New glycosides of acetophenone derivatives and phenylpropanoids from Juniperus occidentalis

Article abstract of DOI:10.1007/s11418-012-0694-3

New glycosides of seven acetophenone derivatives (1-7) and two phenylpropanoids (8, 9), named juniperosides III-XI, have been isolated from the MeOH extract of the leaves and stems of Juniperus occidentalis Hook. (Cupressaceae), together with eleven other known compounds. The structures of these compounds have been successfully elucidated using a variety of spectroscopic techniques.

Full text of DOI:10.1007/s11418-012-0694-3

J Nat Med (2013) 67:359–368
DOI 10.1007/s11418-012-0694-3
ORIGINAL PAPER
New glycosides of acetophenone derivatives and phenylpropanoids
from Juniperus occidentalis
Yuka Inatomi
Tsutomu Nakanishi
Jin Murata Munekazu Iinuma
•
Hiroko Murata
•
Akira Inada
•
•
•
Frank A. Lang
•
Received: 14 June 2012 / Accepted: 10 July 2012 / Published online: 31 July 2012
Ó The Japanese Society of Pharmacognosy and Springer 2012
Abstract New glycosides of seven acetophenone deriv-
atives (1–7) and two phenylpropanoids (8, 9), named ju-
niperosides III–XI, have been isolated from the MeOH
extract of the leaves and stems of Juniperus occidentalis
Hook. (Cupressaceae), together with eleven other known
compounds. The structures of these compounds have been
successfully elucidated using a variety of spectroscopic
techniques.
and can be found in Northern California, Oregon, Wash-
ington and Nevada. In our previous report, describing the
phytochemical study of plants native to the northwest of
America, we investigated the chemical components of two
Juniperus plants collected in Oregon, including J. occi-
dentalis and J. communis var. depressa. As a consequence
of this investigation, we reported the isolation of two
phenol glucosides, two biflavones and a diterpene from
J. occidentalis [1], and 17 phenylpropanoids, 12 neo-
lignans, 14 flavonoids, 7 catechins, 5 terpenoids, and
2 phenolic compounds from J. communis var. depressa
[2–7]. The current study is focused on the constituents of
J. occidentalis and here we report the isolation of 9 new
compounds (1–9) (Fig. 1), together with 11 known com-
pounds from this particular plant species. Furthermore, the
structural elucidation of these new compounds will be
discussed.
Keywords Juniperus occidentalis ꢀ Cupressaceae ꢀ
Phenol glycoside ꢀ Acetophenone derivative ꢀ
Phenylpropanoid
Introduction
Juniperus occidentalis Hook. (Cupressaceae) is an ever-
green tree which typically grows to 20 m in height. The
tree is well distributed along the west coast of the USA
Results and discussion
The n-BuOH soluble part obtained from the MeOH extract
of the leaves and stems of J. occidentalis was separated
using a combination of silica gel, ODS, and Sephadex
LH-20 column chromatography and HPLC purification, to
isolate the nine new glycosides of the acetophenone
derivatives (1–7), two new phenylpropanoid glucosides
Y. Inatomi (&) ꢀ H. Murata ꢀ A. Inada ꢀ T. Nakanishi
Faculty of Pharmaceutical Sciences, Setsunan University,
45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan
e-mail: inatomi@pharm.setsunan.ac.jp
F. A. Lang
Department of Biology, Southern Oregon University,
1
250 Siskiyou, Ashland, OR 97520-5071, USA
(
8, 9), and 11 known compounds. The known compounds
were identified as junipediol A 8-O-b-D-glucopyranoside
8], juniperoside [9], dihydrosyringin [10], skimmin [11],
0
J. Murata
[
Botanical Gardens, Koishikawa, Graduate School of Science,
The University of Tokyo, 3-7-1 Hakusan, Bunkyo-ku,
Tokyo 112-0001, Japan
apigenin 4 -O-b-D-glucopyranoside [12], kaempferol 3-O-
a-L-rhamnopyranoside [13], quercitrin [13], isoscutellarein
7
-O-b-D-xylopyranoside [2], naringenin 7-O-b-D-glucopy-
M. Iinuma
Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi,
Gifu 501-1196, Japan
ranoside [14], aromadendrin 3-O-a-L-rhamnopyranoside
[15], and aromadendrin 7-O-b-D-glucopyranoside [16] by
123

3
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J Nat Med (2013) 67:359–368
0
0
0
0
0
0
between H-3 /C-1 , C-5 , between H-4 /C-2 , C-6 , and
0
0
0
0
between H-5 /C-1 , C-3 and C-6 -CH (Fig. 2), the structure
3
of the aglycone was identified as being 1-(2-hydroxy-6-
methylphenyl)ethanone. The position of the b-D-glucopyr-
anosyl residue in 1 was determined as follows. The HMBC
0
0
0
0
correlation between the anomeric proton (H-1 ) and C-2
0
0
together with the NOESY cross peak between H-1 and H-3
indicated that the b-D-glucopyranose was connected to the
0
phenolic group (2 -OH) of the aglycone. In conclusion, the
0
0
structure of 1 was defined as 1-(2 -hydroxy-6 -methyl-
0
phenyl)ethanone 2 -O-b-D-glucopyranoside and subse-
quently named juniperoside III.
Compound 2 was isolated as a white amorphous powder,
a] -61.7° (c = 0.85, MeOH), and had a molecular for-
[
D
mula C H O , as evidenced by the quasimolecular ion
2
0 28 11
-
M - H] peak at m/z 443.1553 in the negative ion HR
FAB-MS. The FAB-MS also provided two significant
[
Fig. 1 Structures of compounds 1–9
-
fragments at m/z 311 [M-H-132 (a pentose unit)] and 149
-
[M-H-294 (both pentose and hexose units)] . The IR and
comparison of the physical and spectroscopic data with the
literature values.
