Oblongionosides A-F, megastigmane glycosides from the leaves of Croton oblongifolius Roxburgh

Article abstract of DOI:10.1016/j.phytochem.2012.05.011

From the 1-BuOH-soluble fraction of a MeOH extract of the leaves of Croton oblongifolius Roxburgh, collected in Chiang Mai, Thailand, six megastigmane glycosides, named oblongionosides A-F were isolated together with eight known compounds, and their structures elucidated on the basis of spectroscopic data. Absolute structures were determined by HPLC analyses and application of the modified Mosher's method.

Full text of DOI:10.1016/j.phytochem.2012.05.011

Phytochemistry 80 (2012) 132–136
Contents lists available at SciVerse ScienceDirect
Phytochemistry
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Oblongionosides A–F, megastigmane glycosides from the leaves of Croton
oblongifolius Roxburgh
Yuya Takeshige a, Susumu Kawakami a, Katsuyoshi Matsunami a, Hideaki Otsuka a,
,
⇑
Duangporn Lhieochaiphant b, Sorasak Lhieochaiphant c
a Department of Pharmacognosy, Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
b Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, 239 Huaw Kaew Road, Chiang Mai 50200, Thailand
c Faculty of Pharmacy, Payap University, Superhighway Chiang Mai-Lampang Road, Muang, Chiang Mai 50000, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
From the 1-BuOH-soluble fraction of a MeOH extract of the leaves of Croton oblongifolius Roxburgh, col-
lected in Chiang Mai, Thailand, six megastigmane glycosides, named oblongionosides A–F were isolated
together with eight known compounds, and their structures elucidated on the basis of spectroscopic data.
Absolute structures were determined by HPLC analyses and application of the modified Mosher’s method.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 10 December 2011
Received in revised form 1 March 2012
Available online 8 June 2012
Keywords:
Croton oblongifolius
Euphorbiaceae
Oblongionoside
Megastigmane
glycosidemodified
Mosher’s method
1. Introduction
procedures to afford six new megastigmane glycosides, named
oblongionosides A–F (1–6) along with eight known compounds
Croton oblongifolius Roxburgh (Euphorbiaceae) is a deciduous
tree of about 8 m in height that grows wild in Asian pantropical
areas, such as Thailand, Myanmar and Indonesia. C. oblongifoius is
a traditional folk medicine in Thailand, its flowers being used as
an anthelmintic, its fruit, macerated with 28–40% alcohol, as an
oxytocic for post-labor, and its stem bark or leaves as an antidiar-
rheal and a blood tonic etc. (Chuakul et al., 1997). From various
parts of C. oblongifolius, several types of diterpenoids, labdanes
(Roengsumran et al., 2001), halimane (Roengsumran et al., 2004),
clerodanes (Roengsumran et al., 2002; Youngsa-Ad et al., 2007),
and cembranes (Roengsumran et al., 1998, 1999) have been iso-
lated. The current phytochemical study on the leaves of C. oblo-
ngifolius afforded three pairs of megastigmane glycosides, named
oblongionosides A–F (1–6), and this paper describes their struc-
tural elucidation.
(7–14). The known compounds were identified as kaempferol 3-
O-b- -apiofuranosyl)glucopyranoside 7-O- -rhamno
-(200-O-b-
pyranoside (7) (Hisaeda et al., 2011), kaempferol 3-O-b-
-(200-O-
b- -rhamnopyranosyl)glucopyranoside 7-
-apiofuranosyl, 600-O-
O- -rhamnopyranoside (8) (Hisaeda et al., 2011), kaempferitrin
(9) (Marzouk et al., 2009), clerspide A (10) (Zou et al., 2008), icar-
iside F2 (11) (Wang et al., 1998), canthoside A (12) (Kanchanapoom
et al., 2002), dendranthemoside A (13) (Otsuka et al., 1992), and
glochidionioside D (14) (Otsuka et al., 2003) by comparison of their
spectroscopic data, including optical rotation values, with those
reported in the literature.
