Galloylglucosides from berries of Pimenta dioica

Article abstract of DOI:10.1021/np9906121

Three new galloylglucosides, (4S)-α-terpineol 8-O-β-D-(6-O- galloyl)glucopyranoside (1); (4R)-α-terpineol 8-O-β-D-(6-O- galloyl)glucopyranoside (2), and 3-(4-hydroxy-3-methoxyphenyl)propane-1,2- diol 2-O-β-D-(6-O-galloyl)glucopyranoside (3), were isolated from the berries of Pimenta dioica together with three known compounds, gallic acid (4), pimentol (5), and eugenol 4-O-β-D-(6-O-galloyl)glucopyranoside (6). The structures of 1-3 were elucidated on the basis of MS and NMR spectral data and enzymatic hydrolysis. These galloylglucosides (1-3, 5, and 6) showed radical-scavenging activity nearly equivalent to that of gallic acid (4) against 1,1-diphenyl-2-picrylhydrazyl radical.

Full text of DOI:10.1021/np9906121

J . Nat. Prod. 2000, 63, 749-752
749
Ga lloylglu cosid es fr om Ber r ies of P im en ta d ioica
Hiroe Kikuzaki, Akemi Sato, Yoko Mayahara, and Nobuji Nakatani*
Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, Sumiyoshi, Osaka 558-8585, J apan
Received December 8, 1999
Three new galloylglucosides, (4S)-R-terpineol 8-O-â-D-(6-O-galloyl)glucopyranoside (1); (4R)-R-terpineol
8
-O-â-D-(6-O-galloyl)glucopyranoside (2), and 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol 2-O-â-D-
(6-O-galloyl)glucopyranoside (3), were isolated from the berries of Pimenta dioica together with three
known compounds, gallic acid (4), pimentol (5), and eugenol 4-O-â-D-(6-O-galloyl)glucopyranoside (6).
The structures of 1-3 were elucidated on the basis of MS and NMR spectral data and enzymatic hydrolysis.
These galloylglucosides (1-3, 5, and 6) showed radical-scavenging activity nearly equivalent to that of
gallic acid (4) against 1,1-diphenyl-2-picrylhydrazyl radical.
As a part of studies on the chemical components of spices
and herbs,1 we investigated allspice, that is, berries of
Pimenta dioica (Myrtaceae). The plant contains various
27.6, and 24.5), one methine (δ 44.5), one quaternary (δ
79.8), and two olefinic (δ 134.2 and 121.6) carbons, as well
as six carbons corresponding to a glucose moiety (δ 98.2,
-3
essential oils,4 phenolic acids, flavonoids, and catechins.
,5
6
7
8
78.0, 75.0, 74.5, 71.5, and 64.9). Double resonance H NMR
1
In a previous study, we isolated several phenylpropanoids
from allspice. We now report the isolation and character-
ization of three new galloylglucosides (1-3) from allspice
and a study of their free radical-scavenging effects on 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical.
and HMQC measurements allowed assignments of the
glucose signals and indicated esterification at C-6′ based
on the deshielded 6′-methylene protons [δ 4.52 (dd, J )
11.7, 2.2 Hz) and 4.30 (dd, J ) 11.7, 6.6 Hz)]. The
observation of an anomeric proton signal at δ 4.56 as a
doublet with a coupling constant of 7.6 Hz indicated the
â-configuration. Three methyl (δ 1.52, 1.20, and 1.15), one
olefinic (δ 5.30), and one methine (δ 1.64) proton and three
pairs of geminal proton signals corresponding to three
9
1
methylenes were observed in the H NMR spectrum,
showing the presence of an R-terpinyl moiety. Furthermore,
a fragment ion peak at m/z 315 in the SIMS indicated that
the R-terpinyl moiety formed a glycosidic linkage with a
glucose moiety. The 6′-methylene protons showed HMBC
correlation with the carbonyl carbon (δ 166.7) of a galloyl
group, and the anomeric proton (δ 4.56) showed correlation
with a quaternary carbon (δ 79.8) attributed to C-8 of an
R-terpinyl moiety (Figure 1). Acetylation of 1 gave a
1
hexaacetate (1a ). The H NMR spectrum of 1a showed one
6
H singlet at δ 2.242 and one 3H singlet at δ 2.239, which
Resu lts a n d Discu ssion
were attributed to three phenolic acetate groups, consistent
The EtOAc-soluble fraction obtained from a 70% aqueous
acetone extract of allspice was subjected to repeated column
chromatography using Sephadex LH-20, Si gel, and Chro-
matorex ODS to give three new compounds (1-3) together
with gallic acid (4), pimentol (5), and eugenol 4-O-â-D-(6-
O-galloyl)glucopyranoside (6). Compound 4 was identified
by comparing its TLC behavior and its spectral data with
those of an authentic sample, and identification of 5 and 6
was carried out by comparing their physical and spectral
with a galloyl moiety. Furthermore, three 3H singlets (δ
1
.959, 1.956, and 1.94) were observed and assigned to three
aliphatic acetate groups. The deshielding of H-2′ (δ 4.93),
H-3′ (δ 5.18), and H-4′ (δ 4.98) of the glucose moiety in 1a ,
compared to 1, confirmed that the C-6′ hydroxyl group was
esterified with a galloyl group. Consequently, compound 1
was defined as R-terpineol 8-O-â-D-(6-O-galloyl)glucopyra-
noside.