UV data of 2 were very similar to those of 1. Taken
together, these data led us to assume that 2 was a pentosyl
Compound 1 was isolated as a white amorphous powder,
1
13
derivative of juniperoside III (1). Analysis by H- and C-
[
a] -12.4° (c = 0.17, MeOH), and gave a quasimolecular
D
-
ion [M - H] peak at m/z 311 in the negative ion mode of
the fast atom bombardment mass spectrum (FAB-MS). The
high resolution (HR) mass spectrum of the compound in
the same ionization mode revealed the molecular formula
to be C H O . In addition, the FAB-MS (negative mode)
NMR spectra revealed signals for a b-glucopyranosyl
(J100 00 = 7.8 Hz) and an a-arabinofuranosyl (J1000 000
=
,2
,2
1.5 Hz) moiety. Subsequent HPLC analysis of the acid
hydrolysate revealed the presence of D-glucose and
1
L-arabinose. In the C-NMR spectrum, a significant gly-
3
1
5 20 7
00
spectrum also afforded a significant fragment peak at m/z
49, which was consistent with the loss of a hexosyl unit
cosylation shift was observed for the resonance at the C-6
1
(dC 68.3) relative to the corresponding resonance in 1
(Table 1). Furthermore, the position of the a-L-arabino-
furanosyl moiety in 2 was determined by a HMBC
experiment, which revealed a long-range correlation
-
from the [M - H] ion. UV (209, 278 nm) and IR (3392,
-
1
1
691, 1465, and 1077 cm ) absorptions suggested the
existence of hydroxyl and aromatic ketone groups in 1.
1 13
Analysis by H- and C-NMR spectral data (Table 1)
000
00
between the anomeric proton (H-1 ) and the 6 -carbon. The
0
revealed the presence of a b-glucopyranosyl moiety
structure of 2 was therefore identified as being 1-(2 -hydroxy-
0
0
00
(
J100 00 = 7.8 Hz), and the presence of D-glucose was con-
6 -methylphenyl)ethanone 2 -O-(6 -O-a-L-arabinofuranosyl)-
b-D-glucopyranoside, and subsequently named juniperoside
IV.
,2
firmed by HPLC analysis of the acid hydrolysate. Fur-
1
thermore, analysis by H-NMR spectral data revealed the
presence of an aromatic methyl [d 2.20 (3H, s)], an acetyl
methyl [d 2.55 (3H, s)] and a 1,2,3-trisubstituted benzene
ring [d 6.91 (1H, d, J = 8.0 Hz, H-5 ), 7.08 (1H, d,
Compound 3 was isolated as a white amorphous powder,
[a] -31.8° (c = 1.0, MeOH), and its IR and UV spectra
contained absorption bands indicative of hydroxyl and
D
0
0
0
J = 8.0 Hz, H-3 ), 7.24 (1H, dd, J = 8.0, 8.0 Hz, H-4 )].
Taken together, these results suggested that 1 was an
acetophenone glucoside. A nuclear Overhauser exchange
spectroscopy (NOESY) spectrum of 1 revealed the occur-
rence of a significant cross peak between the aromatic
methyl and the acetyl methyl (Fig. 2), indicating that
methyl and acetyl substituents were located on vicinal
carbons of the benzene ring. The heteronuclear multiple
bond correlation (HMBC) spectrum exhibited correlations
aromatic ketone groups. The HR FAB-MS (negative mode)
-
of 3 provided an [M - H] ion peak at m/z 327.1077. This
mass was indicative of the molecular formula C H O and
15 20 8
corresponded to a substance containing one more oxygen-
atom unit than 1, which has a molecular formula of
C H O . With the exception of the appearance of an
1
5 20 7
oxymethylene [d 5.28 (1H, d, J = 17.6 Hz), 5.38 (1H, d,
H
J = 17.6 Hz), d 75.2] in 3 instead of the acetyl methyl
C
1
13
observed in 1, analysis by H- and C-NMR spectral data
(Table 1) revealed that 3 was structurally similar to 1. These
data suggested that 3 was a glucoside of an acetophenone
derivative. Furthermore, the presence of D-glucose was
confirmed by HPLC analysis of the acid hydrolysate of 3.
0
between the aromatic methyl protons (H -6 -CH ) and
3
3
0
0
0
C-1 , C-5 , and C-6 . Correlations were also observed
0
between H -2 and C-1 and C-1 . From these data, together
3
with the data from further HMBC correlations observed
1
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J Nat Med (2013) 67:359–368
361
1
13
3
Table 1 H- and C-NMR data for 1–4 in CD OD
a
Position
1
2
3
3
4
d
H
d
C
d
H
d
C
d
H
d
C
d
H
d
C
d
H
d
C
1
2
208.1
208.0
32.7 4.82 (d, 17.6)
.96 (d, 17.6)
206.1
d
203.9
203.4
76.3
c
c
2.55 (s)
32.7 2.55 (s)
5.28 (d, 17.6)
5.38 (d, 17.6)
75.2 4.82 (d, 17.6)
5.00 (d, 17.6)
127.6
4
0
1
133.5
155.3
133.4
155.3
126.6
156.9
117.1
161.9
0
2
156.7
0
3
7.08 (d, 8.0)
7.24 (dd, 8.0,
114.2 7.10 (d, 8.0)
114.3 6.69 (d, 7.8)
114.4 6.97 (d, 7.8)
114.2 6.17 (br d, 2.3) 101.4
0
4
131.3 7.26 (dd, 8.0,
7.8)
131.4 7.14 (dd, 7.8,
7.8)
132.5 7.20 (dd, 7.8,
7.8)
131.2
162.7
8
.0)
0
5
6.91 (d, 8.0)
125.6 6.90 (d, 7.8)
125.6 6.72 (d, 7.8)
123.0 6.75 (d, 7.8)
121.9 6.20 (dd, 2.3,
0
111.8
.8)
0
6
136.3
136.3
138.7
137.6
142.9
22.1
0
6
-CH
0
3
2.20 (s)
19.3 2.20 (s)
102.5 4.94 (d, 7.8)
19.1 2.24 (s)
102.4 4.38 (d, 7.8)
19.8 2.32 (s)
104.4 5.12 (d, 7.8)
19.6 2.31 (s)
0
Glc1
4.95 (d, 7.8)
104.6 4.36 (d, 7.8)
104.5
75.2
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
3.43 (dd, 7.8,
74.9 3.41 (dd, 7.8,
9.0)
74.9 3.30 (dd, 7.8,
9.0)
75.2 4.15 (dd, 7.8,
