A monoterpene glucoside and three megastigmane glycosides from Juniperus communis var. depressa

Article abstract of DOI:10.1248/cpb.53.783

A new monoterpene glucoside (1) and three new natural megastigmane glycosides (2 - 4) were isolated along with a known megastigmane glucoside (5) from twigs with leaves of Juniperus communis var. depressa (Cupressaceae) collected in Oregon, U.S.A., and their structures were determined on the basis of spectral and chemical evidence. In addition, the antibacterial activities of the isolated components against Helicobacter pylori were also investigated.

Full text of DOI:10.1248/cpb.53.783

July 2005
Chem. Pharm. Bull. 53(7) 783—787 (2005)
783
A Monoterpene Glucoside and Three Megastigmane Glycosides from
Juniperus communis var. depressa
Tsutomu NAKANISHI,*,a Naoki IIDA,a Yuka INATOMI,a Hiroko MURATA,a Akira INADA,a Jin MURATA,b
f
Frank A. LANG,c Munekazu IINUMA,d Toshiyuki TANAKA,e and Yoshikazu SAKAGAMI
a Faculty of Pharmaceutical Sciences, Setsunan University; 45–1 Nagaotoge-cho, Hirakata, Osaka 573–0101, Japan:
b Botanical Gardens, Koishikawa, Graduate School of Science, The University of Tokyo; 3–7–1 Hakusan, Bunkyo-ku, Tokyo
112–0001, Japan: c Department of Biology, Southern Oregon University; 1250 Siskiyou, Ashland, OR 97520–5071, U.S.A.:
d Gifu Pharmaceutical University; 5–6–1 Mitahora-higashi, Gifu 502–8585, Japan: e Gifu Prefectural Institute of Health
and Environmental Sciences; 1–1 Naka Fudogaoka, Kakamigahara, Gifu 504–0838, Japan: and f Osaka Prefectural
Institute of Public Health; 1–3–69 Nakamichi, Higashinari-ku, Osaka 537–0025, Japan.
Received February 7, 2005; accepted March 15, 2005
A new monoterpene glucoside (1) and three new natural megastigmane glycosides (2—4) were isolated along
with a known megastigmane glucoside (5) from twigs with leaves of Juniperus communis var. depressa (Cupres-
saceae) collected in Oregon, U.S.A., and their structures were determined on the basis of spectral and chemical
evidence. In addition, the antibacterial activities of the isolated components against Helicobacter pylori were also
investigated.
Key words Juniperus communis var. depressa; monoterpene glucoside; megastigmane glycoside; Helicobacter pylori; Cupres-
saceae
1
In a survey of chemical components from useful plants
grown in western North America, we have identified a num-
ber of various types of phenolic compounds (nine phenyl-
propanoids, six neolignans, and seven flavonoids) in their
glycoside form from the aerial parts of Juniperus communis
var. depressa (Cupressaceae) and those results were reported
in our previous papers.1,2) Upon continued chemical investi-
gation of the same plant material, a new monoterpene gluco-
side (1) and three new natural megastigmane glycosides (2—
4) were isolated together with a known megastigmane, cor-
choionoside C (5).3) In the present paper, we describe the iso-
lation, structure elucidation, and biological activity of these
constituents.
revealed the molecular formula to be C16H26O8. The H- and
13C-NMR spectral data (Table 1) suggested 1 to be a b-D-glu-
copyranoside.4,5) On the aglycone, proton signals due to two
tert methyls (d 1.35, 1.39, both s) and an olefinic proton (d
7.27, br dd, Jꢁ3.0, 3.0 Hz) and 10 carbon signals including
two tert methyls (dC 23.3, 24.8) and a carboxyl group (dC
169.9) were observed. The two-dimensional (2D) study using
1H–1H shift-correlation spectroscopy (COSY) indicated that
a menthane-type monoterpene is assigned to the aglycone.
The position of the carboxyl group was determined to be at
C-1 based on the presence of a cross peak between H-2 and
the carboxylic carbon (C-7) in heteronuclear multiple-bond
correlation spectroscopy (HMBC), suggesting that the planar
The n-BuOH-soluble part obtained from the MeOH ex-
tract was separated by a combination of silica gel, octadecyl
silica gel (ODS), and Sephadex LH-20 column chromatogra-
phies, followed by HPLC separation to afford each of the
four new natural terpenic compounds (1—4) and the known
megastigmane.
Table 1. 1H- and 13C-NMR Spectral Data for 1 in C5D5Na)
1
NO.
dH
dC
Compound 1, a white powder, [a]D ꢀ36.3°, gave the
[MꢀH]ꢀ ion peak at m/z 345 in the FAB-MS (negative
mode). The high-resolution (HR) spectrum in the same mode
1
2
3a
b
4a
5a
b
6a
b
7
131.8
139.1
27.8
7.27 (br dd, 3.0, 3.0)
2.47 (br dd, 12.0, 16.0)
2.08 (br d, 16.0)
1.85 (dddd, 12.0, 12.0, 4.8, 1.8)
2.22 (br d, 12.0)
1.26 (dddd, 12.0, 12.0, 12.0, 4.8)
2.42 (m)
2.80 (br d, 16.0)
43.7
23.8
26.0
169.9
79.0
23.3b)
24.8b)
98.6
75.3
78.8
71.9
78.0
63.0
8
9
1.39 (s)b)
10
Glc 1ꢂ
2ꢂ
1.35 (s)b)
5.02 (d, 7.8)
3.98 (dd, 9.0, 7.8)
4.26 (dd, 9.0, 9.0)
4.24 (dd, 9.0, 9.0)
3.91 (ddd, 9.0, 5.2, 2.5)
4.35 (dd, 11.8, 5.2)
4.49 (dd, 11.8, 2.5)
3ꢂ
4ꢂ
5ꢂ
6ꢂ
a) Measured at 600 MHz (1H-NMR) and 150 MHz (13C-NMR). b) Interchange-
able in each column.
