Phenolic glycosides from berries of Pimenta dioica

Article abstract of DOI:10.1021/np0705615

Four new phenolic glycosides, (2-hydroxy-3-methoxy-5-allyl)phenyl β-D-(6-O-E-sinapoyl)glucopyranoside (1), (1′ R,5′R)-5-(5- carboxymefhyl-2-oxocyclopentyl)-3Z-pentenyl β-D-(6-O-galloyl) glucopyranoside (2), (5)-α-terpinyl [α-L-(2-O-galloyl) arabinofuranosyl]-(l→6)-β-D-glucopyranoside (3), and (R)-a-terpinyl [α-L-(2-O-galloyl)arabinofuranosyl]-(l→6)-β-D-glucopyranoside (4), were isolated from the berries of Pimenta dioica together with eight known flavonoids. The structures of 1-4 were elucidated on the basis of MS and NMR data and enzymatic hydrolysis. All four glycosides showed radical-scavenging activity against 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicals.

Full text of DOI:10.1021/np0705615

J. Nat. Prod. 2008, 71, 861–865
861
Phenolic Glycosides from Berries of Pimenta dioica
Hiroe Kikuzaki,*,† Yoshiko Miyajima,† and Nobuji Nakatani‡
DiVision of Food and Health Sciences, Graduate School of Human Life Science, Osaka City UniVersity, 3-3-138, Sugimoto, Sumiyoshi,
Osaka 558-8585, Japan, and The Open UniVersity of Japan, 2-11, Wakaba, Mihama, Chiba 261-8586, Japan
ReceiVed October 4, 2007
Four new phenolic glycosides, (2-hydroxy-3-methoxy-5-allyl)phenyl ꢀ-D-(6-O-E-sinapoyl)glucopyranoside (1), (1′R,5′R)-
5-(5-carboxymethyl-2-oxocyclopentyl)-3Z-pentenyl ꢀ-D-(6-O-galloyl)glucopyranoside (2), (S)-R-terpinyl [R-L-(2-O-
galloyl)arabinofuranosyl]-(1f6)-ꢀ-D-glucopyranoside (3), and (R)-R-terpinyl [R-L-(2-O-galloyl)arabinofuranosyl]-(1f6)-
ꢀ-D-glucopyranoside (4), were isolated from the berries of Pimenta dioica together with eight known flavonoids. The
structures of 1-4 were elucidated on the basis of MS and NMR data and enzymatic hydrolysis. All four glycosides
showed radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals.
Allspice, made from the berries of Pimenta dioica Merr.
belonging to Myrtaceae, is used as a spice in foods, and its essential
oil, which is rich in eugenol, has been used as an antimicrobial
and a digestive agent. In our previous studies, several phenylpro-
panoids and galloylglucosides were isolated from this plant.1–3
Herein, we report the isolation and characterization of four new
phenolic glycosides (1-4) from allspice and their free radical-
scavenging effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals.
The EtOAc-soluble fraction obtained from a 70% aqueous
acetone extract of allspice was subjected to successive column
chromatography using Sephadex LH-20, Si gel, and Chromatorex
ODS to give four new glycosides (1-4) together with eight known
compounds. These known compounds were identified by compari-
son of spectroscopic data as quercetin, quercetin 3-O-galactoside
(hyperoside),4 quercetin 3-O-R-L-arabinoside (avicularin),5 quercetin
3-O-(2-O-galloyl)-ꢀ-D-glucoside,6 quercetin 3-O-ꢀ-D-glucuronide
6′′-methyl ester,7 myricetin, myricetin 3-O-galactoside,8 and (+)-
ampelopsin.9
Compound 1 exhibited an [M - H]- peak at m/z 547.1798 in
agreement with the molecular formula of C27H32O12 by negative
ion HRFABMS measurement. The IR spectrum revealed hydroxy
(3600–3100 cm-1) and ester (1717 cm-1) functions and an aromatic
ring (1604 and 1510 cm-1). The UV spectrum showed an absorption
peak at 328 nm, indicating the presence of a cinnamoyl moiety. In
the 1H NMR spectrum, a two-proton singlet at δ 7.04, a 6H singlet
at δ 3.90, and two 1H doublets at δ 7.63 and 6.48 with a coupling
constant of 15.9 Hz were indicative of a sinapoyl group. The 13C
Furthermore, the deshielded 6′-methylene protons [δ 4.53 (dd, J
) 12.0, 2.2 Hz) and 4.39 (dd, J ) 12.0, 6.6 Hz)] of the glucose
moiety due to the esterification correlated with the carbonyl carbon
(δ 167.1) of the sinapoyl group (Figure 1). Consequently, compound
1 was defined as (2-hydroxy-3-methoxy-5-allyl)phenyl ꢀ-D-(6-O-
E-sinapoyl)glucopyranoside.
