5.45 (ddd, 2.5, 4.1, 7.8)
4.92 (dd, 4.1, 12.6)
3.97 (dd, 2.5, 12.6)
Cp
1
2
3
4
5
6
7
154.3
44.5
83.7
200.2
142.6
163.1
170.6
154.3
44.6
82.8
200.0
142.7
162.4
170.7
5.34 (d, 3.0)
5.39 (d, 3.2)
BPH
1, 1′
2, 2′
3, 3′
4, 4′
5, 5′
6
125.6, 125.7
113.6, 114.7
144.5, 144.3b
135.3, 136.6
145.8, 145.5b
110.7
124.9-126.3
113.7, 114.3
144.4-145.8
135.1, 137.1
144.4-145.8
110.4
6′
6.70 (s)
108.2
167.2
168.6
6.70 (s)
108.2
166.2
167.5
7
7′
HHDP
1, 1′
2, 2′
3, 3′
4, 4′
5, 5′
6
6′
7
7′
115.1, 115.9
124.9-126.3
144.4-145.8
136.1, 136.8
144.4-145.8
108.3
107.6
167.6
168.7
6.83 (s)
6.63 (s)
-OCH2
-CH3
4.18 (dq, 7.1, 14.2)
4.17 (dq, 7.1, 14.2)
63.0
14.1
4.22 (dq, 7.1, 10.8)
4.17 (dq, 7.1, 10.8)
63.0
14.2
1.18 (3H, t, 7.1)
1.20 (3H, t, 7.1)
a Measured in acetone-d6 + D2O, δ in ppm, J in hertz. b Assignments may be interchanged within the same column.
to give ellagic acid (889 mg). Known compounds were identified by
spectroscopic comparison with authentic samples or data described in
the literature.
Carboxyl ellagic acid (1): yellow powder, FAB-MS m/z 347 [M +
H]+, 369 [M + Na]+, 385 [M + K]+; high-resolution (HR)-FAB-MS
347.0030 [M + H]+, (C15H7O10 requires 347.0038); UV λmax nm (log
1
ꢀ) 368 (4.06), 254 (4.69); H NMR (DMSO-d6) δ 7.47 (1H, s, H-5);
13C NMR (DMSO-d6) δ 166.8 (C-8), 159.0 (C-7′), 157.7 (C-7), 148.3
(C-4, 4′), 145.2 (C-3), 139.5 (C-3′), 136.5 (C-2′), 136.1 (C-2), 121.2
(C-5), 112.12 (C-6), 112.07 (C-6′), 110.4 (C-1), 110.4 (C-5′), 107.9
(C-1′).
Whiskey tannin A (3): yellow amorphous powder; [R]D -77.8 (c
0.1, MeOH); IR νmax cm-1 3457, 1746, 1614; UV λmax(MeOH) nm
(log ε) 221 (4.55); FAB-MS m/z 677 [M + H]+, 677 [M + Na]+;
HR-FAB-MS m/z 677.0997 [M + H]+ (calcd for C29H25O19 677.0990);
CD (c 4.4 × 10-5 M) ∆ε (nm) -4.2 (273), -0.8 (255), -17.1 (234);
1H and 13C NMR data, see Table 1.
Figure 2. Selected HMBC and NOESY correlations for 3 and HMBC
correlations for 10.
Whiskey tannin B (4): yellow amorphous powder; [R]D -74.9 (c
0.1, MeOH); IR νmax cm-1 3417, 1731, 1614, 1517; UV λmax(MeOH)
nm (log ε) 218 (4.80); FAB-MS m/z 1001 [M + Na]+; HR-FAB-MS
m/z 1001.0869 [M + Na]+ (calcd for C43H30O27Na 1001.0872); CD (c
(20 cm × 1.5 cm i.d., 20-100% MeOH) column chromatography to
yield dehydrocastalagin (13) (153 mg), 10 (20 mg), ellagic acid (158
mg), and castacrenin F (14) (4 mg), along with recovery of 5 (280
mg). The structure of dehydrocastalagin was determined on the basis
of NMR experiments including HSQC and HMBC spectra and finally
identified by comparison of the NMR data with those described in the
literature. Castacrenin F was identified by comparison of the NMR data
and HPLC with those of authentic samples.
1
3.5 × 10-5 M) ∆ε (nm) -15.0 (265), 10.2 (238); H and 13C NMR
data, see Table 1.