Chart 1
∗ To whom correspondence should be addressed. e-mail: nakanisi@pharm.setsunan.ac.jp
© 2005 Pharmaceutical Society of Japan

784
Vol. 53, No. 7
Table 2. 1H- and 13C-NMR Spectral Data for 2, 2a, 3, and 4 in CD3OD
2a)
2ab)
3a)
4a)
No.
dH
dC
dH
dC
dH
dC
dH
dC
1
46.3
45.5
49.7
53.3
38.8
38.8
2a 2.64 (dd, 17.3, 0.7)
2.35 (dd, 18.0, 2.4)
2.65 (dd, 18.0, 2.8)
1.83 (ddd, 12.0, 3.6, 2.4)
1.49 (dd, 12.0, 12.0)
4.05 (m)
1.85 (ddd, 12.0, 3.5, 2.4)
1.49 (dd, 12.0, 12.0)
4.02 (m)
b
3
4
2.38 (br d, 17.3)
47.5
73.3
47.6
73.8
200.9
127.8
211.3
54.0
5.90 (dd, 1.3, 0.7)
2.43 (dd, 17.6, 2.4, a)
2.78 (d, 17.6, b)
6.73 (d, 8.1)
6.85 (dd, 8.1, 2.0)
6.02 (dd, 15.2, 1.2)
2.34 (br dd, 16.8, 5.8, a) 39.8
2.33 (br dd, 16.8, 5.8, a) 39.9
2.02 (b)c)
2.03 (b)c)
5
6
7
167.2
79.4
129.8
87.5
82.4
125.7
125.1
138.6
25.5
125.1
138.6
25.6
5.75 (dd, 16.0, 1.2)
5.83 (dd, 16.0, 5.6)
2.02c)
2.12 (dt, 13.2, 5.1)
1.45 (m)
2.03c)
2.12 (dt, 13.2, 5.1)
1.45 (m)
8
137.2
6.17 (dd, 15.2, 5.4)
140.7
40.7
40.7
1.53 (m)
1.53 (m)
9
10
11
4.33 (m)
68.7
23.8
74.6
4.38 (m)
68.9
24.0
78.4
3.71 (m)
1.17 (d, 6.3)
1.05 (s)
69.2
23.2
28.8
3.71 (m)
1.17 (d, 6.3)
1.06 (s)
69.2
23.3
28.9
1.24 (d, 6.4)
3.59 (d, 10.0)
3.96 (d, 10.0)
1.06 (s)
1.92 (d, 1.3)
4.14 (d, 7.8)
3.14 (dd, 9.0, 7.8)
3.31 (dd, 9.0, 9.0)
3.27 (dd, 9.0, 9.0)
1.28 (d, 6.4)
3.64 (d, 8.0)
3.91 (dd, 8.0, 2.8)
0.95 (s)
12
13
Glc 1ꢂ
2ꢂ
20.1
19.5
104.6
75.1
78.0
71.5
15.7
19.2
1.07 (s)
1.64 (s)
4.42 (d, 7.8)
3.15 (dd, 9.0, 7.8)
3.36 (dd, 9.0, 9.0)
3.30 (dd, 9.0, 9.0)
30.3
20.0
102.4
75.2
78.1
71.7
1.07 (s)
1.64 (s)
4.41 (d, 7.8)
3.14 (dd, 9.0, 7.8)
3.35 (dd, 9.0, 9.0)
3.28 (dd, 9.0, 9.0)
30.3
20.0
102.6
75.2
78.1
72.0
1.18 (s)
3ꢂ
4ꢂ
5ꢂ
3.21 (ddd, 9.0, 5.6, 2.4) 78.0
3.26 (ddd, 9.0, 5.6, 2.4) 77.9
3.45 (ddd, 9.0, 5.6, 2.4) 76.6
6ꢂ
3.65 (dd, 12.0, 5.6)
3.84 (dd, 12.0, 2.4)
62.7
3.67 (dd, 12.0, 5.6)
3.86 (dd, 12.0, 2.4)
62.8
3.60 (dd, 12.0, 5.6)
4.02 (dd, 12.0, 2.4)
4.95 (d, 1.0)
3.98 (dd, 3.4, 1.0)
3.82 (dd, 5.4, 3.4)
68.0
Ara 1ꢄ
2ꢄ
109.9
83.2
79.0
3ꢄ
4ꢄ
3.97 (ddd, 5.4, 5.4, 3.4) 86.1
5ꢄ
3.64 (dd, 12.0, 5.4)
3.73 (dd, 12.0, 3.4)
63.1
a) At 600 MHz (1H-NMR) and 150 MHz (13C-NMR). b) At 400 MHz (1H-NMR) and 100 MHz (13C-NMR). c) Overlapping with other signals.