NMR spectrum showed six carbons corresponding to a glucose
moiety (δ 104.3, 77.0, 75.0, 74.4, 70.9, and 63.8) and one methylene
(δ 40.2), one O-methyl (δ 56.2), two olefinic (δ 138.4 and 115.3),
and six aromatic (δ 148.8, 146.1, 136.5, 130.9, 112.2, and 108.9)
carbons, which suggested the presence of another phenylpropanoid
moiety as well as the sinapoyl group. Resonances of a 1,2,3,5-
tetrasubstituted aromatic ring [δ 6.68 (1H, d, J ) 1.7 Hz) and 6.55
(1H, d, J ) 1.7 Hz)], a vinyl group [δ 5.89 (1H, ddt, J ) 16.8,
10.0, 6.7 Hz), 4.99 (1H, ddt, J ) 16.8, 3.4, 1.5 Hz), and 4.93 (1H,
ddt, J ) 10.0, 3.4, 1.5 Hz)], an O-methyl group [δ 3.77 (3H, s)],
Compound 2 showed an [M - H]- at m/z 539.1776 correspond-
ing to the molecular formula C25H32O13 in the negative ion
HRFABMS. The IR spectrum revealed absorption bands due to
hydroxy (3600–3000 cm-1), carboxylic hydroxy (3000–2500 cm-1),
ketone (1715 cm-1), carboxylic carbonyl (1710 cm-1), and aromatic
ester (1701 cm-1) functions and an aromatic ring (1614 and 1520
cm-1). The presence of a galloyl group was supported by a two-
1
and a methylene group [δ 3.22 (2H, br d, J ) 6.7 Hz)] in the H
NMR spectrum along with mass fragment ions at m/z 367 [M - H
- 180]- and 179 in the negative ion FABMS spectrum suggested
the presence of a 5-hydroxyeugenol framework. In the HMBC
spectrum, the anomeric proton [δ 4.76 (1H, d, J ) 7.6 Hz)] showed
a correlation with an aromatic carbon (δ 146.1) attributed to C-1.
1
proton singlet at δ 7.07 in the H NMR spectrum and five carbon
signals (δ 168.3, 146.5, 139.8, 121.4, and 110.2) (Table 1). The
1H and 13C NMR spectra indicated the presence of a ꢀ-glucopy-
ranose moiety as in 1. The 6′′-methylene protons (δ 4.52 and 4.40)
showed HMBC correlation with the carbonyl carbon (δ 168.3) of
the galloyl group, indicating the acylation of the C-6′′ hydroxy
function (Figure 2). In the HMQC spectrum, the remaining 12
carbons corresponded to one ketone (δ 222.0), one carboxyl (δ
* To whom correspondence should be addressed. Phone: +81-6-6605-
2812. Fax: +81-6-6605-3086. E-mail: kikuzaki@life.osaka-cu.ac.jp.
† Osaka City University.
‡ The Open University of Japan.
10.1021/np0705615 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/04/2008

862 Journal of Natural Products, 2008, Vol. 71, No. 5
Notes
Furthermore, geminal methylene protons at δ 2.62 and 2.26
correlated with both carboxylic carbon (δ 176.4) and C-5′ (δ 39.0),
which showed that a carboxymethyl group was attached to C-5′.
These NMR data indicated 2 had a 5-(5-carboxymethyl-2-oxocy-
clopentyl)-3Z-pentenol moiety. In the 1H NMR spectrum, H-1′ and
H-5′ resonated at δ 1.91 and 2.24, respectively. Resonances for
H-1′ and H-5′ of the 1′,5′-cis isomer were reported at δ 2.35–2.45
and 2.80,10–13 whereas the trans isomer showed chemical shifts of
H-1′ and H-5′ at δ 2.20 and 2.30, respectively.14 Thus 2 possesses
1′,5′-trans relative configuration. The HMBC correlation of the
anomeric proton at δ 4.31 and the oxymethylene carbon at C-1 (δ
70.5) confirmed the glucose moiety was attached to C-1. Thus,
compound 2 was characterized as trans-1′,5′-5-(5-carboxymethyl-
2-oxocyclopentyl)-3Z-pentenyl ꢀ-(6-O-galloyl)glucopyranoside.
To determine the absolute configuration at C-1′ and C-5′, 2 was
first treated with tannase. After recognizing the production of gallic
acid by HPLC analysis, the enzymatic reaction mixture was
subsequently treated with ꢀ-glucosidase to give 5 and glucose. The
1H NMR spectrum of 5 corresponded to trans-tuberonic acid,14
and the specific rotation ([R]26D -67.1) was consistent with that of
the authentic compound with 1′R, 5′R configuration ([R]24D -67).15
Thus, 2 was concluded to be (1′R,5′R)-5-(5-carboxymethyl-2-
oxocyclopentyl)-3Z-pentenyl ꢀ-D-(6-O-galloyl)glucopyranoside.
