Hirtionosides A-C, gallates of megastigmane glucosides, 3-hydroxyoctanoic acid glucosides and a phenylpropanoid glucoside from the whole plants of Euphorbia hirta

Article abstract of DOI:10.1007/s11418-012-0692-5

From the 1-BuOH-soluble fraction of a MeOH extract of Euphorbia hirta, collected in the Okinawa islands, three new megastigmane glucoside gallates, named hirtionosides A-C, 3-hydroxyoctanoic acid glucosides and a phenylpropanoid glucoside were isolated along with 15 known compounds. The structures of the new compounds were elucidated by detailed analysis of physical data, including one- and two-dimensional NMR spectra, and those of known compounds were determined by comparison of physico-chemical data with those reported in the literature. The absolute structures of the megastigmanes were determined by comparison of NMR data and Cotton effects in the CD spectra. The modified Mosher's method was applied to determine the absolute structure of the chiral center in 3-hydroxyoctanoic acid. The DPPH radical-scavenging properties of megastigmane glucoside gallates were assayed and, as expected, they showed moderate activity.

Full text of DOI:10.1007/s11418-012-0692-5

J Nat Med (2013) 67:350–358
DOI 10.1007/s11418-012-0692-5
ORIGINAL PAPER
Hirtionosides A–C, gallates of megastigmane glucosides,
3-hydroxyoctanoic acid glucosides and a phenylpropanoid
glucoside from the whole plants of Euphorbia hirta
•
•
•
Yuya Nomoto Sachiko Sugimoto Katsuyoshi Matsunami
Hideaki Otsuka
Received: 5 June 2012 / Accepted: 8 July 2012 / Published online: 27 July 2012
Ó The Japanese Society of Pharmacognosy and Springer 2012
Abstract From the 1-BuOH-soluble fraction of a MeOH
extract of Euphorbia hirta, collected in the Okinawa
islands, three new megastigmane glucoside gallates, named
hirtionosides A–C, 3-hydroxyoctanoic acid glucosides and
a phenylpropanoid glucoside were isolated along with 15
known compounds. The structures of the new compounds
were elucidated by detailed analysis of physical data,
including one- and two-dimensional NMR spectra, and
those of known compounds were determined by compari-
son of physico-chemical data with those reported in the
literature. The absolute structures of the megastigmanes
were determined by comparison of NMR data and Cotton
effects in the CD spectra. The modified Mosher’s method
was applied to determine the absolute structure of the chiral
center in 3-hydroxyoctanoic acid. The DPPH radical-
scavenging properties of megastigmane glucoside gallates
were assayed and, as expected, they showed moderate
activity.
Introduction
In our previous paper on Euphorbia supina [1], the isola-
tion of two new megastigmane glucosides and six new
hydroxynitrile glucosides was reported. A closely related
plant, Euphorbia hirta, was also an immigrant from trop-
ical America during the end of the Edo period (around
1850), and currently grows wild in the southern part of
Honshu, and Kyushu and Okinawa. Phytochemical inves-
tigation of E. hirta resulted in the isolation of the gallates
of three new megastigmane glucosides, named hirtiono-
sides A–C (1–3), 3-hydroxyoctanoic acid glucoside (4) and
its methyl ester (5), and a phenylpropanoid glucoside (6),
along with 15 known compounds (7–21). The structures of
the known compounds were identified as (Z)-3-hexenyl
b-D-glucopyranoside (7) [2] and rutinoside (8) [3], benzyl
b-D-glucopyranoside (9) [4], rutinoside (10) [5] and b-D-
(60-O-a-L-arabinopyranosyl)glucopyranoside (11) [6], syrin-
gin (12) [7], linocinnamarin (13) [8], blumenol C glucoside
(14)[9], citrosideA(15)[10],corchoionosideC(16)[11],(R)-
lotaustralin (17) [1, 12], bumaldoside (18) [13], isovitexin
(19) [14], afzelin (20) [15], and methyl b-D-glucopyranosyl
tuberonate(21) [16] bycomparison oftheirspectroscopic data
with those reported in the literature.