data were analyzed with the aid of 2D 1H–1H COSY and het-
eronuclear multiquantum coherence (HMQC) experiments
(Table 2). Based on Table 2, the presence of the b-D-glucopy-
ranosyl moiety was demonstrated as the sugar part of 2.4,5)
On the other hand, the aglycone part contained the following
structural fragments: a tert methyl [d 1.06 (3H, s, H3-12)], a
sec methyl [d 1.24 (3H, d, Jꢁ6.4 Hz, H3-10)], a vinyl methyl
[d 1.92 (3H, d, Jꢁ1.3 Hz, H3-13)], an oxymethylene [d 3.59,
3.96 (both 1H, d, Jꢁ10.0 Hz, H2-11)], a four-carbon chain in-
volving a trans-disubstituted olefin [CHꢁCHCH(OH)CH3],
which is bonded to a quarternary carbon atom [d 5.75 (1H,
dd, Jꢁ16.0, 1.2 Hz, H-7), d 5.83 (1H, dd, Jꢁ16.0, 5.6 Hz, H-
8), d 4.33 (1H, m, H-9), d 1.24 (3H, d, Jꢁ6.4 Hz, H3-10)], an
a,b-unsaturated carbonyl group [dH 5.90 (1H, dd, Jꢁ1.3,
0.7, H-4), dC 200.9 (C-3)]. Based on the detailed study of the
HMBC correlation of 2 (Fig. 1), a megastigmane-type planar
structure as shown in Fig. 1 constituted the aglycone part
structure. This planar structure contained three asymmetric
carbons of C-1, C-6, and C-9, among which the absolute
configuration at C-6 was first determined as follows. The CD
spectrum of 2 exhibited a positive-signed Cotton effect (De
ꢃ9.88) at 245 nm, consistent with the b-orientation of the
side chain including a 7(8)-double bond (thus the 6R config-
uration) according to the reported evidence.7) The NOESY
spectrum of 2 (Fig. 1) gave three significant cross peaks (H-
7/H3-12, H-7/H-2b, and H3-12/H-2b), suggesting that the
tert methyl (C-12) attached at C-1 is b-oriented (thus the 1R
structure of the aglycone is the same as that of a known
monoterpene, oleuropeic acid.6) The absolute configuration
of the aglycone was decided as follows. On enzymatic hy-
drolysis with emulsin from almond (b-glucosidase), 1 gave
the corresponding genuine aglycone which showed a nega-
tive-signed optical rotation of ꢀ51.4°. A comparison of this
observed rotation with the reported values for (ꢀ)-S-oleu-
ropeic acid (ꢀ114°) and its (ꢃ)-R-isomer (ꢃ46.5°) prepared
synthetically from (ꢀ)-b-pinene and (ꢃ)-a-pinene, respec-
tively, via the microbial transformations6) indicated the agly-
cone to be (ꢀ)-S-oleuropeic acid. The cross peaks between
the anomeric proton and each of the two tert methyls in nu-
clear Overhauser enhancement spectroscopy (NOESY), to-
gether with the HMBC correlation between the anomeric
proton and C-8, demonstrated that the glucosyl moiety is
bonded to the alcoholic group (8-OH) of the aglycone
through the glycosidic linkage. In conclusion, 1 is defined as
(ꢀ)-oleuropeic acid 8-O-b-D-glucopyranoside [ꢁ(4S)-4-(1-
b-D-glucopyranosyloxy-1-methyl)ethyl-1-cyclohexene-1-car-
boxylic acid].
Compound 2, a white powder, [a]D ꢃ85.9°, gave a quasi-
molecular ion peak, [MꢀH]ꢀ, at m/z 401 in the FAB-MS
(negative mode). The HR-FAB-MS in the same mode re-
vealed the molecular formula to be C19H30O9. In addition, the
FAB-MS (negative mode) also afforded a significant frag-
ment peak at m/z 239, arising from the loss of a hexosyl unit
from the quasimolecular ion. The 1H- and 13C-NMR spectral

July 2005
785
Fig. 2. Data of Modified Mosher’s Method for 2a and 3a
ostructure of 6-hydroxy-junipeionoloside was not established
and no chiroptical data (optical rotation, CD evidence, etc.)
on this compound was provided in that report.9) Therefore it
is impossible to discuss the identity between 6-hydroxy-ju-
nipeionoloside and 2 at present.