Figure 1. Key HMBC correlations for compound 1.
Table 1. NMR Data (500 MHz, CD3OD) for Compounds 2 and
5a
2
5
position
δC
δH (J in Hz)
δH (J in Hz)
1a
1b
70.5 3.78, m
3.57, dt (14.1, 7.1)
3.54, td (7.0, 0.7)
3.54, td (7.0, 0.7)
2a,b
3
4
5a,b
1′
2′
29.1 2.38, br dt (7.3, 7.1)
128.9 5.46, dtt (10.7, 7.3, 1.5)
128.9 5.36, dtt (10.7, 7.3, 1.5)
26.2 2.32, m
55.1 1.91, dtd (9.5, 4.9, 1.4)
222.0
2.30, dt (7.0, 7.0)
5.48, dtt (11.0, 7.0, 0.7)
5.42, dtt (11.0, 7.0, 0.7)
2.39, dd (7.0, 5.4)
1.99, dtd (10.8, 5.4, 0.7)
Compound 3 exhibited a specific rotation of -36.6 and was found
to have a molecular formula of C28H40O14 based on an [M - H]-
peak at m/z 599.2339 in HRFABMS. The 1H and 13C NMR spectra
of 3 indicated that 3 consisted of a galloyl, a ꢀ-glucopyranose, a
pentofuranose, and monoterpene moieties (Table 2). The observed
3′a
3′b
4′a
4′b
5′
38.7 2.28, m
2.32, m
2.06, ddd (18.8, 11.5, 8.5) 2.08, ddd (18.8, 11.5, 8.8)
28.1 2.20, m
1.49, m
39.0 2.24, m
2.23, m
1.53, m
2.30, m
1
protons due to the monoterpene in the H NMR spectrum were
6′a
6′b
7′
39.9 2.62, dd (18.8, 8.8)
2.26, m
176.4
2.67, dd (18.8, 8.8)
2.32, m
one olefinic (δ 5.30), one methine (δ 1.58), three methyl (δ 1.58,
1.16, and 1.14) protons, and three pairs of geminal protons
corresponding to three methylenes, suggesting the presence of an
R-terpinyl moiety.2 The HMBC correlation between the anomeric
proton of ꢀ-glucopyranose [δ 4.49 (d, J ) 7.6 Hz)] and the C-8
resonance (δ 79.8) indicated that the partial structure was R-terpinyl
ꢀ-glucoside (Figure 3). Furthermore, the chemical shifts of C-9 and
C-10 (δ 23.0 and 25.0) were characteristic of a moiety with 4S
configuration.2,16 The 6′-methylene protons [δ 4.01 (dd, J ) 11.0,
2.7 Hz) and 3.62 (dd, J ) 11.0, 6.4 Hz)] showed an HMBC
correlation with the anomeric carbon of the pentose (δ 106.8) and
the anomeric proton of the pentose (δ 5.18 br s) with C-6′ (δ 67.7),
which confirmed a glycosidic linkage between C-1′′ and C-6′. An
oxymethine proton at δ 5.14 (dd, J ) 2.4, 1.0 Hz) was attributable
1′′
2′′
3′′
4′′
104.6 4.31, d (7.4)
75.1 3.20, dd (9.2, 7.4)
77.9 3.41, dd (9.2, 9.2)
71.7 3.38, dd (9.2, 9.2)
75.5 3.55, ddd (9.2, 5.8, 2.2)
64.8 4.52, dd (11.7, 2.2)
4.40, dd (11.7, 5.8)
139.8
110.2 7.07, s
146.5
121.4
168.3
5′′
6′′a
6′′b
1′′′
2′′′,6′′′
3′′′,5′′′
4′′′
7′′′
a Chemical shifts are shown in δ values (ppm) relative to solvent
peak.