9.0)
75.2 3.31 (dd, 7.8,
9.0)
9
.0)
3.44 (dd, 9.0,
.0)
3.36 (dd, 9.0,
.0)
3.42 (ddd, 9.0,
.5, 2.2)
3.68 (dd, 12.0,
.5)
.87 (dd, 12.0,
.2)
78.3 3.44 (dd, 9.0,
9.0)
78.3 3.39 (dd, 9.0,
9.0)
77.8 4.25 (dd, 9.0,
9.0)
78.4 3.39 (dd, 9.0,
9.0)
77.8
71.5
78.2
62.8
9
71.3 3.34 (dd, 9.0,
9.0)
71.8 3.32 (dd, 9.0,
9.0)
71.5 4.27 (dd, 9.0,
9.0)
71.4 3.31 (dd, 9.0,
9.0)
9
b
78.3 3.61
77.0 3.27 (ddd, 9.0,
5.5, 2.3)
78.2 3.95 (ddd, 9.0,
5.5, 2.3)
78.8 3.28 (ddd, 9.0,
5.5, 2.3)
5
b
62.6 3.61
68.3 3.66 (dd, 12.0,
5.5)
62.7 4.37 (dd, 12.0,
5.5)
62.7 3.66 (dd, 12.0,
5.5)
5
3
4.05 (dd, 11.0,
1.5)
3.85 (dd, 12.0,
2.3)
4.53 (dd, 12.0,
2.3)
3.86 (dd, 12.0,
2.3)
2
0
00
Ara(f)1
4.91 (d, 1.5)
110.1
83.3
0
0
0
0
00
00
00
00
2
3
4
5
3.98 (dd, 3.3,
1
.5)
3.82 (dd, 5.8,
.3)
3.95 (ddd, 5.8,
.8, 3.3)
78.9
85.8
63.1
3
5
b
3.61
.71 (dd, 11.0,
.3)
3
3
a
b
c
d
Measured in C
5 5
D N
Overlapping with other signals
Disappeared after a few hours standing of the MeOH solution
Not observed
00
the NOESY (between H-1 /H -2) and HMBC (between
2
Significant cross peaks were observed in the NOESY
00
spectrum of 3 between the aromatic methyl [d 2.32 (3H, s)]
and each of an oxymethylene (H -2) and an aromatic proton
H-1 /C-2) spectra. Thus, the structure of 3 was elucidated
0
0
to be 1-(2 -hydroxy-6 -methylphenyl)-2-hydroxyethanone
2-O-b-D-glucopyranoside, which was subsequently named
as juniperoside V. The signals attributed to the oxymeth-
2
0
1
H-5 ) (Fig. 2). The spectral evidence from the H-NMR
(
and NOESY spectra, together with detailed analysis of the
HMBC correlations observed in 3 (Fig. 2), indicated that
the aglycone structure of 3 was 1-(2-hydroxy-6-methyl-
phenyl)-2-hydroxyethanone. The position of the b-D-glu-
copyranosyl moiety was established as being at the C-2-OH
because of the presence of the respective cross peaks in
ylene protons (H -2) disappeared over time when the NMR
2
spectra of 3 were measured in methanol-d . Although this
4
solvent is typically used for the analysis of the glycosides of
1 13
acetophenone derivatives, the H and C NMR spectra of 3
were subsequently measured in pyridine-d solutions.
5
123

3
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J Nat Med (2013) 67:359–368
Fig. 2 The key HMBC and
NOESY correlations in 1, 3,
and 4
Compound 4 was isolated as a white amorphous powder,
a]D -34.0° (c = 1.0, MeOH), and the HR FAB-MS
negative mode) gave an [M - H] ion peak at m/z
substituent [-CH(OH)-CH ] on an aromatic ring [d 1.55 (3H,
d, J = 6.8 Hz, H -2), 5.35 (1H, q, J = 6.8 Hz, H-1)], and an
3
3
[
(
-
aromatic methyl [d 2.41 (3H, s)]. Signals indicative of the
3
43.1026. This mass data was indicative of a molecular
presence of both b-glucopyranosyl (J100 00 = 7.8 Hz) and
,2
formula of C H O , which contains one more oxygen
1
a-arabinofuranosyl (J 000 000 = 1.5 Hz) moieties were also
5
20
9
1
,2
13
1
atom than that of 3. In addition, the IR and UV spectra
1
exhibited similar absorption bands to those of 3. The H-
observed in H- and C-NMR spectra (Table 2) and
D-glucose and L-arabinose were identified by chiro-optical
HPLC analysis of the acid hydrolysate of 5. Taken together,
these data suggested that 5 was a pentosyl derivative of
1
3
and C-NMR spectral data (Table 1) revealed the presence
of a b-glucopyranosyl (J100 00 = 7.8 Hz) moiety, and the
,2
0 0
presence of D-glucose was confirmed by HPLC analysis of
1
the corresponding acid hydrolysate. The H-NMR spec-
juniperoside I [(1S)-1-(2 -hydroxy-6 -methylphenyl)ethanol
0
13
2 -O-b-D-glucopyranoside] [1]. The C-NMR signals of 5
(Table 2) were compared with the corresponding signals in
the juniperoside I spectrum [1]. It was clear that 5 contained
an additional a-L-arabinofuranosyl moiety located at the
6-OH of the glucosyl residue of juniperoside I. The position
of the a-L-arabinofuranosyl moiety in 5 was also confirmed
trum revealed the presence of an aromatic methyl [d 2.31
(
3H, s)], an oxymethylene [d 4.82 (1H, d, J = 17.6 Hz),
H
5
.00 (1H, d, J = 17.6 Hz)] and a 1,2,3,5-tetrasubstituted
0
benzene ring [d 6.17 (1H, br d, J = 2.3 Hz, H-3 ), 6.20
(1H, dd, J = 2.3, 0.8 Hz, H-5 )]. These results suggested
that 4 was also the glucoside of an acetophenone deriva-
0
0
00
00
000
00
by HMBC (H-1 /C-6 ) and NOESY (H-1 /H -6 ) corre-
2
tive. NOESY correlations were observed between the
0
lations. The enzymatic hydrolysis of 5 gave an aglycone
(C H O , an amorphous powder, [a] -43°), which was
identified as the aglycone of juniperoside I. The structure of
aromatic methyl protons and H -2, H-5 , and the anomeric
2
9
12
2
D
0
0
proton (H-1 ) and H -2. In addition, the HMBC spectrum
2
0 0
of 4 exhibited several correlations, which are shown in
Fig. 2. Based on these data, the structure of 4 was char-
5 was therefore established as being (1S)-1-(2 -hydroxy-6 -
0
00
methylphenyl)ethanol 2 -O-(6 -O-a-L-arabinofuranosyl)-b-
D-glucopyranoside and subsequently named juniperoside
VII.