D
D
a-L
D
D
a-L
a
-L
Oblongionoside A (1), [
powder and its elemental composition was determined to be
24H42O12 by high-resolution (HR) electrospray-ionization (ESI)
a
]D ꢀ64.2, was obtained as an amorphous
C
time-of-flight (TOF) mass spectrometry (MS). In the IR spectrum,
a strong absorption band at 3367 cmꢀ1 suggested that 1 must
possess glycosidic motifs in its molecule. In the 1H NMR spectrum,
two singlet methyls and two doublet methyls, and two anomeric
protons at dH 4.29 (d, J = 8 Hz) and 5.01 (d, J = 2 Hz) were observed
(Table 1). The latter anomeric proton was assigned to an apiose
moiety, and acid hydrolysis of 1 confirmed the presence of
2. Results and discussion
Air-dried leaves of C. oblongifolius were extracted with MeOH
three times and the concentrated extract was partitioned with
solvents of increasing polarity. The 1-BuOH-soluble fraction was
separated and purified by means of various chromatographic
D
-glucose and D-apiose, as sugar components. Eleven signals in
the 13C NMR spectra were assigned to a terminal apiofuranoe and
a substituted glucopyranose (Table 2). The remaining 13 reso-
nances comprised four methyls, two methylenes, two oxygenated
⇑
Corresponding author. Tel./fax: +81 82 257 5335.
E-mail address: hotsuka@hiroshima-u.ac.jp (H. Otsuka).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.05.011

Y. Takeshige et al. / Phytochemistry 80 (2012) 132–136
133
Table 1
1H NMR spectroscopic data for oblongionosides A–F (1–6) (400 MHz, CD3OD).
H
2
1
2
3
4
5
6
1.57 ddd 12.4, 4.4, 1.8
1.69 dd 12.4, 12.1
1.56 ddd 12.4, 4.4, 1.8
1.69 dd 12.4, 12.1
1.15 dd 12.4, 12.3
1.79 ddd 12.3, 4.0, 2.4
1.15 dd 12.4, 12.3
1.79 ddd 12.3, 4.0, 2.4
1.16 dd 12.4, 11.9
1.81 ddd 12.4, 4.0, 2.4
1.15 dd 12.4, 11.9
1.81 ddd 12.4, 4.0, 2.4
3
4
3.89 dddd 12.1, 11.9, 4.6, 3.89 dddd 12.1, 11.9, 4.6, 3.80 dddd 12.4, 11.9, 4.4, 3.80 ddd 12.4, 11.9, 4.4, 3.85 dddd 12.1, 11.9, 4.4, 3.85 dddd 12.1, 11.9, 4.4,
4.4 4.4 4.0 4.0 4.0 4.0
1.16 ddd 12.3, 12.1, 11.7 1.16 ddd 12.3, 12.1, 11.7
1.49 ddd 12.8, 12.4, 11.9 1.49 ddd 12.8, 12.4, 11.9 1.03 ddd 12.5, 12.3, 11.9 1.02 ddd 12.5, 12.3,
11.9
1.84 dddd 12.4, 4.6, 3.8,
1.8
1.84 dddd 12.4, 4.6, 3.8,
1.8
2.04 dddd 12.3, 4.4, 3.8,
2.4
2.04 dddd 12.3, 4.4, 3.8, 2.24 dddd 12.3, 4.4, 3.5,
2.24 dddd 12.3, 4.4, 3.5,
2.4
2.4
2.4
5
6
7
1.97 dqd 12.8, 6.4, 3.8
–
5.55 dd 15.7, 0.9
–
1.95 dqd 12.8, 6.4, 3.8
–
5.55 dd 15.7, 0.9
–
1.46 m
0.55 ddd 11.0, 4.9, 2.4