1
to H-2′′ based on H-1H COSY and HMBC measurements. The
downfield shift of H-2′′ and the HMBC correlation between H-2′′
and the carbonyl carbon of the galloyl group (δ 166.0) indicated
esterification at C-2′′. The anomeric proton of the pentose (H-1′′)
appeared at δ 5.18 as a broad singlet. In addition, correlations were
observed between H-1′′ and H-3′′ [δ 4.15 (dd, J ) 6.0, 2.4 Hz)]
and between H-2′′ and H-4′′ [δ 4.10 (ddd, J ) 6.0, 5.5, 4.2 Hz)]
in the NOESY spectrum, suggesting that the pentofuranose might
be R-arabinofuranose.17
To confirm the structure of the pentose moiety, 3 was treated
with tannase to give 6 and gallic acid. Compound 6 showed an [M
+ H]+ peak at m/z 449.2389 in the HRFABMS spectrum, which
was 152 mass units, corresponding to C7H4O4, smaller than that of
3. The two-proton singlet observed in the 1H NMR spectrum of 3,
attributed to a galloyl group, was absent in the spectrum of 6. In
addition, the 2′′-methine proton was shielded in 6 [δ 3.98 (dd, J )
3.2, 1.2 Hz)] compared to 3. Acetylation of 6 gave a hexaacetyl
derivative (6a). In the 1H NMR spectrum of 6a, the signals due to
the pentose moiety showed the same coupling constants (J1′′,2′′ < 1
Hz, J2′′,3′′ ) 1.7 Hz, J3′′,4′′ ) 5.1 Hz) as those of synthetic R-terpinyl
2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-R-L-arabinofuranosyl)-ꢀ-
D-glucopyranoside, and the proton and carbon chemical shifts of
the sugar parts in 6a were consistent with those of the synthetic
one,16 which confirmed that pentofuranose was R-L-arabinofuranose.
Figure 2. Key HMBC correlations for compound 2.
176.4), two olefinic (δ 128.9 × 2C), one oxymethylene (δ 70.5),
five methylene (δ 39.9, 38.7, 29.1, 28.1, and 26.2), and two methine
1
(δ 55.1 and 39.0) carbons. In the H NMR spectrum, two olefinic
proton signals at δ 5.46 (dtt, J ) 10.7, 7.3, 1.5 Hz) and 5.36 (dtt,
J ) 10.7, 7.3, 1.5 Hz) indicated the presence of a cis double bond.
1
The proton sequence in the H-1H COSY spectrum showed the
presence of a framework of -OCH2CH2CHdCHCH2CHCH-
CH2CH2-. In the HMBC spectrum, vicinal methylene protons [δ
2.28 (H-3′a) and 2.20 (H-4′a)] correlated with the carbonyl carbon
at δ 222.0, and H-3′a also correlated with a methine carbon at δ
55.1, indicating the presence of a cyclopentanone structure.

Notes
Journal of Natural Products, 2008, Vol. 71, No. 5 863
Table 2. NMR Data (500 MHz) for Compounds 3 and 4 in CD3COCD3 and 6 in CD3ODa
3
4
6
position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3a
3b
4
134.2
121.7
27.5
134.1
121.7
27.5
134.9
121.9
28.1
5.30, m
1.98, m
1.72, m
1.58, dddd (11.8, 11.8,
4.9, 2.0)
1.96, m
1.15, m
1.87, br d (15.6)
1.87, m
1.58, br s
5.30, m
2.03, m
1.73, m
1.54, dddd (11.5, 11.5,
5.0, 2.0)
1.93, m
1.16, m
1.89, m
1.84, m
5.35, m
2.05, m
1.78, m
1.67, dddd (12.2, 12.2,
4.6, 2.2)
2.03, m
1.22, m
2.06, m
1.90, m
44.7
24.5
31.6
44.7
24.4
31.6
45.0
25.1
32.1
5a
5b
6a
6b
7
8
9
10
1′
2′
3′
4′
5′
6′a
6′b
1′′
2′′
3′′
4′′
23.6
79.8
23.0
25.0
98.1
75.0
78.1
72.0
75.9
67.7
23.5
79.6
23.8
24.2
98.0
75.0
78.2
72.1
76.0
67.6
1.58, br s
23.6
81.2
23.0
25.2
98.5
75.3
78.2
72.0
76.2
68.2
1.62, br s
1.14, s
1.16, s
4.49, d (7.6)
1.13, s
1.16, s
4.49, d (7.6)
1.17, s
1.22, s
4.46, d (7.6)
3.14, dd (8.8, 7.6)
3.40, dd (8.8, 8.8)
3.29, dd (8.8, 8.8)
3.47, ddd (8.8, 6.4, 2.7)
4.01, dd (11.0, 2.7)
3.62, dd (11.0, 6.4)
5.18, br s
5.14, dd (2.4, 1.0)
4.15, dd (6.0, 2.4)
4.10, ddd (6.0, 5.5, 4.2)
3.77, dd (11.5, 4.2)
3.71, dd (11.5, 5.5)
3.13, dd (9.0, 7.6)
3.39, dd (9.0, 9.0)
3.28, dd (9.0, 9.0)
3.46, ddd (9.0, 6.4, 2.7)
4.01, dd (11.0, 2.7)
3.62, dd (11.0, 6.4)
5.20, br s
5.14, dd (2.2, 0.7)
4.14, dd (4.2, 2.2)
4.10, ddd (6.6, 5.6, 4.2)
3.77, dd (11.7, 5.6)
3.71, dd (11.7, 6.6)
3.12, dd (9.0, 7.6)
3.34, dd (9.0, 9.0)
3.27, dd (9.0, 9.0)
3.39, ddd (9.0, 5.9, 2.4)
3.97, dd (11.0, 2.4)
3.54, dd (11.0, 5.9)
4.91, d (1.2)
3.98, dd (3.2, 1.2)
3.81, dd (5.9, 3.2)
3.94, ddd (5.9, 5.1, 3.4)
3.73, dd (12.0, 3.4)
3.63, dd (12.0, 5.1)
106.8
85.2
77.0
85.9
62.6
106.9
85.1
77.0
86.0
62.7
109.9
83.2
79.0
85.9
63.0
5′′a
5′′b
1′′′
2′′′,6′′′
3′′′,5′′′
4′′′
7′′
139.1
110.0
146.1
121.1
166.0
139.1
110.0
146.1
121.2
166.0
7.12, s
7.11, s
a Chemical shifts are shown in δ values (ppm) relative to solvent peak.