data with those in the previous reports.1
0-12
Compound 2 exhibited an optical rotation of +31.0° and
+
an [M + H] peak at m/z 469.2077, indicating that 2 had
Compound 1 exhibited an optical rotation of -7.0°and
1
+
the same molecular formula (C23
H
33
O
10) as 1. The H and
an [M + H] peak at m/z 469.2053 in good agreement with
1
3
C NMR, SIMS, and UV data of 2 were very close to those
of 1, except for the optical rotation. These data suggested
that 2 was a diastereomer of 1. Comparing the H and
the molecular formula C23
H
33
1
O10. The IR spectrum revealed
-
hydroxyl (3600-3100 cm ) and conjugated ester (1697,
1
13
-
1
C
1
234, and 1083 cm ) functions and an aromatic ring (1613
-
1
NMR spectra of 1 and 2, some differences were observed
in the signals corresponding to the R-terpinyl moiety. The
C-4 methine proton was observed at δ 1.64 in 1, while the
corresponding signal in 2 was observed at δ 1.60. The C-7
methyl proton appeared at δ 1.52 in 1, while in 2 it
appeared at δ 1.57. In the 13C NMR of 1, C-9 and C-10 were
observed at δ 22.9 and 25.1, while they were observed at δ
and 1536 cm ). The presence of a galloyl group was
1
supported by a two-proton singlet at δ 7.14 in the H NMR
spectrum and five characteristic carbon signals (δ 166.7,
1
3
1
(
45.9, 138.6, 121.8, and 109.8) in the C NMR spectrum
Tables 1 and 2). The remaining 16 signals indicated three
methyl (δ 25.1, 23.5, and 22.9), three methylene (δ 31.5,
2
3.9 and 24.1 in the case of 2. These findings suggested
*
To whom correspondence should be addressed. Tel.: +81-6-6605-2812.
Fax: +81-6-6605-3086. E-mail: nakatani@life.osaka-cu.ac.jp.
that 1 and 2 were epimers at C-4.
1
0.1021/np9906121 CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/29/2000

7
50 J ournal of Natural Products, 2000, Vol. 63, No. 6
Kikuzaki et al.