Compound 3, a white powder, [a]D ꢀ48.5°, gave the mol-
ecular formula of C19H34O7 based on the [MꢀH]ꢀ ion peak
at m/z 373.2217 in the HR-FAB-MS (negative mode). The
1H- and 13C-NMR analysis was performed with the aid of 2D
techniques such as 1H–1H COSY, NOESY, HMQC, and
HMBC and as a result, all protons and carbons were assigned
as shown in Table 2. These established assignments (Table 2)
suggested that 3 is constituted of a b-D-glucopyranosyl
residue4,5) and a megastigmane aglycone with a planar stuc-
ture shown for 3a in Fig. 2. Furthermore, the presence of the
NOESY cross peak between H-1ꢂ and H-3 and HMBC corre-
lation between H-1ꢂ and C-3 demonstrated the glucosyl moi-
ety to be connected to the 3-OH group of the aglycone
through a glycosidic linkage. The absolute configurations at
C-3 and C-9 of the aglycone were determined as follows. On
enzymic hydrolysis, 3 gave the corresponding genuine agly-
cone (3a), which was subjected to the Mosher’s procedure in
the same manner as in the case of 2a. A difference between
the 3,9-di-(S)-MTPA ester (3b) and 3,9-di-(R)-MTPA ester
(3c) in chemical shifts was indicative of the 3R, 9S configu-
rations (Fig. 2).8) Based on the above-mentioned combined
evidence, 3 is now defined as (3R,9S)-megastigman-5-en-3,9-
diol 3-O-b-D-glucopyranoside. Recently, synthetic (3R,9S)-
megastigman-5-en-3,9-diol 3-O-b-D-glucopyranoside has
Fig. 1. Selected Correlations in COSY, HMBC and NOESY of 2
configuration). Finally, the absolute configuration of the 9-
sec OH group on the side chain of 2 was determined as fol-
lows. Enzymatic hydrolysis of 2 with cellulase afforded an
artificial aglycone (2a), instead of the desired genuine agly-
cone. The HR-EI-MS spectrum of 2a showed that the molec-
ular formula of 2a is C13H20O4, consistent with that of the
genuine aglycone. To elucidate the structure of 2a, the 13C-
NMR chemical shifts of 2a were compared with those of the
aglycone part of 2 as follows (Table 2). Characteristic signals
due to a methylene carbon (d 54.0, C-4) and a quarternary
carbon bearing an ethereal oxygen (d 87.5, C-5) were ob-
served in 2a instead of those due to the 4(5)-double bond
carbons (d 127.8, C-4 and d 167.2, C-5) observed in 2. Fur-
thermore, the carbonyl carbon (C-3) in 2a resonated down-
field by 10.4 ppm (d 211.3) compared with that (C-3; d
200.9) in 2, indicative of the presence of a saturated carbonyl
group in 2a instead of the a,b-unsaturated carbonyl in 2.
This spectral evidence suggested that the primary alcohol
(11-OH) liberated by the enzymic hydrolysis of 2 is linked to
one (C-5) of the double bond carbons to form the five-mem-
1
bered ether ring in 2a. The H-NMR data of 2a (Table 2)
were also consistent with the established structure of 2a
shown in Fig. 2 (at this stage, the absolute configuration at C-
9 in 2a is uncertain). The remaining structural problem, i.e.,
the absolute configuration at C-9 of 2a was determined ac-
cording to the modified Mosher’s method.8) Treatment of 2a
with (R)-(ꢀ)- and (S)-(ꢃ)-a-methoxy-a-(trifluoromethyl)-
phenylacetyl chloride (MTPA chloride) gave the correspond-
ing 9-(S)-MTPA ester (2b) and 9-(R)-MTPA ester (2c), re-
spectively. Differences between 2b and 2c in 1H-NMR chem-
ical shifts, i.e., Dd values of dSꢀdR, depicted in Fig. 2, sug-
gested that the absolute configuration at C-9 in 2a (accord-
ingly, in 2) is S.8) Finally, the position of the glucosyl residue
on the aglycone was made clear on the basis of the following
NOESY and HMBC experiments (Fig. 1). The NOESY cross
peak between H-1ꢂ and H2-11 and the HMBC correlation be-
tween H-1ꢂ and C-11 indicated that the glucose is connected
to the hydroxy group at C-11 on the aglycone. Based on the
accumulated evidence, 2 is defined as (1R,6R,9S)-6,9,11-
trihydroxy-4,7-megastigmadien-3-one 11-O-b-D-glucopyra-
noside. Recently, a megastigmane glucoside designated 6-hy-
droxy-junipeionoloside, with the same planar structure as 2,
was isolated from Juniperus phoenicea.9) However, the stere-
1
been reported and its H- and 13C-NMR data coincided with
those of 3.10) However, 3 was the first isolation from a natural
source.
Compound 4, a white powder, [a]D ꢀ47.1° had the molec-
ular formula of C24H42O11, which was decided based on the
[MꢀH]ꢀ ion at m/z 505.2651 in HR-FAB-MS (negative
1
mode). In a similar manner as in the case of 3, the H- and
13C-NMR spectra were analyzed in detail and as a result, all
protons and carbons of 4 were assigned as shown in Table 2.