Table 3. DPPH Radical Scavenging Activity of Compounds
1-4 and Related Compoundsa
IC50 (µM)
1
2
3
4
15.0 ( 0.3e
11.2 ( 0.7d
10.5 ( 0.4c
11.6 ( 0.5d
8.0 ( 0.5b
25.7 ( 0.7f
27.5 ( 0.3g
gallic acid
sinapic acid
eugenol
a The concentration of DPPH radical was 100 µM in EtOH. The
absorbance of the reaction mixture at 520 nm against a blank of EtOH
without DPPH was measured by a multilabel counter after 180 min.
b–g Values with different letters are significantly different (p < 0.05).
Figure 3. Key HMBC correlations for compound 3.
On the basis of all the above data, compound 3 was concluded
to be (S)-R-terpinyl [R-L-(2-O-galloyl)arabinofuranosyl]-(1f6)-ꢀ-
D-glucopyranoside.
Experimental Section
Compound 4 exhibited a specific rotation of +14.1 and an [M
- H]- peak at m/z 599.2350 corresponding to the same molecular
General Experimental Procedures. Melting points were measured
using a Yanagimoto micromelting point apparatus and were uncorrected.
Optical rotations were measured using a Jasco P1030 polarimeter
(Tokyo, Japan). UV spectra were recorded on a Shimadzu UV-2500PC
UV–vis spectrophotometer (Kyoto, Japan). IR spectra were run on a
1
formula (C28H40O14) as 3. The H and 13C NMR data of 4 were
similar to those of 3. A difference in specific rotations suggested
1
that 4 was a diastereomer of 3. Comparing the H and 13C NMR
1
Jasco FT/IR685V (Tokyo, Japan). The H, 13C, and 2D NMR spectra
spectra of 3 and 4, differences were due to the protons of C-3-C-6
and the C-9 and C-10 resonances, corresponding to the R-terpinyl
moiety (Table 2). In the 13C NMR spectrum of 4, C-9 and C-10
were observed at δ 23.8 and 24.2, which were in good agreement
with those of (R)-R-terpinyl glucoside as previously reported.2 Thus,
compound 4 was identified as (R)-R-terpinyl [R-L-(2-O-galloyl)ara-
binofuranosyl]-(1f6)-ꢀ-D-glucopyranoside.
The radical-scavenging properties of compounds 1-4 together
with gallic acid, sinapic acid, and eugenol were evaluated against
DPPH radicals in a spectrophotometric assay.18,19 As shown in
Table 3, compound 1 showed higher activity than eugenol and
sinapic acid, the constituent units of 1. Compounds 2-4, which
possess galloyl groups, were more active than 1, but slightly less
active than gallic acid.
were recorded on a Varian Unity Plus 500 (500 MHz, Varian Inc.,
Palo Alto, CA) spectrometer. FABMS (matrix: glycerol) and
HRFABMS were measured on a JEOL JMS-700T mass spectrometer
(Tokyo, Japan). 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
(Jasco) connected to a UV detector (Jasco) operating at 280 nm. The
column was a Mightysil RH-18 (5 µm, 4.6 × 250 nm). For measuring
the DPPH radical scavenging activity, a Wallac 1420 Arvosx multilevel
counter (Perkin-Elmer Life Sciences Inc., Boston, MA) was employed.
Sinapic acid and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased
from Wako Pure Chemical Industries, Ltd. Gallic acid and eugenol
were previously isolated from allspice.1,2

864 Journal of Natural Products, 2008, Vol. 71, No. 5
Notes
Plant Material. Commercially available berries of Pimenta dioica
Merr., Myrtaceae, from Jamaica were kindly supplied by Taiyo
Corporation, Osaka, Japan. A voucher specimen (PD-0302) was
deposited at the Graduate School of Human Life Science, Osaka City
University.