Ta ble 1. 1H NMR Data of Compounds 1, 2, 7, and 8 in (CD3)2CO at 500 MHza
H
1
2
7
8
2
3
4
5
5
6
6
7
9
1
1
2
3
4
5
6
6
2
5.30 (m)
2.02 (m)
1.64 (dddd, 12.0, 12.0, 4.4, 2.2)
5.32 (m)
2.03 (m)
1.60 (dddd, 12.0, 12.0, 4.6, 2.0)
5.34 (m)
2.02 (m)
1.64 (dddd, 12.0, 12.0, 4.6, 2.0)
5.35 (m)
2.03 (m)
1.60 (dddd, 11.7, 11.7, 4.9, 2.2)
eq
ax
eq
ax
eq
1.15 (dddd, 12.0, 12.0, 12.0, 5.4) 1.18 (dddd, 12.0, 12.0, 12.0, 3.9) 1.19 (dddd, 12.0, 12.0, 12.0, 5.1) 1.21 (dddd, 11.7, 11.7, 11.7, 3.9)
1.98 (m)
1.90 (m)
1.97 (m)
1.92 (m)
2.00 (m)
2.00 (m)
1.99 (m)
1.95 (m)
1.82 (br d, 16.6)
1.52 (br s)
1.86 (br d, 16.4)
1.57 (br s)
1.88 (br d, 16.6)
1.61 (br s)
1.89 (br d, 15.9)
1.61 (br s)
b
b
b
b
1.15 (s)
1.16 (s)
1.19 (s)
1.16 (s)
1.21 (s)
1.18 (s)
1.20 (s)
b
b
b
0
′
′
′
′
′
′a
′b
1.20 (s)b
4.56 (d, 7.6)
4.543 (d, 7.6)
4.50 (d, 7.6)
3.12 (dd, 9.0, 7.6)
4.50 (d, 7.6)
3.12 (dd, 8.5, 7.6)
3.19 (dd, 8.8, 7.6)
3.45 (apparent triplet, 8.8)
3.39 (apparent triplet, 8.8)
3.60 (ddd, 8.8, 6.6, 2.2)
4.52 (dd, 11.7, 2.2)
4.30 (dd, 11.7, 6.6)
3.18 (dd, 8.8, 7.6)
3.45 (apparent triplet, 8.8)
3.37 (apparent triplet, 8.8)
3.59 (ddd, 8.8, 7.1, 2.0)
4.536 (dd, 11.7, 2.0)
4.28 (dd, 11.7, 7.1)
7.14 (s)
3.39 (apparent triplet, 9.0)
3.32 (apparent triplet, 9.0)
3.28 (ddd, 9.0, 6.6, 2.9)
3.79 (dd, 11.5, 2.9)
3.62 (dd, 11.5, 6.6)
3.39 (apparent triplet, 8.5)
3.31 (apparent triplet, 8.5)
3.28 (ddd, 8.5, 6.1, 2.7)
3.79 (dd, 11.2, 2.7)
3.61 (dd, 11.2, 6.1)
′′, 6′′ 7.14 (s)
a
Chemical shifts are shown in δ values (ppm) relative to solvent peak. Multiplicity and coupling constant(s) in Hz are in parentheses.
b
Assignments are interchangeable in each column.
1
3
Ta ble 2. C NMR Data of Compounds 1, 2, 7, and 8 in
(
Sch em e 1. Enzymatic Hydrolysis of Compounds 1 and 2
a
CD3)2CO at 125 MHz
C
1
2
7
8
1
2
3
4
5
6
7
8
9
1
1
2
3
4
5
6
1
2
3
4
7
134.2
121.6
27.6
44.5
24.5
31.5
23.5
79.8
22.9
25.1
98.2
75.0
78.0
71.5
74.5
64.9
138.6
109.8
145.9
121.8
166.7
134.1
121.7
27.5
44.5
24.4
31.6
23.5
79.7
23.9
24.1
98.1
75.0
78.1
71.7
74.5
64.9
138.6
109.8
145.9
121.9
166.6
134.3
121.7
27.6
44.6
24.5
31.6
23.6
79.7
23.0
25.0
98.1
75.1
78.2
72.0
76.9
63.2
134.2
121.8
27.5
44.6
24.5
31.7
23.5
79.5
23.9b
24.1b
98.1
75.1
78.2
72.0
76.9
63.2
b
b
b
b
b
b
0
′
′
′
′
′
′
′′
′′,6′′
′′,5′′
′′
′′
a
Chemical shifts are shown in δ values (ppm) relative to
b
solvent peak. Assignments are interchangeable in each column.
F igu r e 1. HMBC correlations for compounds 1 and 3.
widely in fruits and vegetables and is known as a precursor
of flavor component.1
3,14
However, to our knowledge, there
The absolute configuration of 1 and 2 at C-4 was
has been no report dealing with the full structure elucida-
tion of R-terpinyl glucoside.
determined by enzymatic hydrolysis. Treatment of 1 and
25
2
with tannase gave 7 ([R]21
D
-40.6°) and 8 ([R]
D
+36.1°),
Compounds 7 and 8 were separately hydrolyzed with
â-glucosidase to give 9 and 10, respectively (Scheme 1).
respectively, along with gallic acid (4) (Scheme 1). Each
compound showed an [M + H] peak at m/z 317 in the
SIMS spectrum, which was 170 mass units smaller than
+
1
Both 9 and 10 showed the same H NMR spectrum and
1
were identified as R-terpineol by comparison with the H
those of 1 and 2. The two proton singlets observed in the
H NMR spectra of 1 and 2, attributed to a galloyl group,
were absent in the spectra of 7 and 8. In addition, the 6′-
methylene resonances were shielded in 7 and 8 compared
to 1 and 2 (Table 1). R-Terpinyl glucoside is distributed
NMR spectrum of an authentic sample. Compound 9
1
25
showed a negative optical rotation ([R]
D
-78.3°), while
+71.0°).