Based on the assignments, the presence of b-D-glucopyra-
nosyl and a-L-arabinofuranosyl moieties4,5) as the sugar part
of 4 were revealed along with megastigmane-type aglycone
with the same plane structure as the aglycone (3a) of 3. The
subsequent NOESY and HMBC studies suggested that
a-L-arabinofuranosyl-(1→6)-b-D-glucopyranosyl moiety is
bonded to the 3-quasi-equatorial hydroxy group of the agly-
cone (Fig. 3). Due to a shortage in the isolated amounts of 4,
Mosher’s procedure could not be applied to determine the ab-
solute configurations at C-3 and C-9 of the aglycone. How-
1
ever, a detailed comparison of the H- and 13C-NMR data of
4 with those of 3 (Table 2) suggested that 4 must correspond

786
Vol. 53, No. 7
JNM-ECA 600 (1H at 600 MHz and 13C at 150 MHz) or a JEOL JNM-GX
400 (1H at 400 MHz and 13C at 100 MHz) spectrometer. Chemical shifts are
given in d values (ppm) relative to tetramethylsilane (TMS) as an internal
standard. FAB- and HR-FAB-MS spectra in negative mode (matrix, tri-
ethanolamine or glycerin), along with EI- and HR-EI-MS spectra, were ob-
tained with a JEOL JMS-700T spectrometer. Optical rotations were deter-
mined on a JASCO DIP-140 polarimeter and CD spectra were recorded on a
JASCO J-820 spectropolarimeter. GLC was carried out on a Shimadzu GC-
7AG under the following conditions: capillary column, TC-1 (0.32 mm
i.d.ꢅ30 m, GL Sciences Inc.); detector, hydrogen flame ionization detector;
column temperature, 230 °C; injection temperature, 250 °C; and carrier gas,
N2. For column chromatography, silica gel 60 (230—400 mesh, Merck),
Chromatorex ODS DM1020T (100—200 mesh, Fuji Silysia), and Sephadex
LH-20 (Amersham Biosciences) were used. 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 refrac-
tometer and a JAIGEL-ODS or a JAIGEL-GS 310 column, and also on a
JASCO PU-2086 instrument with an RI-2031 differential refractometer and
a TSK gel ODS-80 TS column. The b-glucosidase (almond emulsin) and
cellulase used in this work were commercially obtained from Sigma-Aldrich
Japan Co. and Nagase Co. Ltd., respectively.
Fig. 3. Selected Correlations in COSY, HMBC and NOESY of 4
Table 3. MIC Values of 1, 2, 3, and 5 against Helicobacter pylori
MIC (mg/ml) H. pylori
Compound
NCTC11637
NCTC11916
OCO1
1
2
3
100
100
50
100
100
50
100
100
50
Plant Material Twigs with leaves of J. communis var. depressa were
collected in July 1997, in Oregon, U.S.A. A voucher specimen (Murata J. et
al., No. 053) was deposited in the Herbarium, Botanical Gardens, The Uni-
versity of Tokyo (TI), Japan.
5
ꢆ200
100
100
ꢆ200
100
100
ꢆ200
50
Nat. hinokitiol
Syn. hinokitiol
50
Extraction and Isolation The dried and cut materials (2.4 kg) were ex-
tracted three times with MeOH (18 lꢅweekly) at room temperature. The
MeOH solution was evaporated in vacuo to afford a dark greenish extract
(488 g), an aliquot (202 g) of which was partitioned between n-hexane and
MeOH. The MeOH-soluble part (130 g) was further partitioned between n-
BuOH and water. The resulting n-BuOH extract (76 g) was chromatographed
on silica gel and eluted with CHCl3–MeOH–H2O (7 : 3 : 1, a lower phase) to
give 10 fractions (for each fraction, the abbreviations from A to J are used).
Fraction F (6.2 g) was subsequently fractionated with silica gel column chro-
matography [CHCl3–MeOH–H2O (9 : 3 : 1, a lower phase)] into two frac-
tions. The latter fraction obtained was further separated with Sephadex LH
20 column chromatography (eluted with MeOH) and subsequent ODS col-
umn chromatography (eluted with 50% MeOH) into three subfractions. Sub-
fraction 2 was further purified with HPLC (JAIGEL-ODS column; 50%
MeOH as an eluent) to give 3 (40.8 mg). Subfraction 3 was purified by re-
peated HPLC separation [JAIGEL-GS column (eluted with 50% MeOH),
followed by a TSK gel ODS-80Ts column (50% MeOH)] to yield known 5
(56.2 mg). Fraction H (10.8 g) was divided into 13 fractions (from H-1 to H-
13) with ODS column chromatography eluted with 50% MeOH. Fraction H-
6 (1.96 g) was applied to a Sephadex LH-20 column eluted with 80% MeOH
to afford two fractions. The first fraction obtained from fraction H-6 was fur-
ther separated on an ODS column (eluted with 50% and 70% MeOH, suc-
cessively) to give four subfractions. Subfraction 1 was further purified by re-
peated HPLC separation using JAIGEL-ODS (eluted with 50% MeOH) and
JAIGEL-GS (eluted with 50% MeOH) columns to give 1 (124.7 mg). Sub-
fraction 3 was purified on a Sephadex LH-20 column (eluted with MeOH)
and HPLC separation (JAIGEL-GS column; 50% MeOH as an eluting
agent) to afford 4 (4.2 mg). Fraction H-8 (1.77 g) was subjected to ODS col-
umn chromatography eluted with 50% MeOH to be divided into six frac-
tions. The first fraction obtained was purified by HPLC separation using
JAIGEL ODS (eluted with 50% MeOH) and subsequent JAIGEL-GS (eluted
with 50% MeOH) columns to give 2 (62.9 mg).
1
to 6ꢂ-O-a-L-arabinofuranoside of 3. Furthermore, the H-
chemical shifts of the methylene signals due to C-7 of 4 (d
2.03, 2.12 ppm, 1H each) were compared with those of syn-
thetic (3R,9R)- and (3R,9S)-megastigman-5-en-3,9-diol 3-O-
b-D-glucopyranosides [d 1.92, 2.21 ppm, 1H each in the
(3R,9R)-isomer and d 2.02 and 2.12 ppm, 1H each in the
(3R,9S)-isomer, respectively],10) suggesting that 4 has the ab-
solute configurations of (3R,9S). In conclusion, 4 is defined
as (3R,9S)-megastigman-5-en-3,9-diol 3-O-[a-L-arabinofura-
nosyl-(1→6)]-b-D-glucopyranoside.