0
acterized as the 4 -hydroxy derivative of 3 and therefore
0
0
0
identified as being 1-(2 ,4 -dihydroxy-6 -methylphenyl)-2-
hydroxyethanone 2-O-b-D-glucopyranoside, and subse-
quently named juniperoside VI.
Compounds 6 and 7 were both isolated as white amor-
phous powders. The negative ion HR FAB-MS of both
-
compounds gave quasimolecular ion [M - H] peaks at
Compound 5 was isolated as a white amorphous powder,
a] -62.1° (c = 1.0, MeOH), and its HR FAB-MS (nega-
[
m/z 329, indicating that they both had a molecular formula
of C H O . In addition, 6 and 7 both gave a fragment ion
D
-
tive mode) afforded an [M - H] ion peak at m/z 445.1713,
1
5 22 8
-
peak at m/z 167 [M-H-162 (hexosyl unit)] . The H- and
1
which corresponded to a molecular formula of C H O .
20 30 11
1
3
The FAB-MS of 5 also gave two fragments at m/z 313 [M-H-
1
C-NMR spectral data of 6 and 7 (Table 2) were very
similar to those of the juniperosides I and II [1], respec-
tively, with the exception of the signals corresponding to
-
32 (pentose unit)] and 151 [M-H-132-162 (hexose
-
1
unit)] . The H-NMR data revealed that the aglycone unit
contained a 1,2,3-trisubstituted benzene ring [d 6.89 (1H, br
the 1,2,3,4-tetrasubstituted benzene ring [d 6.64 and 6.95
H
0
0
d, J = 7.3 Hz, H-5 ), 7.08 (1H, br d, J = 8.0 Hz, H-3 ), 7.11
0
(d, J = 8.8 Hz) in 6; d 6.63 and 7.06 (d, J = 8.8 Hz) in 7]
H
(
1H, dd, J = 7.3, 8.0 Hz, H-4 )], a 1-hydroxyethyl
relative to the 1,2,3-trisubstituted benzene ring in
1
23

J Nat Med (2013) 67:359–368
363
1
13
3
Table 2 H- and C-NMR data for 5–7, 6a and 7a in CD OD
Position
5
6
7
6a, 7a
d
H
d
C
d
H
d
C
d
H
d
C
d
H
d
C
1
2
5.35 (q, 6.8)
1.55 (d, 6.8)
66.4 5.38 (q, 6.8)
22.8 1.53 (q, 6.8)
66.6 5.19 (q, 6.8)
23.0 1.55 (d, 6.8)
67.2 5.23 (q, 6.8)
22.8 1.42 (d, 6.8)
69.0
22.8
0
1
134.0
124.2
134.9
123.5
152.2
129.9
122.4
149.2
0
2
157.1
135.3
0
3
7.08 (br d, 8.0)
7.11 (dd, 8.0, 7.3)
6.89 (br d, 7.3)
116.5
152.5
0
b
b
4
129.1 6.64 (d, 8.8)
127.1 6.95 (d, 8.8)
138.0
114.8 6.63 (d, 8.8)
116.9 7.06 (d, 8.8)
150.1
114.8 6.44 (d, 8.8) 115.0
116.9 6.54 (d, 8.8) 115.1
0
5
0
6
151.7
11.7 2.10 (s)
105.1
75.4
150.1
11.3
0
6
-CH
0
3
2.41 (s)
20.2 2.26 (s)
104.1 4.79 (d, 7.8)
75.0 3.39 (dd, 7.8, 9.0)
78.1 3.46 (dd, 9.0, 9.0)
71.8 3.42 (dd, 9.0, 9.0)
76.9 3.34 (ddd, 9.0, 5.5, 2.3)
68.4 3.69 (dd, 12.0, 5.5)
3.86 (dd, 12.0, 2.3)
110.2
12.2 2.20 (s)
0
Glc1
4.88 (d, 7.8)
104.7 4.66 (d, 7.8)
75.0 3.47 (dd, 7.8, 9.0)
78.2 3.42 (dd, 9.0, 9.0)
71.3 3.37 (dd, 9.0, 9.0)
78.3 3.39 (ddd, 9.0, 5.5, 2.3)
62.5 3.71 (dd, 12.0, 5.5)
3.93 (dd, 12.0, 2.3)
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
3.50 (dd, 7.8, 9.0)
3.45 (dd, 9.0, 9.0)
3.37 (dd, 9.0,9.0)
78.4
71.6
a
3.57
78.1
3.60 (dd, 11.0, 6.2)
62.8
4
.05 (dd, 11.0, 1.5)
0
00
Ara(f)1
4.90 (d, 1.5)
0
0
0
0
00
00
00
00
2
3
4
5
3.98 (dd, 3.3, 1.5)
3.80 (dd, 5.6, 3.3)
3.91 (ddd, 5.6, 5.6, 3.3)
3.58 (dd, 11.8, 5.6)
83.3
78.9
85.8
63.0
3
.69 (dd, 11.8, 3.3)
a
Overlapping with other signals
Interchangeable
b
Fig. 3 The key HMBC and
NOESY correlations in 6–9
juniperosides I and II. The NOESY and HMBC correla-
tions observed in 6 and 7 (Fig. 3) demonstrated that the
gross aglycone structures in the two compounds were
consistent with 1-(3,6-dihydroxy-2-methylphenyl)ethanol,
aglycones 6a and 7a, which both had the same molecular
1 13
formula (C H O ). Although the EI-MS and H- and C-
9
12 3
NMR spectra of 6a and 7a were the same, the optical
rotations [6a: [a]D -43.1° (c 0.22, MeOH), 7a: [a]D
?42.5° (c 0.09, MeOH)] were opposite in sign. The signs
of the optical rotations of 6a and 7a were compared with
those of the aglycones present in the juniperosides I and II
0
suggesting that 6 and 7 were the 5 -hydroxy derivatives of
juniperosides I and II, respectively. The enzymatic hydro-
lysis of 6 and 7 afforded D-glucose and the corresponding
123

3
64
J Nat Med (2013) 67:359–368
[
1] [i.e. (1S)- and (1R)-1-(2-hydroxy-6-methylphenyl)eth-
anol, (1S-isomer: [a]D -42.5°, 1R-isomer: [a]D ?43.8°)]
and it was concluded that 6a and 7a corresponded to the
groups [d 3.83 (6H, s)]. In addition, HMBC correlations
(Fig. 3) were observed between each of H -8, H -9/C-7,
2
2
and between H-7/C-1, C-2, and C-6. These results sug-
gested that 8 was a phenylpropanoid glucoside. The two
methoxyl groups were determined to be at C-3 and C-5 on
(1S)- and (1R)-isomers, respectively. Thus, the complete
structures of juniperoside VIII (6) and of juniperoside IX
0
0
0
(
7) were defined as (1S)- and (1R)-1-(2 ,5 -dihydroxy-6 -
the basis of NOESY (OCH /H-2, H-6) and HMBC corre-
3
0
0
methylphenyl)ethanol 2 -O-b-D-glucopyranoside, respec-
tively (The numbering of benzene ring in 6 and 7 was taken
in the same manner as in compound 1–5).