1.22 2H m
1.46 m
1.50 m
0.80 ddd 11.2, 5.1, 2.0
1.24 m
1.50 m
0.81 ddd 11.2, 5.1, 2.0
1.24 m
0.55 ddd 11.0, 4.9, 2.4
1.22 2H m
–
–
1.46 m
1.46 m
8
5.73 dd 15.7, 6.0
–
5.73 dd 15.7, 6.0
–
1.34 m
1.57 m
1.34 m
1.57 m
1.34 m
1.53 m
1.34 m
1.53 m
9
4.29 dqd 6.4, 6.0, 0.9
4.29 dqd 6.4, 6.0, 0.9
1.24 3H d 6.4
0.99 3H s
0.90 3H s
0.82 3H d 6.4
–
3.65 dqd 6.4, 6.2, 5.7
1.14 3H d 6.2
0.84 3H s
0.97 3H s
1.00 3H d 6.4
–
3.66 dqd 6.4, 6.2, 5.7
1.14 3H, d 6.2
0.84 3H s
0.96 3H s
0.99 3H d 6.4
–
3.66 dqd overlapped
1.15 3H d 6.2
0.84 3H s
3.66 overlapped
1.15 d 6.2
0.85 3H s
10 1.24 3H d 6.4
11 0.97 3H s
12 0.87 3H s
13 0.85 3H d 6.4
–
1’
2’
3’
4’
5’
60
0.98 3H s
0.98 3H s
3.43 dd 10.8, 6.8
3.72 dd 10.8, 3.1
4.35 d 7.7
3.13 dd 9.0, 7.7
3.34 dd 9.0, 9.0
3.26 overlapped
3.26 overlapped
3.65 dd 11.7, 5.7
3.86 dd 11.7, 1.8
3.43 dd 10.8, 6.8
3.69 dd 10.8, 3.1
4.35 d 7.7
3.13 dd 9.0, 7.7
3.34 dd 9.0, 9.0
3.26 overlapped
3.27 overlapped
3.65 dd 11.7, 5.7
3.86 dd 11.7, 1.8
4.34 d 7.9
4.34 d 7.9
4.32 d 7.7
4.32 d 7.7
3.13 dd 9.0, 7.9
3.26 dd 9.0, 9.0
3.36 dd 9.0, 9.0
3.40 ddd 9.0, 6.2, 2.0
3.61 dd 11.2, 6.2
3.97 dd 11.2, 2.0
5.01 d 2.4
3.14 dd 9.0, 7.9
3.26 dd 9.0, 9.0
3.34 dd 9.0, 9.0
3.41 ddd 9.0, 6.2, 2.0
3.61 dd 11.2, 6.2
3.97 dd 11.2, 2.0
5.02 d 2.4
3.11 dd 8.9, 7.7
3.30 overlapped
3.24 dd 9.7, 8.9
3.39 ddd 9.7, 6.2, 2.0
3.60 dd 11.2, 6.2
3.96 dd 11.2, 2.0
5.00 d 2.4
3.12 dd 8.9, 7.7
3.30 overlapped
3.24 dd 9.7, 8.9
3.39 ddd 9.7, 6.2, 2.0
3.60 dd 11.2, 6.2
3.96 dd 11.2, 2.0
5.00 d 2.4
100
200
400
3.89 d 2.4
3.76 d 9.5
3.89 d 2.4
3.77 d 9.5
3.85 d 2.4
3.76 d 9.7
3.88 d 2.4
3.76 d 9.7
3.96 d 9.5
3.57 d 11.5
3.61 d 11.5
3.97 d 9.5
3.57 d 11.5
3.61 d 11.5
3.97 d 9.7
3.56 d 11.2
3.59 d 11.2
3.97 d 9.7
3.56 d 11.2
3.59 d 11.2
500
Table 2
13-methyl was also in the equatorial orientation. The megastig-
mane skeleton, which has these functional groups, was found in
turpinionoside A (15), isolated from Turpinia ternata (Yu et al.,
2002), and dendranthemoside A (13), isolated from Dendranthema
shiwogiku zeylanicum (Otsuka et al., 1992) (Fig. 1). The NMR
spectroscopic data for the aglycones of these compounds were
essentially indistinguishable. Since in the HMBC spectrum the
anomeric proton (dH 5.01) of the apiofuranose moiety showed a cor-
relation cross peak with the C-60 of the glucopyranose, the structure
13C NMR spectroscopic data for oblongionosides A–F (1–6 and 5a) (100 MHz, CD3OD).