Keywords Euphorbiua hirta ꢀ Euphorbiaceae ꢀ
Megastigmane glucoside ꢀ Hirtionoside ꢀ Modified
Mosher’s method ꢀ Radical scavenging activity
Results and discussion
Y. Nomoto ꢀ S. Sugimoto ꢀ K. Matsunami ꢀ H. Otsuka
Department of Pharmacognosy, Graduate School of Biomedical
and Health Sciences, Hiroshima University, 1-2-3 Kasumi,
Minami-ku, Hiroshima 734-8553, Japan
From the 1-BuOH-soluble fraction of a MeOH extract of
Euphorbia hirta, collected in Okinawa, six new compounds
were isolated along with 15 known ones by a combination
of various kinds of chromatography. The structures of the
new compounds were elucidated as follows.
H. Otsuka (&)
Department of Natural Products Chemistry, Faculty of
Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi,
Asaminami-ku, Hiroshima 731-0153, Japan
e-mail: hotsuka@hiroshima-u.ac.jp
Hirtionoside A (1), [a]D -11.3, was isolated as an
amorphous powder and its elemental composition was
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351
determined to be C26H34O12 by observation of a quasi-
molecular ion peak [M?Na]? at 561.1934 on high-resolution
(HR)-electrospray ionization (ESI)-mass spectrometry (MS).
The IR spectrum showed absorption bands at 3408, 1730 and
1651 cm-1, which were assignable to those for hydroxy and
carbonyl groups, and a double bond, respectively, and the UV
absorption band at 273 nm suggested the presence of a con-
corchoionoside C (1b = 16). The esterified position of gal-
late was assigned as the 60-position of the glucopyranoside
from the HMBC correlation from H2-60 to the carbonyl car-
bon. Therefore, the structure of hirtionoside A (1) was elu-
cidated to be corchoionoside C 60-O-gallate, namely 9-epi-
mallophenol B [18] (Fig. 1).
Hirtionoside B (2), [a]D ?17.7, and hirtionoside C (3),
[a]D ?25.4, were isolated as amorphous powders and their
elemental compositions were determined to be C26H36O11
and C26H34O11 by HR-ESI–MS, respectively. The NMR
spectroscopic data of 2 and 3 showed high similarity to
those of hirtioside A (1), except for replacement of the
hydroxy group at the 6-position by a hydrogen atom in 2
and 3, and the trans double bond between C-7 and C-8 was
reduced to a single bond in 3. Gallic acid was also esterified at
the hydroxy group at the 60-position. The absolute configu-
rations at the 6-position were confirmed by the CD spectrum
[9, 19], and those at the 9-position were determined by com-
parisonof13CNMRdata[9].Therefore,thestructuresof2 and
3 were assigned as (4Z,6R,7E,9S)-6,9-dihydroxymegastigma-
4,7-dien-3-one 9-O-b-D-glucopyranoside 60-O-gallate and
blumenol C 60-O-gallate, respectively.
1
jugated system. The H NMR spectrum exhibited signals
assignable to two singlet methyls, two doublet methyls [dH
1.27 (d, J = 6.4 Hz) and 1.90 (d, J = 1.4 Hz)], a trans
double bond [dH 5.71 (dd, J = 15.7, 6.9 Hz) and 5.81 (d,
J = 15.7 Hz)], oxymethine (dH 4.42), and an aromatic signal
fortwoprotons and an olefinic proton(dH 5.83), alongwith an
anomeric proton (dH 4.28) and sugar ring protons (Table 1).