3600–3100, 1692, 1604, 1525 cm-1; FABMS m/z 599 [M - H]-, 169;
HRFABMS m/z 599.2339 [M - H]- (calcd for C28H39O14, 599.2340).
Enzymatic Hydrolysis of 3. A solution of 3 (4.6 mg) in H2O (2
mL) was preincubated at 30 °C for 2 h; then 2 mg of tannase (49 units/
mg, Aspergillus oryzae, Wako) was added, and the sample solution
was incubated at 30 °C for 1 h. HPLC analysis [solvent, CH3CN-0.1%
HOAc in H2O (30:70, v/v); flow rate, 0.5 mL/min] showed that 3 (tR
17.0 min) disappeared and gallic acid (tR 7.8 min) was formed. The
reaction mixture was concentrated in Vacuo, and then 1 mL of MeOH
was added to the residue. After sonication and subsequent filtration,
the filtrate was purified on Sephadex LH-20 CC (CH3OH) to give 6
(2.4 mg); δH and δC (Table 2); [R]29D -41.3 (c 0.24, MeOH); FABMS
m/z 449 [M + H]+, 313, 295; HRFABMS m/z 449.2389 [M + H]+
(calcd for C21H37O10, 449.2387).
Acetylation of 6. A solution of 6 (1.6 mg) in pyridine (0.3 mL) and
Ac2O (0.3 mL) was allowed to stand overnight at room temperature.
The reaction mixture was poured into cold 2 N HCl and then extracted
with EtOAc. The organic layer was washed with saturated NaCl(aq),
dried over anhydrous Na2SO4, and evaporated to dryness to give 6a
(2.4 mg): 1H NMR [CDCl3, 500 MHz] δ 5.33 (1H, m, H-2), 5.20 (1H,
dd, J ) 9.8, 9.5 Hz, H-3′), 5.072 (1H, br d, J ) 1.7 Hz, H-2′′), 5.070
(1H, br s, H-1′′), 5.01 (1H, dd, J ) 9.7, 9.5 Hz, H-4′), 4.96 (1H, ddd,
J ) 5.1, 1.7, 0.7 Hz, H-3′′), 4.93 (1H, dd, J ) 9.8, 8.1 Hz, H-2′), 4.67
(1H, d, J ) 8.1 Hz, H-1′), 4.41 (1H, dd, J ) 12.0, 3.2 Hz, H-5′′a),
4.28 (1H, ddd, J ) 5.6, 5.1, 3.2 Hz, H-4′′), 4.21 (1H, dd, J ) 12.0, 5.6
Hz, H-5′′b), 3.75 (1H, dd, J ) 11.0, 2.2 Hz, H-6′a), 3.65 (1H, ddd, J
) 9.7, 6.1, 2.2 Hz, H-5′), 3.59 (1H, dd, J ) 11.0, 5.9 Hz, H-6′b), 2.14
(3H, s, 3′′-OCOCH3), 2.10 (3H, s, 5′′-O OCOCH3), 2.08 (3H, s, 2′′-
OCOCH3), 2.03 (3H, s, 4′-OCOCH3), 2.02 (3H, s, 2′-OCOCH3), 2.00
(3H, s, 3′-OCOCH3), 1.99 (1H, m, H-6a), 1.96 (1H, m, H-3a), 1.90
(1H, m, H-6b), 1.85 (1H, ddddd, J ) 12.7, 2.4, 2.4, 2.4, 0.7 Hz, H-5eq),
1.72 (1H, m, H-3b), 1.63 (3H, br s, H-7-CH3), 1.54 (1H, dddd, J )
11.7, 11.7, 4.4, 2.4 Hz, H-4), 1.18 (1H, dddd, J ) 12.0, 11.7, 11.7, 5.6
Hz, H-5ax), 1.17 (3H, s, 10-CH3), 1.12 (3H, s, 9-CH3); 13C NMR
[CDCl3, 125 MHz] δ 170.7 (5′′-OCOCH3), 170.5 (3′-OCOCH3), 170.3
(3′′-OCOCH3), 169.5 (4′-OCOCH3, 2′′-OCOCH3), 169.2 (2′-OCOCH3),
134.2 (C-1), 120.3 (C-2), 105.7 (C-1′′), 95.1 (C-1′), 81.0 (C-2′′), 80.4
(C-8, C-4′′), 77.2 (C-3′′), 73.2 (C-3′), 72.8 (C-5′), 71.5 (C-2′), 69.2
(C-4′), 65.9 (C-6′), 63.3 (C-5′′), 43.9 (C-4), 30.9(C-6), 26.7 (C-3), 24.9
(C-10), 23.5 (C-5), 23.4 (C-7), 22.1 (C-9), 20.8, 20.77, 20.76, 20.74,
20.73, 20.70 (2′, 3′, 4′, 2′′, 3′′, 5′′-OCOCH3).