Compound 5 was defined as formula 5 based on our struc-
tural elucidation using HR-FAB-MS, H- and 13C-NMR, and
1
CD spectral analyses. Furthermore, it was identical with a
known megastigmane, corchoionoside C [ꢁ(6S,9S)-roseo-
side A] by comparison of the optical rotation and H- and
1
13C-NMR spectral data.3)
Antibacterial activities of 1, 2, 3, 5, and hinokitiol against
three strains of Helicobacter pylori (NCTC11637,
NCTC11916, and OCO1) were evaluated by measurement of
the minimum inhibitory concentration (MIC value),11) and
the results are shown in Table 3. One (3) of the new
megastigmane glucosides showed potent inhibition (MIC
valueꢁ50 mg/ml) comparable to those of natural and syn-
thetic hinokitiol as positive controls. Both the new monoter-
pene glucoside (1) and the other new megastigmane gluco-
side (2) also had mild anti-H. pylori activity (MIC
valueꢁ100 mg/ml). However, the MIC value of the known
megastigmane glucoside (5) was greater than 200 mg/ml, in-
dicative of no activity. The inhibitory activities of natural
products against H. pylori have been investigated in earnest
for about the last 10 years, but it is reported here for the first
time that the glucosides of lower terpenes such as 1, 2, and 3
show potent or mild anti-H. pylori activity. Furthermore, we
expect that the present results may contribute to development
of new anti-H. pylori drugs.
1: A white powder, [a]D ꢀ36.3° (cꢁ0.54, MeOH). HR-FAB-MS (nega-
tive mode) m/z: 345.1544 (Calcd for C16H25O8, [MꢀH]ꢀ: 345.1550). 1H-
and 13C-NMR data are given in Table 1.
2: A white powder, [a]D ꢃ85.9° (cꢁ0.32, MeOH). FAB- and HR-FAB-
MS (negative mode) m/z: 401.1805 (Calcd for C19H29O9, [MꢀH]ꢀ:
401.1811), 239 [MꢀHꢀ162]ꢀ. CD (cꢁ1.08ꢅ10ꢀ4 mol/l, MeOH) De (l
nm): ꢃ9.88 (245). 1H- and 13C-NMR data are given in Table 2.
3: A white powder, [a]D ꢀ48.5° (cꢁ1.05, MeOH). HR-FAB-MS (nega-
tive mode) m/z: 373.2217 (Calcd for C19H33O7, [MꢀH]ꢀ: 373.2226). 1H-
and 13C-NMR data are given in Table 2.
4: A white powder, [a]D ꢀ47.1° (cꢁ0.23, MeOH). HR-FAB-MS (nega-
1
tive mode) m/z: 505.2651 (Calcd for C24H41O11, [MꢀH]ꢀ: 505.2649). H-
and 13C-NMR data are given in Table 2.
5: A white powder, [a]D ꢃ64.1° (cꢁ1.01, MeOH). HR-FAB-MS (nega-
tive mode) m/z: 385.1858 (Calcd for C19H29O8, [MꢀH]ꢀ: 385.1862). CD
(cꢁ9.3ꢅ10ꢀ5 mol/l, MeOH) De (l nm): ꢃ9.94 (242). The 1H- and 13C-
Experimental
1H- and 13C-NMR spectra were measured on a GE-Omega 600 or JEOL

July 2005
787
NMR spectral data of 5 were in agreement with those reported for cor-
choionoside C [ꢁ(6S,9S)-roseoside A].3)
0.909 (3H, s, H3-11), 0.934 (3H, s, H3-12), 1.350 (3H, d, Jꢁ6.3 Hz, H3-10),
1.491 (3H, s, H3-13), 1.560 (1H, dd, Jꢁ12.0, 12.0 Hz, H-2b), 1.578 (2H, m,
H2-8), 1.779 (1H, ddd, Jꢁ12.0, 3.6, 2.4 Hz, H-2a), 1.891 (2H, m, H2-7),
1.999 (1H, br dd, Jꢁ16.6, 10.0 Hz, H-4b), 2.250 (1H, br dd, Jꢁ16.6, 5.8 Hz,
H-4a), 5.099 (1H, m, H-9), 5.218 (1H, m, H-3).
Enzymatic Hydrolysis of 1 A solution of 1 (20.0 mg) in water (2 ml)
was incubated with b-glucosidase (almond emulsin; 10 mg) at 37 °C for
17 h. The reaction mixture was poured into a large amount of water and ex-
tracted with AcOEt. The solvent of the extract was evaporated to dryness
under reduced pressure to give the corresponding aglycone 1a (8.5 mg), a
white powder, [a]D ꢀ51.4° (cꢁ0.58, MeOH). HR-FAB-MS (negative mode)
m/z: 183.1029 (Calcd for C10H15O3, [MꢀH]ꢀ: 183.1021). 1H-NMR
(400 MHz, pyridine-d5) d: 1.30 (6H, s, H3-9, H3-10), 1.40 (1H, m, H-5), 1.71
(1H, m, H-4), 2.20 (2H, m, H-3, H-5), 2.46 (2H, m, H-3, H-6), 2.90 (1H,
br d, Jꢁ16.0 Hz, H-6), 7.38 (1H, br dd, Jꢁ3.0, 3.0 Hz, H-2). 13C-NMR
(100 MHz, pyridine-d5) d: 24.0 (C-9 or C-10), 26.2 (C-5), 27.1 (C-10 or C-
9), 27.8 (C-6), 28.0 (C-3), 45.1 (C-4), 70.9 (C-8), 131.8 (C-1), 139.4 (C-2),
169.9 (C-7).