lations (Fig. 3). The NOESY (H-1 /H -8) and HMBC
2
0
(H-1 /C-8) correlations indicated that the b-D-glucopyran-
osyl moiety was connected at C-8-OH. The structure of 8
was therefore identified as 3-hydroxy-2-(4-hydroxy-3,5-
dimethoxyphenyl)propyl b-D-glucopyranoside and subse-
quently named juniperoside X.
Compound 8 was isolated as a white amorphous powder,
[
a] -4.0° (c = 0.57, MeOH), with the FAB-MS (negative
D
- -
mode) revealing [M - H] and [M-H-162] ion peaks at
m/z 389 and 227, respectively. Furthermore, the HR FAB-
MS revealed the molecular formula to be C H O . The
Compound 9 was isolated as a white amorphous powder,
[a]D -12.0° (c = 0.47, MeOH), and the HR FAB-MS
(negative mode) showed an [M - H] ion peak at m/z
1
7 26 10
1
13
-
H- and C-NMR spectral data (Table 3) showed the
presence of a b-glucopyranose (J1 = 7.8 Hz); this
0
0
389.1444. This mass corresponded to a molecular formula
1
of C H O , which was the same as that of 8. The H-
,2
absolute configuration was confirmed to be D by HPLC
1
analysis of the acid hydrolysate. The H-NMR and COSY
1
13
7 26 10
and C-NMR spectral data (Table 3) were also similar to
those of 8. The HMBC analysis was conducted in a manner
similar to that of 8 and indicated that 9 was also a phe-
nylpropanoid glucoside, with the only difference being the
position of one of the two methoxyl groups on the agly-
cone, which was at C-4 in 9 as opposed to C-3 in 8 (Fig. 3).
The presence of D-glucose was confirmed by HPLC anal-
ysis of the acid hydrolysate 9. In conclusion, the structure
of 9 was identified as 3-hydroxy-2-(3-hydroxy-4,5-dime-
thoxyphenyl)propyl b-D-glucopyranoside, and subse-
quently named juniperoside XI. The two phenylpropanoid
glucosides (8 and 9) contained an asymmetric carbon
spectra of 8 revealed the presence of a 1,2,3,5-tetrasubsti-
tuted benzene ring [d 6.59 (2H, s, H-2, 6)], a 1,3-dihydr-
oxyisopropyl group [d 2.99 (1H, m, H-7), 3.81 (1H, dd,
J = 9.6, 7.6 Hz, H-8a), 4.12 (1H, dd, J = 9.6, 6.0 Hz,
H-8b), 3.78 (1H, dd, J = 10.8, 6.4 Hz, H-9a), and 3.89
(
1H, dd, J = 10.8, 6.4 Hz, H-9b)] and two methoxyl
1
13
3
Table 3 H- and C-NMR data for 8, 9 in CD OD
Position
8
9
d
H
d
C
d
H
d
C
(C-7), arising from the glucosidation at the 8-hydroxyl
1
2
3
4
5
6
7
8
132.9
138.1
110.0
151.4
136.5
154.4
105.2
49.7
group of an achiral aglycone. The absolute configuration at
position C-7 in 8 and 9, however, remains to be
determined.
6.59 (s)
106.7 6.44 (d, 2.0)
149.2
135.4
To the best of our knowledge, the aglycones of 5–7 and
the 1-phenylethanol derivatives obtained from the reduc-
tion of the corresponding acetophenone derivatives are
structurally unique compounds. Furthermore, the phenol
glycosides 5–7 must be novel and rare types of naturally
occurring plant components. The juniperosides VII–IX
contain particularly original skeletons that are similar to
those of the phenylethanoids and have so far not been
reported.
149.2
6.59 (s)
106.7 6.47 (d, 2.0)
49.7 2.96 (m)
72.3 3.80 (dd, 9.6, 7.6)
4.10 (dd, 9.6, 6.0)
65.0 3.76 (dd, 10.8, 6.4)
2.99 (m)
3.81 (dd, 9.6, 7.6)
72.2
4
.12 (dd, 9.6, 6.0)
3.78 (dd, 10.8, 6.4)
.89 (dd, 10.8, 6.4)
9
64.8
a
3
3.87
3
4
5
-OCH
3
3
3
3.83 (s)
56.8
-OCH
3.75 (s)
60.9
56.4
104.8
75.1
78.0
71.7
78.2
62.8
-OCH
0
3.83(s)
56.8 3.82 (s)
Experiments
Glc 1
0
4.31 (d, 7.8)
104.8 4.30 (d, 7.8)
75.1 3.19 (dd, 7.8, 9.0)
78.0 3.35 (dd, 9.0, 9.0)
71.7 3.28 (dd, 9.0, 9.0)
2
3.19 (dd, 7.8, 9.0)
3.34 (dd, 9.0, 9.0)
3.27 (dd, 9.0, 9.0)
0
General
3
0
4
1
13
H- and C-NMR spectra were measured on a JEOL JNM-
0
a
a
5
3.2
78.2 3.28
ECA 600 spectrometer at 600 and 150 MHz, respectively.