Extraction and Isolation. Dried and ground berries of P. dioica
(1890 g) were successively extracted with n-hexane (6 × 3 L) and
70% aqueous Me2CO (6 × 3 L) at room temperature. For each
extraction, the plant material was soaked in the solvent and allowed to
stand overnight. Acetone from the combined 70% aqueous Me2CO
extract was evaporated in Vacuo, and the resulting aqueous residue was
partitioned consecutively with n-hexane and EtOAc to obtain n-hexane-
soluble, EtOAc-soluble, and H2O-soluble fractions. The EtOAc-soluble
fraction (43.9 g) was subjected to Sephadex LH-20 column chroma-
tography (CC) using 2-propanol to give five fractions. Fraction 4 (10.7
g) was rechromatographed over ODS gel (CH3CN-H2O, 3:7) to give
10 fractions, A-J. Fraction B (3.38 g) was subjected to Sephadex LH-
20 CC using CH3OH to yield fractions B1-B6. Fraction B2 (240 mg)
was purified by Sephadex LH-20 CC (Me2CO-H2O, 7:3) followed by
ODS CC (CH3CN-H2O, 13:87) to give compound 2 (22 mg). Fraction
B3 (547 mg) was purified by Sephadex LH-20 CC using CH3OH
repeatedly to afford quercetin 3-O-ꢀ-glucuronide 6′′-methyl ester7 (8.5
mg). Fraction B4 (977 mg) was rechromatographed over ODS gel
(CH3CN-H2O, 15:85) followed by Sephadex LH-20 (CH3OH) to give
hyperoside4 (68.4 mg), myricetin 3-O-galactoside8 (8.7 mg), and (+)-
ampelopsin9 (5.9 mg). Fraction C (1.78 g) was subjected to Sephadex
LH-20 CC using CH3OH followed by ODS CC (CH3CN-H2O, 15:
85) to give avicularin5 (11.0 mg), hyperoside (165.5 mg), (+)-
ampelopsin (4.0 mg), quercetin 3-O-(2-O-galloyl)-ꢀ-D-glucoside6 (7.2
mg), and myricetin (3.7 mg). Fraction E (675 mg) was rechromato-
graphed on Si gel (CH2Cl2-CH3OH, 9:1) to give 10 fractions, E1-E10.
Fraction E8 (72 mg) was further subjected to repeated column
chromatography over ODS gel (CH3CN-H2O, 25:75) to afford 3 (20
mg) and 4 (8.6 mg). Fraction H (70 mg) was subjected to Sephadex
LH-20 CC eluted with Me2CO to give 1 (10.0 mg) and quercetin (22.0
mg).
Compound 1: colorless viscous liquid; [R]25 -21.2 (c 0.82,
D
MeOH); UV (MeOH) λmax (log ꢀ) 283 (3.75), 328 (3.94) nm; IR (Nujol)
1
νmax 3600–3100, 1717, 1604, 1510 cm-1; H NMR [(CD3)2CO, 500
MHz] δ 7.63 (1H, d, J ) 15.9 Hz, H-7′′), 7.04 (2H, s, H-2′′, 6′′), 6.68
(1H, d, J ) 1.7 Hz, H-6), 6.55 (1H, d, J ) 1.7 Hz, H-4), 6.48 (1H, d,
J ) 15.9 Hz, H-8′′), 5.89 (1H, ddt, J ) 16.8, 10.0, 6.7 Hz, H-8), 4.99
(1H, ddt, J ) 16.8, 3.4, 1.5 Hz, H-9a), 4.93 (1H, ddt, J ) 10.0, 3.4,
1.5 Hz, H-9b), 4.76 (1H, d, J ) 7.6 Hz, H-1′), 4.53 (1H, dd, J ) 12.0,
2.2 Hz, H-6′a), 4.39 (1H, dd, J ) 12.0, 6.6 Hz, H-6′b), 3.90 (6H, s,
3′′-OCH3, 5′′-OCH3), 3.77 (3H, s, 3-OCH3), 3.74 (1H, ddd, J ) 9.3,
6.6, 2.0 Hz, H-5′), 3.56 (1H, dd, J ) 9.9, 9.0 Hz, H-3′), 3.52 (1H, dd,
J ) 9.0, 7.6 Hz, H-2′), 3.47 (1H, dd, J ) 9.3, 9.0 Hz, H-4′), 3.22 (2H,
br d, J ) 6.7 Hz, H-7a,b); 13C NMR [(CD3)2CO, 125 MHz] δ 167.1
(C-9′′), 148.8 (C-3), 148.6 (C-3′′, C-5′′), 146.1 (C-1, C-7′′), 139.2 (C-
4′′), 138.4 (C-8), 136.5 (C-2), 130.9 (C-5), 125.8 (C-1′′), 115.4 (C-
8′′), 115.3 (C-9), 112.2 (C-6), 108.9 (C-4), 106.6 (C-2′′, C-6′′), 104.3
(C-1′), 77.0 (C-3′), 75.0 (C-5′), 74.4 (C-2′), 70.9 (C-4′), 63.8 (C-6′),
56.4 (3′′-OCH3, 5′′-OCH3), 56.2 (3-OCH3), 40.2 (C-7); FABMS m/z
547 [M - H]-, 367 [M - H - 180]-, 341 [M - H - 206]-, 223, 205,
179, 164; HRFABMS m/z 547.1798 (calcd for C27H31O12, 547.1815).