The four aromatic signals [dC 110.2 (d) 9 2, 121. 5 (s), 139.8
(s) and 146.5 (s) 9 2] for six carbons and a carbonyl group
observed in the 13C NMR spectrum implied the presence of a
symmetrically tetrasubstituted aromatic ring, such as a gal-
loyl moiety [17] (Table 2). The 13C NMR spectrum also
exhibited six carbons for a hexopyranose, which was identi-
fied as D-glucose on HPLC analysis of the hydrolyzate of 1
using a chiral detector. The remaining 13 signals were
essentially superimposable with those reported for corcho-
ionosideC (16), whichwasco-isolatedfromthetitleplantand
originally isolated from Corchorus olitorius [11]. Mild
alkaline hydrolysis of 1 gave methyl gallate (1a) and
3-Hydroxyheptanoic acid O-b-D-glucopyranoside (4),
[a]D -22.4, was isolated as an amorphous powder and its
elemental composition was determined to be C14H26O8 by
HR-ESI–MS. The IR spectrum showed strong absorption
Table 1 1H NMR spectroscopic data for hirtionosides A–C (1–3) (CD3OD, 400 MHz). Number of protons, multiplicities and coupling constants
in Hz are in parentheses
H
2
1
2
3
2.16 (1H, d, 17.0)
2.47 (1H, d, 17.0)
5.83 (1H, br s)
–
2.02 (1H, d, 16.5)
2.34 (1H, d, 16.5)
5.85 (1H, br s)
1.96 (1H, d, 17.3)
2.34 (1H, d, 17.3)
5.78 (1H, br s)
4
6
7
2.65 (1H, br d, 9.0)
5.53 (1H, dd, 15.4, 9.0)
1.97 (1H, m)
5.81 (1H, d, 15.7)
1.54 (1H, m)
1.90 (1H, m)
8
5.71 (1H, dd, 15.7, 6.9)
5.52 (1H, dd, 15.4, 7.0)
1.59 (1H, m)
1.68 (1H, m)
9
4.42 (1H, dq, 6.9, 6.4)
1.27 (3H, d, 6.4)
4.36 (1H, m)
3.79 (1H, m)
10
11
12
13
10
20
30
40
50
60
1.26 (3H, d, 6.4)
1.19 (3H, d, 6.4)
0.99 (3H, s)
1.00 (3H, s)
0.98 (3H, s)
0.95 (3H, s)
0.89 (3H, s)
1.06 (3H, s)
1.90 (3H, d, 1.4)
1.92 (3H, d, 1.2)
2.00 (3H, d, 1.2)
4.35 (1H, d, 7.8)
3.20 (1H, dd, 9.1, 7.8)
3.35 (1H, dd, 9.1, 9.1)
3.38 (1H, dd, 9.1, 9.1)
3.54 (1H, ddd, 9.1, 6.5, 2.1)
4.40 (1H, dd, 11.7, 6.5)
4.53 (1H, dd, 11.7, 2.1)
7.08 (2H, s)
4.28 (1H, d, 7.6)
4.29 (1H, d, 7.3)
3.24 (1H, dd, 8.6, 7.6)
3.32 (1H, dd, 8.6, 8.6)
3.38 (1H, dd, 8.6, 8.6)
3.40 (1H, ddd, 8.6, 5.8, 2.0)
4.36 (1H, dd, 118, 5.8)
4.56 (1H, dd, 11.8, 2.0)
7.02 (2H, s)
3.24 (1H, dd, 8.0, 7.3)
3.37 (1H, dd, 8.0, 8.0)
3.34 (1H, dd, 8.0, 8.0)
3.48 (1H, ddd, 8.0, 6.7, 2.1)
4.34 (1H, dd, 11.9, 6.7)
4.59 (1H, dd, 11.9, 2.1)
7.07 (2H, s)
200, 600
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Table 2 13C NMR spectroscopic data for hirtionosides A–C (1–3)
(CD3OD, 100 MHz)
C
1
2
3
1
42.3
50.6
37.1
48.6
37.3
48.1
2
3
201.3
127.1
166.6
79.8
202.0
126.2
165.2
56.7
202.4
125.4
169.9
52.6
4
5
6
7
133.8
133.6
74.8
131.4
136.9
74.8
26.8
8
37.7
9
78.1
10
11
12
13
10
22.2
22.1
21.9
23.4
27.0
29.0
24.6
28.1
27.4
19.5
23.8
24.9
101.3
74.9
101.0
74.9
104.0
75.3
20
30
78.2
78.3
78.2
40
71.9
72.1
72.0
50
75.6
75.7
75.4
60
64.8
64.9
65.0
100
200,600
300,500
400
700
121.5
110.2
146.5
139.8
168.2
121.5
110.2
146.5
140.2
168.2
121.6
110.3
146.5
140.0
168.3
bands at 3405 and 1716 cm-1 for hydroxyl and carbonyl
groups, respectively. One triplet methyl, fairly deshielded