Chemical shifts are given in d values (ppm) relative to a
tetramethylsilane (TMS) internal standard. The EI- and
HR-EI-MS spectra (at 30 eV), together with the FAB- and
0
6
3.65 (dd, 12.0, 5.4)
62.8 3.66 (dd, 12.0, 5.4)
a
3.87
3
.86 (dd, 12.0, 2.4)
a
Overlapping with other signals
1
23

J Nat Med (2013) 67:359–368
365
HR-FAB-MS spectra in the negative mode (matrix: tri-
ethanolamine), were obtained on a JEOL JMS-700T
spectrometer. Optical rotations were determined on a
JASCO P-1020 polarimeter. IR and UV spectra were
measured on JASCO FT/IR-410 and Shimadzu UV-1600
UV/VIS spectrophotometers, respectively. Silica gel 60
(9:3:1, a lower phase)]. Fraction G (1.6 g) was divided
4
into seven fractions (frs. G4-1–G4-7) using ODS column
chromatography (eluted with 50 % MeOH). Fraction G4-2
was purified with Sephadex LH-20 column chromatogra-
phy (eluted with 60 % MeOH) to give aromadendrin 7-O-
b-D-glucopyranoside (6.6 mg) and another crude fraction.
The crude fraction was further purified with HPLC (JAI-
GEL-GS column; 50 % MeOH) to give dihydrosyringin
(4.9 mg). Fraction G4-3 was subjected to Sephadex LH-20
column chromatography (eluted with MeOH) to give is-
oscutellarein 7-O-b-D-xylopyranoside (3.3 mg). Fraction
G4-4 was subjected to HPLC purification (JAIGEL-GS
column; eluted with MeOH) to give naringenin 7-O-b-D-
glucopyranoside (4.1 mg), whereas fraction G4-5 gave
(
(
(
230–400 mesh, Merck), Chromatorex ODS DM1020T
100–200 mesh, Fuji Silysia), and Sephadex LH-20
Amersham Biosciences) were used for column chroma-
tography. Kiesel gel 60 F254 (Merck) and RP-18 F254
Merck) were used for analytical TLC. Preparative HPLC
(
was performed on a JAI LC-918 instrument with an RI-50
differential refractometer and a JAIGEL-ODS or a JAI-
GEL-GS 310 column. A JASCO PU-2086 instrument was
also used with an RI-2031 differential refractometer and a
TSK gel ODS-80 T or a COSMOSIL 5C -MS-II column.
0
apigenin 4 -O-b-D-glucopyranoside (8.3 mg) and quercitrin
(15.4 mg) following HPLC purification (JAIGEL-GS col-
umn; eluted with MeOH). Fraction H (19.1 g) was subse-
quently fractionated into eight fractions (from H to H )
S
18
Plant material
1
8
using silica gel column chromatography [CHCl –MeOH–
3
The leaves and stems of J. occidentalis were collected in
July 1997, in Oregon, USA. A voucher specimen (Murata
J. et al., No. 97071) was deposited in the Herbarium of the
Botanical Gardens at The University of Tokyo (TI) in
Japan.
H O (7:3:1, a lower phase)]. Fraction H (1.3 g) was sep-
2
3
arated into five further fractions (frs. H3-1–H3-5) by ODS
column chromatography (eluted with 40 % MeOH). Frac-
tion H3-2 was further purified by HPLC (COSMOSIL 5C18-
MS-II column; eluted with 50 % MeOH), followed by
HPLC (JAIGEL-GS column; eluted with 60 % MeOH) to
Extraction and isolation
afford 4 (5.5 mg). Fraction H (2.5 g) was further divided
4
in 12 fractions (frs. H4-1–H4-12) following ODS column
chromatography eluted with 40 % MeOH. Fraction H4-3
was further purified with HPLC (JAIGEL-ODS column;
eluted with 40 % MeOH), followed by HPLC (JAIGEL-GS
column; eluted with 50 % MeOH) to afford 6 (123.0 mg).
The dried leaves and stems of J. occidentalis (5.1 kg) were
extracted three times with MeOH (18 L 9 weekly) at room
temperature. The MeOH solution was then evaporated in
vacuo to afford a dark greenish extract (1050 g) and an
aliquot (300 g) of the extract was partitioned between
n-hexane and 90 % MeOH. The 90 % MeOH layer was
collected and evaporated in vacuo to give a residue
Fraction H (5.8 g) was subjected to ODS column chro-
5
matography eluted with 50 % MeOH, followed by HPLC
purification (JAIGEL-GS column; eluted with MeOH) to
(
217.8 g) which was further partitioned between n-BuOH
afford skimmin (2.8 mg). Fraction H (3.7 g) was subse-
6
and water. The n-BuOH layer was then collected and
evaporated in vacuo to give a residue (174.6 g) which was
subsequently chromatographed on silica gel [eluted suc-
cessively with CHCl –MeOH (10:1), CHCl –MeOH–H O
quently fractionated into five further fractions (frs. H6-1–
H6-5) by ODS column chromatography (eluted with 50 %
MeOH). Fraction H6-2 was divided into five sub-fractions
following ODS column chromatography eluted with 40 %
MeOH and the second sub-fraction was subjected to a
combination of HPLC purifications (JAIGEL-ODS col-
umn; eluted with 30 % MeOH and JAIGEL-GS; eluted
with 30 % MeOH) to give 1 (12.9 mg), 8 (5.7 mg), 9
(4.7 mg), and junipediol A 8-O-b-D-glucopyranoside
(3.2 mg). The third sub-fraction was purified by HPLC
(JAIGEL-ODS column; eluted with 30 % MeOH), fol-
lowed by HPLC (JAIGEL-GS column; eluted with 60 %
MeOH) to afford 7 (73.4 mg), whereas the fourth sub-
fraction was further purified with HPLC (JAIGEL-ODS
column; eluted with 40 % MeOH), followed by HPLC
(JAIGEL-GS column; eluted with 60 % MeOH) to afford 2
(5.2 mg), 3 (11.2 mg), 5 (8.5 mg) and aromadendrin 3-O-
a-L-rhamnopyranoside (7.1 mg). Fraction H6-5 was further
3
3
2
(
7:3:1, a lower phase), CHCl –MeOH–H O (6:4:1) and
3 2
MeOH] to give 12 fractions; each fraction was labeled
according to an alphabetic sequence from A to L. Fraction
F (9.1 g) was subsequently divided into three further
fractions (from F1 to F ) following silica gel column
3
chromatography eluted with CHCl –MeOH–H O (9:3:1, a
3
2
lower phase). Fraction F (3.6 g) was divided into nine
2
further fractions (frs. F2-1–F2-9) following purification by
Chromatorex ODS column chromatography (eluted with
5
0 % MeOH). Fraction F2-1 was purified by HPLC (TSK-
gel-ODS 80Ts; eluted with 50 % MeOH) to give junip-
eroside (78.9 mg). Fraction G (8.0 g) was subsequently
fractionated into seven fractions (from G to G ) using
1
7
silica gel column chromatography [CHCl –MeOH–H O
3
2
123

3
66
J Nat Med (2013) 67:359–368
purified with HPLC (JAIGEL-GS column; eluted with
Enzymatic hydrolysis of 5
MeOH) to give kaempferol 3-O-a-L-rhamnopyranoside
(
17.0 mg).