Compound 4: white powder (acetone-benzene); mp 128–129 °C;
δH and δC (Table 2); [R]25 +14.1 (c 0.12, MeOH); FABMS m/z 599
D
[M - H]-, 169; HRFABMS m/z 599.2350 [M - H]- (calcd for
C28H39O14, 599.2340).
Determination of the Scavenging Effect on DPPH Radicals.19
To 75 µL of the EtOH solution of each test compound at different
final concentrations (3.125, 6.25, 12.5, 25.0, and 50.0 µM) in a 96-
well flat-bottom microplate was added 75 µL of an EtOH solution of
DPPH radical (final concentration, 100 µM). Neat EtOH (75 µL) was
used as a control. After the reaction mixtures were slightly shaken and
held for 180 min at room temperature in a multilabel counter, the
absorbance of test compounds was measured at 520 nm against a blank
of EtOH without DPPH. DPPH radical scavenging activity was
calculated according to the following equation: DPPH radical scaveng-
ing activity (%) ) [(absorbance of control – absorbance of test
compound)/absorbance of control] × 100. IC50 was defined as the
concentration of compounds that showed 50% DPPH radical scavenging
activity. All analyses were carried out in triplicate, and the values were
averaged. A factorial analysis of variance (ANOVA) with multiple
comparisons and linear regression were used. Significance was estab-
lished at p < 0.05.
Compound 2: colorless, viscous liquid; [R]20 -27.7 (c 0.30,
D
MeOH); UV (MeOH) λmax (log ꢀ) 276 (4.14) nm; IR (film) νmax
3600–3000, 3000–2500, 1715, 1710, 1701, 1614, 1520, 1322, 1232,
1039, 874 cm-1; δH and δC (Table 1); FABMS m/z 539 [M - H]-,
169; HRFABMS m/z 539.1776 (calcd for C25H31O13, 539.1765).
Enzymatic Hydrolysis of 2. A solution of 2 (5 mg) in H2O (3 mL)
was incubated with 5 mg of tannase (49 units/mg, Aspergillus oryzae,
Wako) at 30 °C for 1.5 h. HPLC analysis [solvent: CH3CN-0.1%
HOAc in H2O (15:85, v/v); flow rate: 0.7 mL/min] showed that 1 (tR
26.5 min) disappeared and gallic acid (tR 6.2 min) was formed. Then,
ꢀ-glucosidase (5 mg) was added to the reaction mixture followed by
incubation at 37 °C for 3 h. The reaction mixture was extracted with
EtOAc (3 × 3 mL) and the EtOAc extract (3.1 mg) was chromato-
graphed on Si gel (CH2Cl2-CH3OH ) 85:15) to obtain gallic acid
and 5 (2.1 mg): δH (Table 1); FABMS m/z 215 [M - H]-; [R]26D -67.1
Acknowledgment. This research was supported financially by
Grants-in-Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology of Japan (MEXT). We thank
Ms. T. Maekawa for measurement of the NMR spectra and Ms. R.
Miyake for measurement of the MS spectra.
References and Notes
(1) Kikuzaki, H.; Hara, S.; Kawai, Y.; Nakatani, N. Phytochemistry 1999,
52, 1307–1312.
(2) Kikuzaki, H.; Sato, A.; Mayahara, Y.; Nakatani, N. J. Nat. Prod. 2000,
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(3) Miyajima, Y.; Kikuzaki, H.; Hisamoto, M.; Nakatani, N. BioFactors
2004, 21, 301–303.
(c 0.21, MeOH) [lit.15, [R]24 -67].
D
Compound 3: white powder (acetone-benzene); mp 122–123 °C;
δH and δC (Table 2); [R]25 -36.6 (c 0.78, MeOH); IR (Nujol) νmax
D

Notes
Journal of Natural Products, 2008, Vol. 71, No. 5 865
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