C
1
2
3
4
5
5a
6
1
2
3
4
5
6
7
8
9
10
11
12
13
1’
2’
3’
4’
5’
60
100
200
300
400
500
40.4
42.7
76.0
38.2
35.5
78.2
40.4
42.7
75.9
38.2
35.4
78.3
36.8
48.6
76.1
44.9
35.1
54.3
26.4
42.7
69.2
23.4
21.4
31.4
21.5
102.9
75.2
78.1
71.9
76.9
68.7
111.0
78.2
80.6
75.1
65.9
36.8
48.5
76.1
44.9
35.2
54.3
26.3
42.6
69.0
23.5
21.4
31.4
21.6
102.9
75.1
78.1
71.9
76.9
68.7
111.0
78.2
80.6
75.2
65.9
36.6
48.5
76.1
39.1
42.8
48.5
26.0
41.9
68.9
23.5
21.2
31.2
65.9
102.9
75.2
78.1
71.8
77.9
62.9
36.7
36.6
48.5
76.1
39.2
42.7
48.5
26.0
42.0
69.1
23.4
21.2
31.2
66.0
102.9
75.2
78.1
71.8
77.9
62.9
51.8 (ꢀ3.3)a
67.6 (+8.5)a
40.8 (ꢀ1.7)a
42.8
48.5
26.0
42.0
69.0
23.6
21.4
31.3
66.0
133.8
135.6
69.2
24.2
25.9
25.2
16.5
102.8
75.1
78.1
71.9
76.8
68.7
111.0
78.1
80.6
75.0
65.8
133.7
135.5
69.2
24.1
25.8
25.1
16.5
102.7
75.0
78.0
71.8
76.8
68.7
110.9
78.0
80.5
75.0
65.8
of 1 was expected to be the 60-O-b-
D-apiofuranoside of turpiniono-
side A or dendranthemoside A. The absolute stereochemistry at the
9-position of 1 was independently determined by application of the
modified Mosher’s method (Ohtani et al., 1991) to the aglycone (1a)
of 1, it being assigned as S, as shown in Fig. 2. Therefore, the
structure of oblongionoside A (1) was elucidated to be (3S,5R,6S,
7E,9S)-megastigm-7-ene-3,6,9-triol 3-O-b-
D
-(60-O-b-
D-apiofuranosylturpinionoside
D-apiofurano-
syl)glucopyranoside, namely 60-O-b-
A, as shown in Fig. 1.
Oblongionoside B (2), [
a]
ꢀ67.3, was obtained as an amor-
D
phous powder and its elemental composition was the same as that
of oblongionoisde A. The NMR spectroscopic data for 1 and 2
shown in Tables 1 and 2 are essentially the same. Since oblongion-
osides A (1) and B (2) were isolated on the same preparative HPLC
run at different retention times, 31 min and 24 min, respectively,
they are unambiguously different compounds. Thus, the only pos-
sible explanation was that 2 is an epimer at the 9-position (Otsuka
et al., 1995; Matsunami et al., 2010). Therefore, the structure of
a
D
d5a–5.
methines, one methine, one oxygenated tertiary carbon, one qua-
ternary carbon and a trans double bond. These carbons must make
up a megastigmane skeleton, with three hydroxy groups at the 3, 6
and 9 positions and the trans double bond between the 7- and
8-positions. The proton coupling constants indicated that the
hydroxyl group at the 3-position is in the equatorial orientation,
and the coupling constant of H-5 (J = 12, 7, 4 Hz) suggested the
oblongionoside
B
(2) was assigned as (3S,5R,6S,7E,9R)-meg-
-(60-O-b-
-apiofuranosyl)glucopy-
-apiofuranosylglochidionionoside A, as
astigm-7-ene-3,6,9-triol 3-O-b-
D
D
ranoside, namely 60-O-b-
D
shown in Fig. 1.
Oblongionosides C (3), [
also isolated as amorphous powders on the same preparative HPLC
a
]
ꢀ20.8, and D (4), [
a
]
ꢀ21.6, were
D
D

134
Y. Takeshige et al. / Phytochemistry 80 (2012) 132–136
OMTPA
+0.087
+0.066
+0.098
+0.194
– 0.084
+0.121
OMTPA
MTPAO
– 0.01
+0.01
Fig. 3. Results of the modified Mosher’ method for oblongionoside E (5). Figures are
d values.
D
(60-O-b-
in Fig. 1.