Pyrolysis of Castalagin. Castalagin (5) (1.0 g) was first dissolved in
a small amount of MeOH and then dried under reduced pressure. Dried
solid was heated in a microwave oven at 190 °C for 70 min. The
resulting mixture was separated by MCI gel CHP-20P (23 cm × 2.5
cm i.d., 0-100% MeOH, 10% stepwise elution) and Sephadex LH-20

7308 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Fujieda et al.
Figure 3. Structures of pyrolysis products of 5.
Figure 4. Possible production mechanism for 3 and 4.
Compound 10: yellow amorphous powder, [R]D -2.6 (c 0.05,
MeOH); MALDI-TOF-MS m/z 495 [M + H]+, 517 [M + Na]+; IR
mg/L), 3-O- and 6-O-galloyl glucoses were also isolated by
similar chromatographic separation (data not shown) together
with ellagic acid. Isolation of these simple galloyl glucoses was
somewhat surprising because the occurrence of these compounds
in oak wood has not been reported so far. Brevifolin carboxylic
acid is an oxidation product of the HHDP group of ellagitannins
(11). 3-Methoxy-4-hydroxyphenol 1-O-(6′-O-galloyl)-ꢀ-D-glu-
copyranoside is a phenolic glycoside originally isolated from
acorns of Quercus mongolica and the bark of Quercus acutis-
sima (12). Castacrenin B (7) is a dehydrative degradation
product of castalagin (5), a major oak wood ellagitannin, and
was first isolated from heart wood of the Japanese chestnut tree
(3). Castalagin and related ellagitannins that were reported to
be present in the original oak wood (1) were not detected in
any of the four whiskey samples examined in this study.
ν
max cm-1 3389, 1724, 1605; UV λmax (MeOH) nm (log ε) 370 (4.02),
1
251 (4.69); H NMR (DMSO-d6) δ 7.57 (1H, s, H-5′), 7.20 (1H, d, J
) 5.6 Hz, H-3′′), 6.87 (1H, d, J ) 5.6 Hz, H-4′′); 13C NMR (DMSO-
d6) δ 161.2 (C-1′′), 160.1 (C-7′), 158.5 (C-7), 148.5 (C-4′), 146.8, 144.7,
140.0, 139.0, 136.9 (C-2, C-3, C-4, C-6′′, C-7′′), 142.5 (C-3′′), 139.7
(C-3′), 139.4 (C-5′′), 137.4 (C-2′), 124.2 (C-5), 121.9 (C-4a′′), 117.4
(C-8′′), 113.9 (C-6), 113.7 (C-6′), 113.0 (C-8a′′), 111.2 (C-5′), 109.4
(C-1′), 109.0 (C-1), 102.3 (C-4′′). Anal. Calcd for C23H10O13 ·9/4H2O:
C, 51.65%; H, 2.73%. Found: C, 51.95%; H, 3.12%.
RESULTS AND DISCUSSION
The total polyphenol content of four commercial Japanese
whiskeys was compared by the Folin-Ciocalteu method (4).
The highest value of 417 ( 44.1 mg/L was observed in a vatted
whiskey that contained a malt whiskey aged in charred new
oak barrels. A representative Japanese single-malt whiskey also
contained a high concentration of polyphenols, 236.4 ( 1.2 mg/
L. Concentrations of the remaining two blended whiskeys were
relatively low (82.2 ( 17.7 and 78.7 ( 16.8 mg/L). The whiskey
that showed the highest polyphenol concentration was selected
for further chemical study on whiskey polyphenols. The whiskey
was directly passed through a Diaion HP20SS column to remove
nonpolar constituents, and then the fractions that contained
polyphenols were successively partitioned with Et2O and EtOAc.