Thus, it was concluded that 9 was (4S)-R-terpineol ([R]
10 exhibited a positive optical rotation ([R]25
D
D
15
-100.5°) 15 and 10 was (4R)-R-terpineol ([R]
+101.8°).
D

Galloylglucosides from Pimenta Berries
J ournal of Natural Products, 2000, Vol. 63, No. 6 751
Ta ble 3. Scavenging Effects of Compounds 1-6 on the DPPH Radicala
radical-scavenging efficacies expressed as percentage at concentrationb
compound
80.0 µM
40.0 µM
20.0 µM
10.0 µM
5.0 µM
2.5 µM
c
c
c
c
c
c
c
d
c
c
c
c
c
c
c
d
efg
df
48.4 ( 0.64f
26.2 ( 0.72e
de
e
1
2
3
4
5
6
95.4 ( 0.06
95.3 ( 0.02
95.6 ( 0.02
95.7 ( 0.20
95.8 ( 0.26
95.7 ( 0.05
95.8 ( 0.07
68.0 ( 2.04
95.4 ( 0.05
95.3 ( 0.05
95.3 ( 0.11
94.8 ( 0.25
95.7 ( 0.09
95.2 ( 0.07
95.7 ( 0.18
47.5 ( 3.60
88.9 ( 1.38
90.1 ( 0.46
14.8 ( 0.66
12.9 ( 0.55
17.7 ( 0.80
19.0 ( 0.30
12.2 ( 0.41
15.0 ( 1.49
8.3 ( 0.66
6.3 ( 1.92
46.2 ( 1.74d
24.5 ( 0.32e
92.4 ( 0.37d
56.9 ( 0.33c
61.6 ( 1.04
53.6 ( 1.11e
de
33.1 ( 0.40c
34.9 ( 0.15
26.8 ( 0.93e
cd
cd
c
86.8 ( 0.69g
de
e
91.2 ( 0.91c
cd
d
de
f
95.2 ( 0.21
60.9 ( 1.50g
23.4 ( 1.69
16.7 ( 1.14h
31.7 ( 0.63f
h
R-tocopherol
BHT
48.8 ( 0.92
14.0 ( 0.63
11.8 ( 1.75f
i
f
28.6 ( 0.66
a
The concentration of DPPH ethanolic solution was 100 µM. b The results are expressed in % ) [(A in the absence of compound - A
in the presence of compound)/A in the absence of compound] × 100. Values with standard deviations are from three independent
c-i
experiments.
Values in each column with the different superscripts are significantly different (p < 0.05).
Based on all the above data, compound 1 was identified
as (4S)-R-terpineol 8-O-â-D-(6-O-galloyl)glucopyranoside
and 2 as (4R)-R-terpineol 8-O-â-D-(6-O-galloyl)glucopyra-
noside.
important role in the activity, and the ortho-diphenols
additionally increase the capacity for quenching DPPH
radicals.1
8,19
Galloylglucosides (1-3, 5, and 6) exhibited
almost the same activity as gallic acid (4), which had been
reported to be one of the strong scavengers.