A solution of 5 (4.0 mg) in water (2 ml) was incubated
with cellulase (10 mg) at 37 °C for 48 h. The reaction
mixture was then poured into a large amount of water and
extracted with ethyl acetate (EtOAc). The organic extract
was evaporated to dryness in vacuo and the resulting res-
idue purified using a Sep-Pak Silica cartridge column
(using CHCl3 as the eluent) to give the aglycone 5a
(0.9 mg) as a white amorphous powder, [a]D -43.0°
Juniperoside III (1)
White amorphous powder, [a]D -12.4° (c 0.17, MeOH).
FAB- and HR FAB-MS (negative ion mode) m/z: 311.1130
-
M - H] (calcd for C H O : 311.1131), 149 [M-H-
[
1
-1
5 19 7
-
Glc] . IR m
1
(film) cm : 3392, 2920, 1691, 1465, 1257,
max
(
c 0.09, MeOH). Compound 5a was identified as the
077. UV kmax (MeOH) nm (loge): 209 (4.02), 230 (sh,
.44), 278 (3.01). H- and C-NMR data are given in
1
13
aglycone of juniperoside I by comparison of the physical
and spectral data with those of an authentic sample.
3
Table 1.
Juniperoside VIII (6)
Juniperoside IV (2)
White amorphous powder, [a]D -33.9° (c 0.84, MeOH).
FAB- and HR FAB-MS (negative ion mode) m/z: 329.1239
White amorphous powder, [a]D -61.7° (c 0.85, MeOH).
FAB- and HR FAB-MS (negative ion mode) m/z: 443.1553
-
M - H] (calcd for C H O : 329.1236), 167 [M-H-
[
-
M - H] (calcd for C H O : 443.1553), 311 [M-H-
15 21
-1
8
[
2
0
27 11
-
-
-
-1
(film) cm : 3392,
Glc] . IR mmax (film) cm : 3393, 2917, 1074. UV kmax
MeOH) nm (loge): 205 (4.36), 213 (sh, 4.01), 287 (3.57).
H- and C-NMR data are given in Table 2.
Ara] , 149 [M-H-Glc-Ara] . IR m
max
(
1
2
921, 1684, 1457, 125, 1074. UV kmax (MeOH) nm (loge):
13
1 13
09 (4.04), 232 (sh, 3.48), 278 (3.03). H- and C-NMR
2
data are given in Table 1.
Enzymatic hydrolysis of 6
Juniperoside V (3)
A solution of 6 (6.0 mg) in water (2 ml) was incubated
with b-glucosidase (almond emulsin; 10 mg) at 37 °C for
White amorphous powder, [a]D -31.8° (c 1.0, MeOH).
FAB- and HR FAB-MS (negative ion mode) m/z: 327.1077
2
4 h. The reaction mixture was then poured into a large
amount of water and extracted with EtOAc. The organic
extract was evaporated to dryness in vacuo to give agly-
cone 6a (2.2 mg) as a white amorphous powder. The
aqueous phase was passed through an OASIS HLB car-
tridge column and the resulting solution was concentrated
to give a sugar fraction, which was analyzed by HPLC
under the following conditions: a COSMOSIL Sugar-D
column (4.6 mm i.d. 9 250 mm, Nacalai Tesque, Inc.)
was eluted with CH CN–H O (4:1) at a flow rate of 1.0 ml/
-
M - H] (calcd for C H O : 327.1080), 165 [M-H-
[
15 19 8
-1
-
Glc] . IR m
(film) cm : 3366, 2916, 1577, 1288, 1074.
max
UV kmax (MeOH) nm (loge): 202 (3.99), 213 (3.95), 252
1 13
3.36), 285 (3.13). H- and C-NMR data are given in
(
Table 1.
Juniperoside VI (4)
3
2
White amorphous powder, [a] -34.0° (c 1.0, MeOH). HR
FAB-MS (negative ion mode) m/z: 343.1026 [M - H]
-
min. Subsequent detection and determination of the optical
rotation were achieved by using a JASCO OR-2090 Plus.
Identification of the D-glucose present in the sugar fraction
was achieved by the comparison of the retention times (tR)
and optical rotations with those of an authentic sample.
tR (min): 11.6 (D-glucose, positive optical rotation).
D
-
(
calcd for C H O : 343.1029), 311 [M-H-Glc] . IR m
1
1
5
19
9
max
-
(
film) cm : 3374, 2919, 1607, 1242, 1074. UV kmax
MeOH) nm (loge): 203 (4.21), 220 (4.01), 229 (sh, 3.89),
(
1 13
77 (3.86). H- and C-NMR data are given in Table 1.
2
6a: white amorphous powder, [a]D -43.1° (c 0.22,
Juniperoside VII (5)
MeOH), EI- and HR EI-MS m/z ( %): 168.0788 (calcd
?
?
for C H O , M : 168.0786, 25), 150 ([M-H O] , 100).
9
12
13
3
2
1
White amorphous powder, [a]D -62.1° (c 1.0, MeOH).