Most of the polyphenols remained in the aqueous layer. The
Et2O and EtOAc layer contained ellagic acid, which was the
major polyphenol of the whiskey. Lyoniresinol (6), a lignan
composed of two sinapylalchohol units, was isolated from the
EtOAc layer. Chromatographic separation of the aqueous layer
yielded gallic acid, ellagic acid (2) (5), 6-O-galloyl glucose (7),
3-O-galloyl glucose (8), 2,3-di-O-galloyl glucose (10), 2,3-(S)-
HHDP-D-glucose (9), brevifolin carboxylic acid (11), 3-meth-
oxy-4-hydroxyphenol 1-O-(6′-O-galloyl)-ꢀ-D-glucopyranoside
(12), and castacrenin B (3), together with three new compounds
named carboxyl ellagic acid (1) and whiskey tannins A (2) and
B (3). The known compounds were identified by comparison
of their NMR spectroscopic data with those from authentic
samples or with those described in the literature. From another
Japanese single-malt whiskey (total polyphenol ) 236.4 ( 1.2
Carboxyl ellagic acid (1) was obtained as a yellow powder,
which was hardly soluble in water and MeOH. The UV
absorption at 254 and 368 nm was similar to that of ellagic
acid (2). FAB-MS showed [M + H]+, [M + Na]+, and [M +
K]+ at m/z 347, 369, and 385, respectively, which indicated
that the molecular weight was 346. The molecular weight was
44 mass units larger than that of 2, and the molecular formula,
C15H6O10, was confirmed by HR-FAB-MS. The 1H NMR
spectrum resembled that of 2 and showed an aromatic proton
singlet at δ 7.47 (H-5′). The 13C NMR spectrum was also related
to that of 2. However, in contrast to the symmetrical structure
of 2, the spectrum of 1 showed signals attributable to six
oxygenated aromatic carbons [δ 148.3 (2C, C-4, 4′), 145.2 (C-
3′), 139.6 (C-3), 136.5 (C-2), 136.1 (C-2′)], six nonoxygenated
aromatic carbons [δ 121.2 (C-5′), 112.1 (C-6′), 112.0 (C-6),
110.4 (2C, C-5, 1′), 107.9 (C-1)], and three carboxyl carbons
[δ157.7 (C-7), 159.0 (C-7′), 166.8 (C-8)]. An unusual upfield
shift of two of the three carboxyl carbon signals indicated that
these were ascribable to R,ꢀ,γ,δ-unsaturated δ-lactone, similar
to those of 2. When the molecular weight was taken into
account, the remaining carboxyl carbon signal (δ 166.8) was
assignable to the R,ꢀ-unsaturated carboxyl group. The location
of the carboxyl group was concluded to be at C-5 of 2, on the
basis of the results of th HMBC experiment: the H-5′ signal
was correlated with C-1′, C-2′, C-3′, C-4′, and C-6′, and the

Structure of Whiskey Polyphenols
J. Agric. Food Chem., Vol. 56, No. 16, 2008 7309
remaining aromatic and carboxyl carbons did not correlate with
any proton signals. Thus, the structure of 1 was deduced to be
5-carboxyl ellagic acid.
HMBC correlation illustrated in Figure 2, the glucose C-3
hydroxyl group formed another lactone ring with the carboxyl
group attached to the hexahydroxybiphenyl moiety (BPH C-7).
This implied that the glucose C-3 and the biphenyl moiety were
placed on the same side of the molecule. Assuming that 3 was
derived from 6 and, therefore, that the glucose was the D-form,
on the basis of the similarity of the planar structure of 3 and 6,
the configuration at Cp-2 methine carbon was deduced to be R
as shown in Figure 2. The configuration of the quaternary
carbon Cp-3 was determined to be R because the Cp-2 methine
proton showed NOESY correlations with the ethoxyl protons
(Figure 2). On the basis of these observations, the structure of
whiskey tannin A was concluded to be represented by the
formula 3. Atropisomerism of the BPH group could not be
determined clearly in this study. The CD spectrum showed a
large negative Cotton effect at 234 nm and a small positive
Cotton effect at 255 nm, which suggested that the biphenyl bond
was R-configuration (17). However, this result was inconsistent
with the configurations reported for 5 and 6 (18). The CD curve
of 3 was different from those of typical ellagitannins and was
apparently affected by the coexisting unsaturated carbonyl of
the cyclopentenone ring.
Whiskey tannin A (3) was obtained as a yellow amorphous
powder and gave a blue coloration with the FeCl3 reagent. The
molecular formula C29H24O19 was determined by HR-FAB-MS.