Oblongionoside F (5), [
powder and its elemental composition was determined to be
19H36O8 by HR-ESI-TOF-MS. The 1H NMR spectrum exhibited two
D-apiofuranosyl)glucopyranosides, respectively, as shown
a
]D ꢀ26.9, was isolated as an amorphous
C
singlet methyls, one doublet methyl and an anomeric proton at dH
4.35 (d, J = 8 Hz). Of the 19 13C NMR signals, six were assignable as
those of terminal glucopyranose, the remaining 13 resonances com-
prising three methyls, four methylenes, one oxygenated methylene,
two methines, two oxygenated methines and a quaternary carbon.
The results of COSY spectrum suggested that primary hydroxy func-
tion group was at C-13 and this was confirmed by correlations in the
HMBC spectrum from H2-13 to C-4, C-5 and C-6. Other NMR
spectroscopic evidence indicated that oblongionoside F (5) was a
megastigmane having three hydroxy groups at the 3-, 9- and 13-
positions. The typical coupling pattern of H-3 placed the hydroxy
group at this position in the equatorial orientation, and the coupling
constants between H-5 and H-6 (J = 11 Hz) also placed the primary
alcohol and the side-chain in the equatorial orientation. Oblongion-
oside E (5) was enzymatically hydrolyzed to give an aglycone (5a),
to which the modified Mosher method was applied, the obtained
data being shown in Fig. 3. The
shows that the 9-position has the S-configuration and the
for the 2-position show that the 3-position is also S. However, the
D
d value for the 10-position clearly
Fig. 1. Structure of isolated compounds and reference compounds.
D
d values
D
d
values for the 4-position were conflicting, probably due to the effect
of the extra MTPA at the 13-position. Since the upfield shift at the 2-
position was apparently larger than that at the 4-position (Table 2),
OMTPA
+0.043
+0.060
+
+0.088
+0.103
the empirical rule, the b-D-glucopyranosylation-induced shift trend
S
+
-0.045
rule between 5 and 5a (Kasai et al., 1977), also supports that the
absolute configuration at the 3-position is S. Therefore, the structure
of oblongionoside E (5) was elucidated to be (3S,5R,6S,9S)-megastig-
OH
S
+0.013
MTPAO
-0.073
-0.088
+0.025
mane-3,9,13-triol 3-O-b-
D
-glucopyranoside, as shown in Fig. 1.
ꢀ18.1, was obtained as an amor-
Oblongionoside F (6), [
a]
D
phous powder and was found to be a similar compound to 5, hav-
ing the same elemental composition, and its NMR spectral data
were also indistinguishable from those of 5. However, 6 was a def-
initely different compound from 5, since they were separated on
the same preparative HPLC run at different retention times, i.e., 6
and 5 were obtained from the peaks at 22 min and 25 min, respec-
tively. Therefore, the structure of 6 was assigned as an isomer of 5
as to the 9-position, namely (3S,5R,6S,9R)-megastigmane-3,9,13-
Fig. 2. Results of the modified Mosher’ method for oblongionoside A (1). Figures are
d values.
D
run at different retention times, 138 min and 131 min, respectively.
Their elemental compositions were determined to be the same, i.e.,
24H44O12 by HR-ESI-TOF-MS. NMR spectra of 3 and 4 were similar
C
to those of 1 and 2, except for the replacement of the hydroxyl
triol 3-O-b-D-glucopyranoside, as shown in Fig. 1.
group by a hydrogen atom and the double bond was reduced to a
single bond. Acid hydrolyses of 3 and 4 equally gave
D-apiose and
D-glucose as sugar components. A similar compound, symplocos-
ionoside A (16), has been isolated from Symplocos cochinchinensis
var. philippinensis, the structure of which was elucidated unambig-
uously, including the absolute stereochemistry (Cai et al., 2011).
The double bond between the 7- and 8-positions of symplocosion-
oside A (16) was catalytically reduced to give dihydrosymplocos-
ionoside A (16a), which must be identical with either 3 or 4. On
HPLC analysis, 16a appeared at the retention time of 131 min. Thus,
16a is an identical compound to oblongionoside D (4). Therefore,
the structures of oblongionosides C (3) and D (4) were assigned as
3. Conclusion
Six new megastigname glycosides were isolated from the leaves
of C. oblongifoius and their structures were carefully elucidated.