FAB- and HR FAB-MS (negative ion mode) m/z: 445.1713
H- and C-NMR data are given in Table 2.
-
M - H] (calcd for C H O : 445.1710, 313 [M-H-
[
Juniperoside IX (7)
2
0 29 11
-
-
-1
(film) cm : 3391,
Ara] , 151 [M-H-Glc-Ara] . IR m
max
2
925, 1584, 1070. UV k
(MeOH) nm (loge): 203 (4.13),
09 (sh, 3.90), 270 (2.81). H- and C-NMR data are given
White amorphous powder, [a]D -38.2° (c 0.52, MeOH).
FAB- and HR FAB-MS (negative ion mode) m/z: 329.1235
max
1
13
2
-
[M - H] (calcd for C H O : 329.1236), 167 [M-H-
in Table 2.
1
5 21 8
1
23

J Nat Med (2013) 67:359–368
367
-
-1
(film) cm : 3365, 2918, 1074. UV kmax
Glc] . IR m
rotations with those of the authentic samples. t (min): 11.6
R
max
(
1
MeOH) nm (loge): 205 (4.46), 212 (sh, 3.98), 287 (3.44).
H- and C-NMR data are given in Table 2.
(D-glucose, positive optical rotation), 8.6 (L-arabinose,
positive optical rotation).
13
Acknowledgments This work was supported in part by a Grant-in-
Aid for International Scientific Research (No. 09041194) from the
Ministry of Education, Culture, Sports, Science, and Technology in
Japan.
Enzymatic hydrolysis of 7
A solution of 7 (3.0 mg) in water (2 ml) was subjected to
enzymatic hydrolysis as described for 6 to give 7a (0.9 mg)
and a sugar fraction. HPLC analysis of the sugar fraction
under the same conditions as those used in the example of 6
References
revealed the presence of D-glucose. t (min): 11.6 (D-glu-
R
cose, positive optical rotation).
1. Nakanishi T, Inatomi Y, Murata H, Iida N, Inada A, Lang FA,
Murata J (2002) Phytochemical study on American plants I. Two
new phenol glucosides, together with known biflavones and
diterpene, from leaves of Junperus occidentalis Hook. Chem
Pharm Bull 50:1358–1361
2. Nakanishi T, Iida N, Inatomi Y, Murata H, Inada A, Murata J,
Lang FA, Iinuma M, Tanaka T (2004) Neolignan and flavonoid
glycosides in Juniperus communis var. depressa. Phytochemistry
7
a: white amorphous powder, [a]D ?42.5° (c 0.09,
MeOH)], EI- and HR EI-MS m/z ( %): 168.0793 (calcd for
?
?
1
C H O , M : 168.0786, 15), 150 ([M-H O] , 100). H-
9
12
3
2
1
and C-NMR data are given in Table 2.
3
Juniperoside X (8)
6
5:207–213
3
4
5
6
. Nakanishi T, Iida N, Inatomi Y, Murata H, Inada A, Murata J,
Lang FA, Iinuma M, Tanaka T (2004) New neolignan and phe-
nylpropanoid glycosides in Juniperus communis var. depressa.
Heterocycles 63:2573–2580
. Inatomi Y, Iida N, Murata H, Inada A, Murata J, Lang FA,
Iinuma M, Tanaka T, Nakanishi T (2005) A pair of new atrop-
isomeric cupressuflavone glucosides isolated from Juniperus
communis var. depressa. Tetrahedron Lett 46:6533–6535
. Nakanishi T, Iida N, Inatomi Y, Murata H, Inada A, Murata J, Lang
FA, Iinuma M, Tanaka T, Sakagami Y (2005) A monoterpene
glucoside and three megastigmane glycosides from Juniperus
communis var. depressa. Chem Pharm Bull 53:783–787
White amorphous powder, [a]D -4.0° (c 0.57, MeOH).
FAB- and HR FAB-MS (negative ion mode) m/z: 389.1451
-
M - H] (calcd for C H O : 389.1448), 227 [M-H-
[
1
7 25 10
-1
-
Glc] . IR m
(film) cm : 3385, 2925, 1616, 1115. UV
max
kmax (MeOH) nm (loge): 207 (4.47), 220 (sh, 3.94), 272
1 13
3.22). H- and C-NMR data are given in Table 3.
(
Juniperoside XI (9)
. Iida N, Inatomi Y, Murata H, Inada A, Murata J, Lang FA,
Matsuura N, Nakanishi T (2007) A new flavone xyloside and
two new flavan-3-ol glucosides from Juniperus communis var.
depressa. Chem Biodivers 4:32–42
White amorphous powder, [a]D -12.0° (c 0.47, MeOH).
FAB- and HR FAB-MS (negative ion mode) m/z: 389.1444
-
M - H] (calcd for C H O : 389.1448), 227 [M-H-
[
1
7 25 10
-1
-
Glc] . IR m
(film) cm : 3400, 2919, 1590, 1101. UV
7. Iida N, Inatomi Y, Murata H, Murata J, Lang FA, Tanaka T,
Nakanishi T, Inada A (2010) New phenylpropanoid glycosides
from Juniperus communis var. depressa. Chem Pharm Bull
max
kmax (MeOH) nm (loge): 207 (4.36), 219 (sh, 3.96), 270
1 13
3.17). H- and C-NMR data are given in Table 3.
(
5
8:742–746
8
. Comte G, Allaris DP, Chulia AJ, Vercauteren J, Pinaud N (1997)
Three phenylpropanoids from Juniperus phoenicea. Phytochem-
istry 44:1169–1173
Determination of the sugar configurations in 1–5, 8,
and 9
9
. Comte G, Chulia AJ, Vercauteren J, Allaris DP (1996) Phenyl-
propane glycosides from Juniperus phoenicea. Planta Med
Each glycoside (ca. 1 mg) was added to a 1 M HCl solu-
tion (1.0 ml) and heated at 95 °C for 3 h. The mixtures
were then cooled to ambient temperature, neutralized via
the addition of Amberlite IRA-93ZU (Organo Co., Ltd.)
and passed through an OASIS HLB cartridge column. The
resulting solution was then concentrated in vacuo and dried
to give a sugar fraction, which was analyzed by HPLC
under the following conditions: a COSMOSIL Sugar-D
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23