+
19
Compound 3 showed an [M + H] at m/z 513 and a stable
fragment ion peak at m/z 315 in the positive SIMS. The
spectra of 3 were very similar to those of 1, except for the
signals attributed to the monoterpene moiety, and indi-
cated that 3 was also a related 6-O-galloylglucose com-
pound. An aglycon moiety having six aromatic (δ 148.0,
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured using a Union PM-101 automatic digital
polarimeter. UV spectra were recorded on a Shimadzu UV-
2
500PC UV-vis spectrophotometer. IR spectra were run on a
1
3
6
45.7, 130.6, 122.6, 115.5, and 113.8), one methylene (δ
8.3), one methoxyl (δ 56.1), one oxygenated methylene (δ
4.8), and one oxygenated methine (δ 83.9) carbons was
1 13
Perkin-Elmer 1800 instrument. The H, C, and 2D NMR
spectra were recorded on a Varian Unity plus 500 (500 MHz)
spectrometer. Positive SIMS (matrix: glycerol) and EIMS (70
eV) were measured on a Hitachi M-2000 mass spectrometer,
and HRSIMS were obtained by a Hitachi M-4100 mass
spectrometer [internal standard: poly(ethylene glycol)]. Si gel
60 (70-230 mesh, Merck), Sephadex LH-20 (Pharmacia), and
Chromatorex ODS DM1020T (100-200 mesh, Fuji Silysia
Chemical) were used for column chromatography, and Si gel
60 F254 plates (Merck) and ODS plates (Merck) were used for
TLC. HPLC analysis was carried out with a pump and a
system controller (Hitachi) connected to a UV detector (Hita-
chi) operating at 280 nm. The column (4.6 × 250 nm) was a
1
3
revealed by C NMR and HMQC measurements. Three
aromatic protons were observed in the H NMR spectrum
1
at δ 6.94, 6.71, and 6.69, corresponding to a 1,2,4-trisub-
stituted phenyl group. An NOE between the proton at δ
6
.94 and the methoxy protons at δ 3.81 (3H, s) indicated
this to be a 4-hydroxy-3-methoxyphenyl group. A 2H-broad
doublet signal at δ 2.77 assignable to benzylic methylene
protons was coupled with an oxymethine proton at δ 3.89
(tt), the latter was also coupled with a 2H-broad doublet
signal at δ 3.52 corresponding to an oxymethylene group.
These data suggested that the aglycon moiety was a 3-(4-
hydroxy-3-methoxyphenyl)propane-1,2-diol, which had pre-
5
-µm Develosil ODS HG-5 (Nomura Chemicals), a mixture of
MeCN, H O, and HOAc (29:70:1, v/v/v) was used as a solvent
2
9
with a flow rate of 0.75 mL/min. For measuring the DPPH
radical scavenging activity, a UV-2500PC UV-vis spectro-
photometer (Shimadzu) was employed. DPPH, R-tocopherol,
and BHT were purchased from Wako Pure Chemical Indus-
tries, Ltd.
viously been isolated from allspice. The shielded anomeric
proton (δ 4.39) compared with 5 (δ 4.74)11 and 6 (δ 4.93)
suggested that a glucosyl unit was linked with an aliphatic
hydroxyl, not with a phenolic hydroxyl group. The C-8
oxymethine carbon of 3 resonated at lower field (δ 83.9)
than that of the previously reported aglycon (δ 74.2),9
suggesting that the glucose moiety was attached to C-8.
This was confirmed by HMBC, in which an anomeric proton
showed correlation with C-8, and H-8 (δ 3.89) showed
correlation with C-1′ (δ 103.9) (Figure 1). Thus, compound
12
P la n t Ma ter ia l. Berries of P. dioica from J amaica were
kindly supplied by Taiyo Koryo Co., Ltd., Osaka, J apan.
Extr a ction a n d Isola tion . Dried and ground berries of P.
dioica (500 g) were successively extracted with n-hexane (6 ×
1.5 L), CH
2
Cl
2
(6 × 1.5 L), and 70% aqueous Me CO (6 × 1.5
2
L) at room temperature. For each extraction, the plant
material was soaked in the solvent and allowed to stand
3
was characterized as 3-(4-hydroxy-3-methoxyphenyl)-
2
overnight. Acetone from the combined 70% aqueous Me CO
extract was evaporated in vacuo, and the resulting aqueous
residue was partitioned with EtOAc to give the EtOAc-soluble
and H O-soluble fractions. The EtOAc-soluble fraction (6.3 g)
2
was subjected to Sephadex LH-20 column chromatography
using 2-propanol to give six fractions. Fraction 2 was rechro-
propane-1,2-diol 2-O-â-D-(6-O-galloyl)glucopyranoside. De-
termination of the absolute configuration at C-8 has not
been achieved due to the small amount of 3 obtained. The
aglycon of 3, 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-
9
diol, was a mixture of R and S isomers in a 1:4 ratio.
However, compound 3 might have either configuration at
C-8 because its NMR spectrum showed no signals corre-
sponding to C-8 diastereoisomers.