Enzymatic Hydrolysis of 2 A solution of 2 (14.9 mg) in water (2 ml)
was incubated with cellulase (20 mg) at 37 °C for 43 h. The reaction mixture
was poured into a large amount of water and extracted with AcOEt. The sol-
vent of the extract was evaporated to dryness under reduced pressure to give
an artifact aglycone 2a (3.2 mg), a white powder, [a]D ꢃ27.9° (cꢁ0.17,
MeOH). HR-EI-MS m/z: 222.1252 (Calcd for C13H18O3, [MꢀH2O]ꢃ:
222.1256). 1H- and 13C-NMR data are given in Table 2.
Determination of Configurations of Each Component Glucose in 1—4
and the Component Arabinose in 4 A solution of each of 1—4 (each
2.0 mg) in 2 M HCl–EtOH (1 : 1; 0.5 ml) was stirred under reflux for 2 h. The
solution was extracted with EtOAc and water. The resulting aqueous layer
was neutralized with Amberlite IRA-93ZU (Organo Co., Ltd.) and evapora-
tion of the water gave a monosaccharide residue that was subjected to the
preparation of the corresponding thiazolidine derivative, followed by
trimethylsilylation and GLC analysis, according to the reported procedure.5)
The D-configuration for glucose obtained from each of 1—4 and the L-con-
figuration for arabinose from 4 were determained, respectively, based on a
direct comparison with D- and L-standards of glucose (tR: D-Glc, 13 min 15 s;
tR: L-Glc, 13 min 54 s) and arabinose (tR: D-Ara, 8 min 13 s; tR: L-Ara, 7 min
39 s).
Bioassay. Bacteria and Broth Helicobacter pylori NCTC11637, H.
pylori NCTC11916, and H. pylori OCO1 were used in the present assay.
Brucella broth and Brucella agar (Becton Dickinson Co., Ltd.) were used for
preincubation of H. pylori and for the measurement of MIC, respectively.
Measurements of MIC of 1, 2, 3, and 5 MIC values were determined
using the agar dilution method.11) Each of the samples (1, 2, 3, and 5, and
both natural and synthetic specimens of hinokitiol as positive standards) was
dissolved individually in dimethylsulfoxide (0.5 ml) and added to sterilized
distilled water, giving a sample solution of 200 mg/ml that was diluted with
sterilized distilled water according to the two-fold dilution method to afford
100, 50, and 25 mg/ml of solutions, respectively. To each sample solution
(1 ml) at various concentrations in each Petri dishes, sterilized albumin solu-
tion (250 mg/50 ml; 1 ml) and sterilized Brucella agar (8 ml) were added and
mixed. H. pylori suspension 50 ml (about 1.0ꢅ107 cells/ml) was inoculated
into each agar plate. The agar plates were placed in a cultivation jar
equipped with AnaeroPack® Campylo (Mitsubishi Gas Co., Ltd.). After cul-
tivation at 37 °C for 2 d under slightly aerobic conditions, survival or death
of the bacteria was judged and the MIC was determined.
Preparation of (S)-MTPA Ester (2b) and (R)-MTPA Ester (2c) from
2a To a solution of 2a (1.6 mg) in pyridine (0.5 ml), (R)-(ꢀ)-MTPA chlo-
ride (40 ml) was added and the reaction mixture was allowed to stand for
25 h at room temperature. After the solvent was evaporated in vacuo, the re-
sulting residue was extracted with a mixture of ether and water. The ether
layer was washed with brine and dried over MgSO4. After evaporation of
ether, the residue was purified with silica gel column chromatography eluted
with a mixture of n-hexane and acetone (2 : 1) to give the corresponding 9-
(S)-MTPA ester (2b) (3.0 mg), a colorless oil. 1H-NMR (400 MHz, CD3OD)
d: 0.953 (3H, s, H3-12), 1.111 (3H, s, H3-13), 1.416 (3H, d, Jꢁ6.4 Hz, H3-
10), 2.358 (1H, dd, Jꢁ18.0, 2.4 Hz, H-2), 2.408 (1H, dd, Jꢁ17.6, 2.4 Hz, H-
4), 2.595 (1H, dd, Jꢁ18.0, 2.8 Hz, H-2), 2.644 (1H, d, Jꢁ17.6 Hz, H-4),
3.640 (1H, d, Jꢁ8.0 Hz, H-11), 3.908 (1H, dd, Jꢁ8.0, 2.8 Hz, H-11), 5.684
(1H, m, H-9), 6.170 (1H, dd, Jꢁ15.2, 6.8 Hz, H-8), 6.247 (1H, d,
Jꢁ15.2 Hz, H-7). A similar treatment of 2a (1.6 mg) with (S)-(ꢃ)-MTPA
chloride (40 ml) gave the corresponding 9-(R)-MTPA ester (2c) (3.0 mg), a
Acknowledgments This work was supported in part by a Grant-in-Aid
for Scientific Research (No. 09041194) from the Ministry of Education,
Culture, Sports, Science, and Technology in Japan. Thanks are due to Dr.