Since pairs of oblongionosides, A (1) and B (2), C (3) and D (4),
and E (5) and F (6), are isolated on the same preparative HPLC runs,
they were easily recognized to be different compounds, although
the pairs exhibited essentially the same NMR spectroscopic data.
This is interesting, and further accumulation of empirical data will
be helpful for establishing a rule that for all such pairs, 9-S isomers
(3S,5R,6S,9S) and (3S,5R,6S,9R)-megastigmane-3,9-diol 3-O-b-D-

Y. Takeshige et al. / Phytochemistry 80 (2012) 132–136
135
(2, 4 and 6) were regularly eluted more slowly than 9-R-isomers (1,
3 and 5) under the same HPLC conditions.
14 (4.42 mg) in fractions 24–28, 39–46, and 47–49, respectively.
The 1 and 2 mixture was separated by HPLC (ODS) with MeOH–
H2O (3:7) to give 2 (24.9 mg) and 1 (8.4 mg) from the peaks at
24 min and 31 min, respectively. The crude 13 was purified by
HPLC (ODS) with MeOH–H2O (1:3) to give 13 (8.0 mg) from the
peak at 23 min. The crude 14 was purified by HPLC with MeOH–
H2O (3:7) to give 14 (2.5 mg) from the peak at 24 min.
4. Experimental
4.1. General experimental procedures
The residue (124 mg) in 109–131 fractions obtained on RPCC
was separated by DCCC to afford a 5 and 6 mixture (18.0 mg) in
fractions 30–34, which were separated by HPLC with MeOH-H2O
(3:7) to give 6 (2.4 mg) and 5 (5.4 mg) from the peaks at 22 min
and 25 min, respectively.
Optical rotations were measured on a JASCO P-1030 polarime-
ter. IR spectra were recorded on a Horiba FT-710 Fourier transform
infrared spectrophotometer. 1H- and 13C-NMR spectra were ob-
tained on a JEOL JNM
a
-400 spectrometer at 400 MHz for 1H and
100 MHz for 13C, with tetramethylsilane (TMS) as an internal stan-
dard. Positive-ion HR-ESI-TOF-MS were recorded on an Applied
Biosystem QSTAR XL spectrometer.
The residue (336 mg) in 12–14 fractions obtained on Diaion HP-
20 CC was subjected to RPCC [
U
¼ 2:5 cm, L = 25 cm, linear gradi-
ent: MeOH–H2O (1:9, 500 mL) ? (9:1, 500 mL), 2.5 g/fraction], and
the residue (35.0 mg) in fractions 83–103 was separated by DCCC
to afford crude 11 (6.3 mg) in fractions 25–32. The crude 11 was
purified by HPLC (ODS) with MeOH–H2O (11:39, v/v) to give 11
(1.1 mg) from the peak at 24 min. The residue (295 mg) in fractions
104–148 RPCC was separated by DCCC to afford crude 12 (30.1 mg)
in fractions 25–32. The crude 12 was purified by HPLC (ODS) with
35% MeOH to give 12 (1.9 mg) from the peak at 10 min. The residue
(9.5 mg) in fractions 149–154 obtained on RPCC was separated by
DCCC to afford a 3 and 4 mixture (3.0 mg) in fractions 40–45,
which were separated by HPLC (ODS) with CH3CN:MeOH:H2O
(5:18:77) to give 4 (1.6 mg) and 3 (1.2 mg) from the peaks at
131 min and 138 min, respectively.
A highly-porous synthetic resin (Diaion HP-20) was purchased
from Mitsubishi Chemical Co. Ltd., (Tokyo, Japan). Silica gel CC
was performed on silica gel 60 (Merck, Darmstadt, Germany) and
reversed-phase (ODS) open CC (RPCC) on Cosmosil 75C18-OPN
(Nacalai Tesque, Kyoto, Japan). The droplet counter-current chro-
matograph (DCCC) (Tokyo Rikakikai, Tokyo, Japan) was equipped
with 300 glass columns (
U
¼ 2 mm, L = 40 cm), and the lower
and upper layers of a solvent mixture of CHCl3–MeOH–H2O–PrOH
(9:12:8:2) were used as the stationary and mobile phases, respec-
tively. Five-gram fractions were collected and numbered according
to their order of elution with the mobile phase. HPLC was per-
formed on ODS (Inertsil ODS-3; GL Science, Tokyo, Japan;
U
¼ 6 mm, L = 250 mm, flow rate; 1.6 mL/min), and the eluate
The residue (1.79 g) in 15–17 fractions obtained on Diaion HP-
was monitored with a refractive index monitor.