2
matographed over ODS gel (MeCN-H O, 3:7) to give nine
fractions (A-I). Fraction B was recrystallized with H O to
2
afford gallic acid (4, 337 mg). Fraction D was rechromato-
The radical-scavenging properties of compounds 1-6
graphed on Si gel (CH
MeCN-H O-HOAc, 15:84:1) to give 3 (3.4 mg). Fraction H
was rechromatographed over Si gel (CH Cl -MeOH, 9:1) to
2 2
Cl -MeOH, 9:1), then over ODS
(
2
were evaluated against the DPPH radical in a spectropho-
tometric assay.1
6,17
2
2
Two antioxidants, R-tocopherol and
give a mixture of 1 and 2, which was further subjected to
repeated column chromatography over ODS gel eluted with
butylated hydroxytoluene (BHT), were used as reference
compounds. As shown in Table 3, compounds 1-6 showed
higher activity than R-tocopherol and BHT. Recent inves-
tigations on the DPPH radical-scavenging effects of phe-
nolic compounds indicated that the presence of an addi-
tional hydroxyl group in the ortho position plays an
MeCN-H
12.0 mg). Fraction 3 was subjected to ODS chromatography
eluted with MeCN-H O (3:7) to give pimentol (5, 611 mg) and
crude compound 6, which was purified by Si gel column
chromatography (CH Cl -MeOH, 9:1) to give 6 (42 mg).
2
O-HOAc, 15:84:1 to separate 1 (28.8 mg) and 2
(
2
2
2

7
52 J ournal of Natural Products, 2000, Vol. 63, No. 6
Kikuzaki et al.
3.89 (1H, tt, J ) 6.1 Hz, 5.6 Hz, H-8), 3.81 (3H, s, 3-OCH ),
(
4S)-r-Ter p in eol 8-O-â-D-(6-O-ga lloyl)glu cop yr a n osid e
1): colorless amorphous powder; δ , see Table 1; δ , see Table
-7.0° (c 1.00, EtOH); UV (EtOH) λmax (log ꢀ 277.0
3
(
2
(
H
C
3.63 (1H, ddd, J ) 9.0 Hz, 6.3 Hz, 2.0 Hz, H-5′), 3.52 (2H, br
d, J ) 5.6 Hz, H-9a,b), 3.43 (1H, apparent triplet, J ) 9.0 Hz,
H-4′), 3.40 (1H, apparent triplet, J ) 9.0 Hz, H-3′), 3.24 (1H,
2
0
; [R]
D
4.05) nm; IR (Nujol) νmax 3600-3100, 1697, 1613, 1536, 1234,
-
1
+
1
083 cm ; SIMS m/z 469 [M + H] , 315, 153, 137; HRSIMS
dd, J ) 9.0 Hz, 7.8 Hz, H-2′), 2.77 (2H, br d, J ) 6.1 Hz,
+
13
m/z 469.2053 [M + H] (calcd for C23
91.1886 [M + Na]+ (calcd for C23
Acetyla tion of 1. A solution of 1 (4.7 mg) in pyridine (1.0
mL) and Ac O (1.0 mL) was allowed to stand overnight at room
H
33
O
10, 469.2071), m/z
3 2
H-7a,b); C NMR [(CD ) CO, 125 MHz] δ 166.6 (C-7′′), 148.0
4
H
32
O
10Na, 491.1892).
(C-3), 145.9 (C-3′′, 5′′), 145.7 (C-4), 138.7 (C-1′′), 130.6 (C-1),
122.6 (C-6), 121.6 (C-4′′), 115.5 (C-5), 113.8 (C-2), 109.9 (C-
2′′,6′′), 103.9 (C-1′), 83.9 (C-8), 77.4 (C-3′), 75.1 (C-5′), 74.7 (C-
2
temperature. The reaction mixture was poured into cold 2 N
HCl, and then extracted with EtOAc. The organic layer was
3
2′), 71.2 (C-4′), 64.8 (C-9), 64.4 (C-6′), 56.1 (OCH ), 38.3 (C-7);
+
+
SIMS m/z 605 [M + G] , 513 [M + H] , 315.
washed with water, dried over anhydrous MgSO
4
, and evapo-
Det er m in a t ion of t h e Sca ven gin g E ffect on DP P H
1
16,17
rated to dryness to give 1a (6.5 mg): H NMR [(CD CO, 500
3
)
2
Ra d ica ls.