Robert P. Adams, Director of the Pacific Center for Molecular Biodiversity,
Bishop Museum, U.S.A., for identification of the plant and to Dr. E. Ishii,
Osaka City Institute of Public Health and Environmental Sciences, for his
kind gift of three strains of H. pylori.
1
colorless oil. H-NMR (400 MHz, CD3OD) d: 0.905 (3H, s, H3-12), 1.088
(3H, s, H3-13), 1.471 (3H, d, Jꢁ6.4 Hz, H3-10), 2.310 (1H, dd, Jꢁ18.0,
2.4 Hz, H-2), 2.392 (1H, dd, Jꢁ17.6, 2.4 Hz, H-4), 2.469 (1H, dd, Jꢁ18.0,
2.8 Hz, H-2), 2.559 (1H, d, Jꢁ18.0 Hz, H-4), 3.622 (1H, d, Jꢁ8.0 Hz, H-11),
3.882 (1H, dd, Jꢁ8.0, 2.8 Hz, H-11), 5.703 (1H, m, H-9), 6.072 (1H, d,
Jꢁ15.2 Hz, H-7), 6.114 (1H, dd, Jꢁ15.2, 6.8 Hz, H-8).
Enzymatic Hydrolysis of 3 A solution of 3 (6.0 mg) in water (2 ml) was
incubated with b-glucosidase (almond emulsin; 10 mg) at 37 °C for 16.5 h.
The reaction mixture was poured into a large amount of water and extracted
with AcOEt. The usual work-up and purification of the AcOEt layer gave the
corresponding genuine aglycone 3a (3.0 mg), a white powder, [a]D ꢀ54.4°
(cꢁ0.30, MeOH). HR-EI-MS m/z: 212.1779 (Calcd for C13H24O2, Mꢃ:
References and Notes
1) Nakanishi T., Iida N., Inatomi Y., Murata H., Inada A., Murata J., Lang
F. A., Iinuma M., Tanaka T., Phytochemistry, 65, 207—213 (2004).
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tsuda H., Chem. Pharm. Bull., 45, 464—469 (1997).
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5) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 34, 1843—1845
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1
212.1776). H-NMR (400 MHz, CD3OD) d: 1.03 (3H, s, H3-11), 1.06 (3H,
s, H3-12), 1.17 (3H, d, Jꢁ6.3 Hz, H3-10), 1.37 (1H, dd, Jꢁ12.0, 12.0 Hz, H-
2b), 1.45 (1H, m, H-8), 1.52 (1H, m, H-8), 1.63 (3H, s, H3-13), 1.68 (1H,
ddd, Jꢁ12.0, 3.6, 2.4 Hz, H-2a), 1.92 (1H, br dd, Jꢁ16.6, 10.8 Hz, H-4b),
2.02 (1H, dt, Jꢁ13.2, 5.1 Hz, H-7), 2.12 (1H, dt, Jꢁ13.2, 5.1 Hz, H-7), 2.18
(1H, br dd, Jꢁ16.6, 5.8 Hz, H-4a), 3.70 (1H, m, H-9), 3.84 (1H, m, H-3).
13C-NMR (100 MHz, CD3OD) d: 20.0 (C-13), 23.3 (C-10), 25.6 (C-7), 28.9
(C-11), 30.4 (C-12), 38.9 (C-1), 40.7 (C-8), 43.0 (C-4), 49.6 (C-2), 65.7 (C-
3), 69.2 (C-9), 125.5 (C-5), 138.4 (C-6).
Preparation of 3,9-Di-(S)-MTPA Ester (3b) and 3,9-Di-(R)-MTPA
Ester (3c) from 3a In a similar manner as in the derivation of 2b and 2c
from 2a, 3a (1.5 mg each) was treated with (R)-(ꢀ)- and (S)-(ꢃ)-MTPA
chloride (40 ml each) to afford 3b (2.0 mg) and 3c (2.2 mg), respectively. 3b:
a colorless oil, 1H-NMR (400 MHz, CD3OD) d: 0.972 (3H, s, H3-11), 1.043
(3H, s, H3-12), 1.291 (3H, d, Jꢁ6.3 Hz, H3-10), 1.500 (1H, dd, Jꢁ12.0,
12.0 Hz, H-2b), 1.618 (3H, s, H3-13), 1.689 (2H, m, H2-8), 1.729 (1H, ddd,
Jꢁ12.0, 3.6, 2.4 Hz, H-2a), 2.080 (2H, m, H2-7), 2.165 (1H, br dd, Jꢁ16.0,
10.0 Hz, H-4b), 2.369 (1H, br dd, Jꢁ16.6, 5.8 Hz, H-4a), 5.111 (1H, m, H-
9), 5.252 (1H, m, H-3). 3c: a colorless oil, 1H-NMR (400 MHz, CD3OD) d:
7) Oritani T., Yamashita K., Tetrahedron Lett., 1972, 2521—2524 (1972).
8) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem. Soc.,
113, 4092—4096 (1991).
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Phytochemistry, 50, 1219—1223 (1999).
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(2003).
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Ohsawa N., Tanami H., Chemotherapy, 29, 76—79 (1981).