1
The H NMR spectrum exhibited an aromatic proton singlet
signal (δ 6.70, H-6′) and aliphatic proton signals attributable
to six methine, two methylene, and one methyl group (Table
1). The presence of an ethoxyl group was indicated by the
appearance of mutually coupled methyl and methylene proton
1
signals. The H-1H COSY correlations of the remaining one
methylene and five methine protons revealed the presence of a
polyalchohol unit, which was closely related to the open-chain
glucose unit of castalin (6) (3, 15). As well as their chemical
shifts, comparison of the coupling constants of 3 (Table 1) with
those of 6 (J1,2 ) 5 Hz, J2,3 ) 1 Hz, J3,4 ) 7 Hz, J4,5 ) 7 Hz,
J5,6a ) 3 Hz, J5,6b ) 6 Hz) strongly suggested that the
polyalcohol moiety was an open-chain glucose (3). In addition,
on the basis of the 1H NMR chemical shifts and HMBC
correlations (Figure 2), it was apparent that hydroxyl groups
at C-2, C-3, and C-5 of the glucose were acylated. However, in
contrast to the three pyrogallol rings of 6, signals arising from
only two pyrogallol moieties were observed in the spectrum of
3. Instead, 3 showed carbon signals due to a carbonyl [δ 200.2
(Cp-4)], a carboxyl [δ 170.6 (Cp-7)], and two olefinic [δ 142.6
(Cp-5) and 154.3 (Cp-1)] and two aliphatic carbons [δ 44.5 (Cp-
2) and 83.7 (Cp-3)]. In the HSQC spectrum, the methine carbon
Whiskey tannin B (4) was isolated as a tan amorphous powder
and exhibited an [M + Na]+ peak at m/z 1001 in FAB-MS,
which was 302 mass units larger than that of 3. The difference
coincided with the molecular mass of 2. The 1H and 13C NMR
spectra were closely related to those of 3 and showed signals
due to an open-chain glucose, an ethoxyl group, and a
cyclopentene ring moiety. The HMBC correlations of the signals
supported the presence of the same partial structures in 3 and
4. In addition, the appearance of two aromatic singlet signals
(δ 6.63, 6.83) in the 1H NMR spectrum and signals arising from
two pyrogallol and two carboxyl carbons in the 13C NMR
spectrum indicated the presence of an additional HHDP group
in 4. This was consistent with the above-mentioned FAB-MS
results. Compared to those of 3, large low-field shifts of glucose
H-4 (δ 5.42) and H-6 (δ 3.97, 4.92) were observed, and these
protons were correlated with the carboxyl group (δ 167.6, 168.7)
of the HHDP moiety in the HMBC spectrum. This observation
showed that the HHDP group was attached to H-4 and H-6 of
the open-chain glucose. In contrast to that of 3, the CD spectrum
of 4 exhibited large positive and negative Cotton effects at 238
and 265 nm, respectively, and this strongly suggested that the
HHDP group had an S-biphenyl bond (17). Thus, the structure
of whiskey tannin B was concluded to be represented by the
formula 4.
A production mechanism of whiskey tannins A and B was
proposed as illustrated in Figure 4. The pyrogallol ring attached
to the glucose C-1 position was oxidized, and subsequent
benzylic acid-type rearrangement after addition of ethanol
formed the ethoxycarbonyl cyclopentenone moiety. To examine
whether this oxidation occurred during the charring process of
barrel making, pyrolysis products of 5 (Figure 3) at 190 °C
were investigated. The major products were identified to be
ellagic acid (2) and dehydrocastalagin (8) (13), which was
accompanied by castacrenin F (9) (14) and a new product 10.
Production of 9 suggested electrophilic substitution of the C-1
of C-glycosidin ellagitannins to the HHDP group or ellagic acid.
1
resonating at δ 44.5 (Cp-2) showed a J correlation peak with
an aliphatic proton singlet at δ 5.34 (Hcp-2). This methine proton
2
3
signal showed J and J HMBC correlation peaks with Cp-1,
Cp-4, Cp-5, and aromatic carbon signals of a pyrogallol ring
(Figure 2). Furthermore, the Hcp-2 and methylene protons of
the aforementioned ethoxyl group were correlated to an ester
carboxyl carbon (δ 170.6, Cp-7). Although the oxygen-bearing
quaternary carbon (δ 83.7, Cp-3) did not show any HMBC
correlation peaks, these spectroscopic observations indicated that
the ethoxyl group was connected to the Cp-3 carbon through
the Cp-7 ester carboxyl carbon. In addition, the HMBC
correlations of the Cp-1-Cp-5 (Figure 2) indicated the presence
of a cyclopentenone ring structure. The connection of the Cp-5
to the glucose H-1 was apparent from the HMBC correlations
of the glucose H-1 with Cp-1, Cp-5, and Cp-4. A homoallyl
long-range coupling (J ) 3.0 Hz) between glucose H-1 and
Cp-2 methine proton was also observed. Chemical shift of the
remaining ester carbonyl carbon signal at δ 163.1 (Cp-6)
indicated that this carbonyl was conjugated with a double bond,
and this ester carbon was shown to form a δ-lactone ring with
the glucose C-2 hydroxyl group, by observation of the HMBC
correlation between this ester carbon and glucose H-2. More-
over, glucose H-3 and H-5 also showed correlation peaks with
ester carbonyl signals at δ 167.2 and 168.6, respectively, and
the latter signal was correlated with the aromatic singlet signal
at δ 6.70. On the basis sof these results, the planar structure
was constructed as shown in Figure 1, and this was supported
by comparison of the spectroscopic data with those of rhoipte-
leanin H (16).