Test compounds were added to an EtOH solution
MHz] δ 7.71 (2H, s, H-2′′, 6′′), 5.25 (1H, m, H-2), 5.18 (1H,
apparent triplet, J ) 9.5 Hz, H-3′), 4.98 (1H, apparent triplet,
J ) 9.5 Hz, H-4′), 4.93 (1H, dd, J ) 9.5 Hz, 8.1 Hz, H-2′), 4.64
of DPPH radical (final concentration was 100 µM), the reaction
mixtures were shaken vigorously on a vortex stirrer and then
2
incubated for 30 min in a H O bath at 25 °C in the dark. The
(
1H, d, J ) 8.1 Hz, H-1′), 4.37 (1H, dd, J ) 12.0 Hz, 2.6 Hz,
absorbance of the remaining DPPH was determined colori-
metrically at 517 nm. The scavenging activity of the tested
compounds was measured as the decrease in absorbance of
the DPPH expressed as a percentage of the absorbance of a
control DPPH solution without test compounds. Six different
concentrations of each of the isolated compounds were pre-
pared for DPPH tests. All analyses were carried out in
triplicate, and the values were averaged. A factorial analysis
of variance (ANOVA) with multiple comparisons was carried
out. Significance was established at p < 0.05.
H-6′a), 4.25 (1H, dd, J ) 12.0 Hz, 6.6 Hz, H-6′b), 3.73 (1H,
ddd, J ) 9.5 Hz, 6.6 Hz, 2.7 Hz, H-5′), 2.242 (6H, s, OAc), 2.239
(3H, s, OAc), 1.959 (3H, s, OAc), 1.956 (3H, s, OAc), 1.94 (3H,
s, OAc), 1.91 (1H, m, H-3eq), 1.88 (1H, m, H-5eq), 1.85 (1H,
m, H-6a), 1.79 (1H, m, H-6b), 1.64 (1H, m, H-3ax), 1.53 (3H,
br s, H-7), 1.51 (1H, m, H-4), 1.11 (1H, dddd, J ) 12.0 Hz,
1
1
2.0 Hz, 12.0 Hz, 5.5 Hz, H-5ax), 1.09, 1.04 (each 3H, s, H-9,
0); EIMS m/z 720 (0.5), 567 (8), 525 (4), 483 (1), 440 (2), 405
(3), 398 (2), 380 (2), 363 (1), 338 (4), 320 (3), 279 (26), 237 (40),
1
95 (21), 153 (20), 152 (26), 136 (100).
Ack n ow led gm en t. This research was supported finan-
cially by Program for Promotion of Basic Research Activities
for Innovative Biosciences. The authors are grateful to Ms.
Tomomi Maekawa for measurement of the NMR spectra. The
authors are thankful to Dr. Kayoko Saiki, Kobe Pharmaceuti-
cal University, for measurement of the HRSIMS spectra.
En zym a tic Hyd r olysis of 1. A solution of 1 (10.7 mg) in
H
2
O (3 mL) was incubated with 5 mg of tannase (49 units/
mg, Aspergillus oryzae, Wako) at 30 °C for 3 h. HPLC analysis
showed that 1 (t 19.9 min) disappeared, and gallic acid (t
.9 min) was yielded. The reaction mixture was extracted with
EtOAc (3 × 3 mL), and the EtOAc extract (10.2 mg) was
chromatographed on Si gel (CH Cl -MeOH, 9:1) to obtain 7
5.5 mg); δ , see Table 1; δ , see Table 2; [R]
MeOH); SIMS m/z 317 [M + H] . A solution of 7 (5.0 mg) in
O (2 mL) was incubated with â-glucosidase (10 mg) at 37
C for 24 h. The reaction mixture was extracted with Et O (3
2 mL), and the organic layer was dried and evaporated in
R
R
3
2
2
2
1
(
H
C
D
-40.6° (c 0.55,
Refer en ces a n d Notes
+
(
1) Kikuzaki, H.; Kawabata, M.; Ishida, E.; Akazawa, Y.; Takei, Y.;
Nakatani, N. Biosci. Biotech. Biochem. 1993, 57, 1329-1333.
2) Kikuzaki, H.; Nakatani, N. Phytochemistry 1996, 43, 273-277.
3) Wang, M.; Kikuzaki, H.; Lin, C.-C.; Kahyaoglu, A.; Huang, M.-T.;
Nakatani, N.; Ho, C.-T. J . Agric. Food Chem. 1999, 47, 1911-1914.
4) Pino, J .; Rosado, A.; Gonzalez, A. Nahrung 1989, 33, 717-720.
H
°
×
2
(
(
2
vacuo to give 9 (1.4 mg). TLC (SiO
2
, n-hexane-acetone, 3:1,
(
1
R
f
0.5) and H NMR of 9 was compared with authentic
(5) Oberdieck, R. Fleischwirtschaft. 1989, 69, 320-330.