20 CC was subjected to RPCC [
U
¼ 5 cm, L = 25 cm, linear gradient:
Emulsin was purchased from Tokyo Chemical Industries Co.
Ltd., (Tokyo, Japan), and crude hesperidinase was a gift from Tokyo
MeOH–H2O (1:9, 2 L) ? (9:1, 2 L), 10 g/fraction], and the residue
(272 mg) in fractions 128–152 was separated by DCCC to afford 8
(34.1 mg) and 7 (25.8 mg) in fractions 16–21 and 25–31, respec-
tively. The residue (354 mg) in fractions 183–197 was recrystal-
lized to afford 9 as crystals (87.7 mg).
Tanabe Pharmaceutical Co. Ltd., (Tokyo, Japan). (R)- and (S)-
a-
methoxy- -trifluoromethylphenylacetic acids (MTPA) were the
a
products of Wako Pure Chemical Industry Co. Ltd., (Tokyo, Japan).
The residue (869 mg) in fractions 18–20 obtained on Diaion HP-
4.2. Plant material
20 CC was subjected to RPCC [
U
¼ 5 cm, L = 25 cm, linear gradient:
MeOH–H2O (1:9, 2 L) ? (9:1, 2 L), 10 g/fraction], and the residue
(39.5 mg) in fractions 189–198 was separated by DCCC to afford
11.0 mg of 10 in fractions 88–98.
Leaves of C. oblongifolius (Euphorbiaceae) were collected in the
Medicinal Plant Garden, Chiang Mai University, Thailand, in Janu-
ary 2009, and a voucher specimen was deposited in the Herbarium
of the Faculty of Pharmaceutical Sciences, Chiang Mai University
(0901-CO-Chiang Mai U.)
4.3.1. Oblongionoside A (1)
Amorphous powder; ½a D24
ꢁ
64.2 (c 0.55, MeOH); IR mmax (film)
cm1: 3367, 2966, 2931, 2879, 1652, 1457, 1367, 1050; For 1H
NMR (CD3OD, 400 MHz) and; 13C NMR (CD3OD, 100 MHz) spectro-
scopic data, see Tables 1 and 2; HR-ESI-TOF-MS (positive-ion
mode) m/z: 545.2567 [MNa] (calcd for C24H42O12Na, 545.2568).
4.3. Extraction and isolation
Air-dried and powdered leaves of C. oblongifolius (1.08 kg) were
extracted with MeOH (9 L) three times. The MeOH extract was con-
centrated to 1 L and then washed with n-hexane (1 L, 19.6 g). The
methanolic layer was concentrated to a viscous gum. The gummy
mass was suspended in H2O (1 L), and then partitioned with EtOAc
(1 L) and 1-BuOH (1 L), successively, to give EtOAc – 38.1 g and 1-
BuOH – 16.4 g soluble fractions, respectively. The remaining H2O
layer was concentrated to give a H2O-soluble fraction (36.8 g).
The 1-BuOH-soluble fraction was subjected to a Diaion HP-20 col-
4.3.2. Oblongionoside B (2)
Amorphous powder; ½a D24
ꢁ
67.2 (c 1.46, MeOH); IR mmax (film)
cm1: 3367, 2965, 2932, 2880, 1648, 1456, 1368, 1157; For 1H
NMR (CD3OD, 400 MHz) and; 13C NMR (CD3OD, 100 MHz) spectro-
scopic data, see Tables 1 and 2; HR-ESI-TOF-MS (positive-ion
mode) m/z: 545.2567 [MNa] (calcd for C24H42O12Na, 545.2568).