As for stereostructure, the 1H-1H coupling constant between
glucose H-1 and H-2 (J ) 6.0 Hz) of 3 indicated that the
configuration of glucose C-1 of this compound was the same
as that of 6 (J ) 5 Hz) and different from that of vescalin (J <
1 Hz), the C-1 epimer of 6. This implied that the glucose C-3
carbon of 3 was oriented to the R-side of the δ-lactone ring
formed between C-2 and Cp-6 (Figure 2). As shown by the
The 1H NMR spectrum of 10 showed an aromatic singlet at
δ 7.57 (H-5′) and two mutually coupled (J ) 5.6 Hz) olefinic
proton signals at δ 6.87 and 7.20. The chemical shift of the
aromatic singlet was similar to those of 1 and 2, and the HMBC
correlations of this signal strongly suggested the presence of
an ellagic acid moiety in 10 (Figure 2). This was supported by

7310 J. Agric. Food Chem., Vol. 56, No. 16, 2008
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the appearance of UV absorption at 370 and 251 nm and by
comparison of the 13C NMR signals with those of 1. The 13C
NMR spectrum showed three carboxyl carbon signals at δ 158.5,
160.1, and 161.2, two of which were attributable to the δ-lactone
of ellagic acid moiety (δ 158.5, C-7; 160.1, C-7′). The remaining
one (δ 161.2, C-1′′) showed HMBC correlations with one of
the olefinic protons at δ 6.87 (H-3′′). In addition, HMBC
correlations of the two olefinic protons shown in Figure 2
revealed a trihydroxyisocoumarin unit. Because this compound
was the degradation product of 5, these spectroscopic data
allowed us to construct the structure of 10, as shown in Figure
3. The [M + H]+ peak at m/z 495 in MALDI-TOF-MS
supported the structure. The two olefinic methine carbons of
the isocoumarin unit originated from the glucose C-1 and C-2.
In molecules 8-10, the pyrogallol ring attached to the glucose
C-1 was not oxidized. No products having structures related to
3 and 4 were detected among the reaction products. Thus, the
results suggested that 3 and 4 in the commercial whiskey were
produced by oxidation of 5 and 6 during the aging process in
barrels. Related oxidation of the pyrogallol ring of 5 in model
aqueous ethanol solutions was demonstrated (19), and the
spectroscopic data of one of the products was closely related to
4. The oxidation product of the in vitro model experiment may
be identical to whiskey tannin B (4).
In conclusion, two new ellagitannins named whiskey tannins
A (3) and B (4) were isolated from Japanese whiskey, which
were produced by oxidation of oak wood ellagitannins during
the aging process in barrels. Another product, carboxyl ellagic
acid (1), was also presumed to be an oxidation product of
ellagitannin, although the origin of the carboxyl group was not
clear. Probably, it came from the ellagic acid moiety attached
to the glucose C-1 of C-glycosidic ellagitannins, such as
castacrenin F (9), or from triphenyl compounds, such as
compound 10. Brevifolin carboxylic acid was also isolated, and
this compound is known to be an oxidation product of HHDP
ester groups (11). In addition, it was very interesting that simple
galloyl glucoses, which have not been reported in oak wood,
were found in two commercial Japanese whiskeys. In this study,
we were conscious of the presence of significant amounts of an
uncharacterized phenolic substance in the whiskey, which was
detected as a broad hump on the baseline upon HPLC analysis
and a broad spot on cellulose TLC. Condensation of ellagitan-
nins with coexisting compounds possibly occurs (20). Further
chemical study on the substance is now in progress.
ACKNOWLEDGMENT
We are grateful to K. Inada, N. Yamaguchi, and Y. Oowatari
(Nagasaki University) for NMR and MS measurements and
elemental analysis.
LITERATURE CITED
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(3) Tanaka, T.; Ueda, N.; Shinohara, H.; Nonaka, G.; Fujioka, T.;
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Received for review April 23, 2008. Revised manuscript received July
1, 2008. Accepted July 1, 2008.
JF8012713