(6) Schulz, J . M.; Herrmann, K. Z. Lebensm.-Unters. Forsch. 1980, 171,
2
5
15
R-terpineol; [R]
D
-78.3° (c 0.14, CHCl ) [lit. -100.5°].
3
1
93-199.
(
4R)-r-Ter p in eol 8-O-â-D-(6-O-ga lloyl)glu cop yr a n osid e
(
7) V o¨ sgen, B.; Herrmann, K. Z. Lebensm.-Unters. Forsch. 1980, 170,
204-207.
(
2
(
2): colorless amorphous powder; δ
H
C
, see Table 1; δ , see Table
2
0
; [R]
D
+31.0° (c 0.21, EtOH); UV (EtOH) λmax (log ꢀ) 276.0
(8) Schulz, J . M.; Herrmann, K. Z. Lebensm.-Unters. Forsch. 1980, 171,
278-280.
+
3.95) nm; SIMS m/z 469 [M + H] , 315, 153, 137; HRSIMS
(
9) Kikuzaki, H.; Hara, S.; Kawai, Y.; Nakatani, N. Phytochemistry 1999,
+
m/z 469.2077 [M + H] (calcd for C23
91.1897 [M + Na]+ (calcd for C23
En zym a tic Hyd r olysis of 2. Compound 2 (8.5 mg) was
treated with tannase in the same manner as 1 (t 20.9 min)
to give 8 (3.1 mg): δ , see Table 1; δ , see Table 2; [R]
c 0.31, MeOH); SIMS m/z 317 [M + H] . Compound 8 (2.5
mg) was hydrolyzed in the same manner as 7 to give 10 (0.5
H
33
O
10, 469.2071), m/z
5
2, 1307-1312.
4
H
32
O
10Na, 491.1892).
(10) Matsumoto, A.; Matsumoto, T.; Tokuda, H. J apan Kokai Tokkyo Koho,
04041499 (Feb. 12, 1992); Chem. Abstr. 1992, 117, 14407.
(
(
(
(
11) Oya, T.; Osawa, T.; Kawakishi, S. Biosci. Biotech. Biochem. 1997,
R
6
1, 263-266.
2
5
H
C
D
+36.1°
12) Tanaka, T.; Orii, Y.; Nonaka, G.; Nishioka, I. Chem. Pharm. Bull.
1993, 41, 1232-1237.
+
(
13) Williams, P. J .; Strauss, C. R.; Wilson, B.; Massy-Westropp, R. A. J .
Agric. Food Chem. 1982, 30, 1219-1223.
1
mg). TLC (SiO
2
f
, n-hexane-acetone, 3:1, R , 0.5) and H NMR
14) Sakho, M. J .; Chassagne, D.; Crouzet, J . J . Agric. Food Chem. 1997,
2
5
of 10 was identical with authentic R-terpineol, [R]
D
+71.0°
4
5, 883-888.
1
5
(
c 0.05, CHCl
-(4-Hyd r oxy-3-m eth oxyp h en yl)p r op a n e-1,2-d iol 2-O-
â-D-(6-O-ga lloyl)glu cop yr a n osid e (3): colorless viscous liq-
3
) [lit. +101.8°].
(15) Beilsteins Handbuch Der Organischen Chemie; Springer-Verlag:
Berlin, 1944; H6, pp 55-57 (E II 6, pp 66-67).
3
(
(
16) Blois, M. S. Nature 1958, 181, 1199-1200.
17) Brand-Willams, W.; Cuvelier, M. E.; Berset, C. Food Sci. Technol.
(London) 1995, 28, 25-30.
2
0
1
uid; [R]
D
-27.7° (c 0.15, MeOH); H NMR [(CD
3 2
) CO, 500
MHz] δ 7.16 (2H, s, H-2′′, 6′′), 6.94 (1H, br d, J ) 1.5 Hz, H-2),
(18) Chen, J . H.; Ho, C.-T. J . Agric. Food Chem. 1997, 45, 2374-2378.
(19) Chevalley, I.; Marston, A.; Hostettmann, K. Phytochemistry 1999, 50,
6
.71 (1H, d, J ) 7.3 Hz, H-5), 6.69 (1H, br dd, J ) 7.3 Hz, 1.5
1
51-154.
Hz, H-6), 4.65 (1H, dd, J ) 12.0 Hz, 2.0 Hz, H-6′a), 4.39 (1H,
d, J ) 7.8 Hz, H-1′), 4.27 (1H, dd, J ) 12.0 Hz, 6.3 Hz, H-6′b),
NP9906121