umn (
U
¼ 32 mm, L = 40 cm, 100 mL/fraction), and eluted with
4.3.3. Oblongionoside C (3)
H2O–MeOH (4:1, 500 mL), (3:2, 500 mL), (2:3, 500 mL), and (1:4,
500 mL), and MeOH (500 mL). The residue (4.38 g) in fractions 2–
4 was subjected to silica gel CC (
fraction) with elution with CHCl3 (2 L), CHCl3–MeOH (49:1, 1 l),
(24:1, 1 L), (23:2, 1 L), (9:1, 1 L), (17:3, 1 L), (4:1, 1 L), (3:1, 1 L),
and (7:3, 1 L) and MeOH (1 L).
Amorphous powder; ½a D24
ꢁ
20.8 (c 0.09, MeOH); IR mmax (film)
cm1: 3363, 2960, 2927, 2874, 1597, 1458, 1369, 1046; For 1H
NMR (CD3OD, 400 MHz) and; 13C NMR (CD3OD, 100 MHz) spectro-
scopic data, see Tables 1 and 2; HR-ESI-TOF-MS (positive-ion
mode) m/z: 531.2771 [MNa] (calcd for C24H44O11Na, 531.2771).
U
¼ 32 mm, L = 57.5 cm, 250 mL/
The residue (665 mg) in fractions 9–11 obtained on Diaion HP-
20 CC was subjected to RPCC [
U
¼ 2:5 cm, L = 25 cm, linear gradi-
4.3.4. Oblongionoside D (4)
ent: MeOH–H2O (1:9, 500 mL) ? (9:1, 500 mL), 2.5 g/fraction], and
the residue (196 mg) in fractions 88–108 was separated by DCCC to
afford a 1 and 2 mixture (53.4 mg), crude 13 (23.8 mg), and crude
Amorphous powder; ½a D24
ꢁ
21.6 (c 0.11, MeOH); IR mmax (film)
cm1: 3366, 2959, 2927, 2875, 1597, 1459, 1369, 1045; For 1H
NMR (CD3OD, 400 MHz) and; 13C NMR (CD3OD, 100 MHz) spectro-

136
Y. Takeshige et al. / Phytochemistry 80 (2012) 132–136
scopic data, see Tables 1 and 2; HR-ESI-TOF-MS (positive-ion
mode) m/z: 531.2773 [MNa] (calcd for C24H44O11Na, 531.2775).
Orbitrap XL mass spectrometer at the Natural Science Center for
Basic Research and Development, Hiroshima University. This work
was supported in part by Grants-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology of Japan, and
the Japan Society for the Promotion of Science. Thanks are also
due to the Takeda Science Foundation for the financial support.
4.3.5. Oblongionoside E (5)
Amorphous powder; ½a D24
ꢁ
26.9 (c 0.36, MeOH); IR mmax (film)
cm1: 3363, 2962, 2929, 2881, 1649, 1457, 1369, 1078; For 1H
NMR (CD3OD, 400 MHz) and; 13C NMR (CD3OD, 100 MHz) spectro-
scopic data, see Tables 1 and 2; HR-ESI-TOF-MS (positive-ion
mode) m/z: 415.2304 [MNa] (calcd for C19H36O8Na, 415.2302).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
05.011.
4.3.6. Oblongionoside F (6)
Amorphous powder; ½a D24
ꢁ
18.1 (c 0.16, MeOH); IR mmax (film)
cm1: 3361, 2960, 2927, 2878, 1651, 1457, 1370, 1075; For 1H
NMR (CD3OD, 400 MHz) and; 13C NMR (CD3OD, 100 MHz) spectro-
scopic data, see Tables 1 and 2; HR-ESI-TOF-MS (positive-ion
mode) m/z: 415.2309 [MNa] (calcd for C19H36O8Na, 415.2302).
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4.6. HPLC analysis for determination of the orientation at position 9 of
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With CH3CN–MeOH–H2O (5:18:77, v/v/v), the retention times
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conditions,a peak appearing at 131 min, which was the same as
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supplementary data.
Acknowledgements
The authors are grateful for access to the superconducting NMR
instrument (JEOL JNM a-400) and the Thermo Fisher Scientific LTQ