methylene protons (dH 2.47 and 2.69), eight aliphatic
protons, a methine proton (dH 4.07), and an anomeric
proton (dH 4.33), along with sugar ring protons were
Fig. 1 Structures of the isolated compounds
that methylation of 4, followed by enzymatic hydrolysis
should have given methyl 3-hydroxyoctanoate, which
indicates it could be a volatile compound. To reduce the
volatility of the aglycone, 4 was first esterified with
p-bromophenacyl bromide to give 4a and then the sugar
moiety was removed with b-glucosidase to give 4b. The b-D-
glucopyranosylation-induce shift-trend rule [21] was applied
to 4a and 4b, and the absolute configuration was judged to be
R (Dd4a–4b at the 2-position was -1.4 ppm and that at the
4-position -2.1 ppm). The results of the modified Mosher’s
method in Fig. 2 confirmed that the absolute configuration at
the 3-position was also R, as expected from the b-D-gluco-
pyranosylation-induce shift-trend rule.
1
observed in the H NMR spectrum. The 13C NMR spec-
trum exhibited six signals assignable to b-glucopyranose,
whose absolute configuration was also assigned as D, and
the remaining eight signals comprised those of one methyl
(dC 14.3), five methylenes, one oxymethine (dC 78.2), and a
carbonyl carbon (dC 175.7) (Table 3). The connectivity of
all protons was established by their COSY and HSQC
spectra, and in the HMBC spectrum, the anomeric proton
crossed with the oxygenated methine carbon, and the
methine proton with C-1, C-2, C-4 and C-5. Thus, the
planar structure of 4 was established to be 3-hydroxyocta-
noic acid b-D-glucopyranoside. The absolute configuration
at the 3-position was confirmed by the b-D-glucopyr-
anosylation-induce shift-trend rule and then the modified
Mosher’s method [20]. To avoid the formation of lactone,
the carboxyl group must first be protected by some
substituent, i.e., esterification, and the second problem is
Methyl 3-hydroxyoctanoate O-b-D-glucopyranoside (5),
[a]D -37.6, was isolated as an amorphous powder and its
elemental composition was determined to be C15H28O8 by
HR-ESI–MS. Its NMR spectroscopic data were similar to
those of 4, except for the presence of methoxy signals
(dH 3.66 and dC 52.1). Methylation of 4 gave a methyl ester
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353
1
Table 3 NMR spectroscopic data for compounds 4 and 5 (CD3OD, 13C: 100 MHz and H: 400 MHz)
4
5
C
H
C
H
1
2
175.7
42.1
174.2
41.9
2.69 (1H, dd, 15.3, 6.7)
2.47 (1H, dd, 15.3, 5.4)
4.07 (1H, tt, 6.7, 5.4)
1.66 (1H, m)
2.71 (1H, dd, 15.3, 6.7)
2.50 (1H, dd, 15.3, 5.5)
4.08 (1H, tt, 6.7, 5.5)
1.66 (1H, m)
3
4
78.2
35.9
77.9
35.8
1.58 (1H, m)
1.58 (1H, m)
5
25.8
32.9
23.5
14.3
104.3
75.2
78.0
71.6
77.7
62.8
1.41 (2H, m)
25.7
32.9
23.5
14.3
104.2
75.2
78.0
71.7
77.7
62.9
1.41 (2H, m)
6
1.54 (2H, m)
1.54 (2H, m)
7
1.33 (2H, m)
1.33 (2H, m)
8
0.91 (3H, t, 6.9)
0.90 (3H, t, 7.0)
10
20
30
40
50
60
4.33 (1H, d, 7.6)
4.31 (1H, d, 7.6)
3.12 (1H, dd, 8.7, 7.6)
3.36 (1H, dd, 8.7, 8.7)
3.30 (1H, dd, 8.7, 8.7)
3.15 (1H, dd, 8.7, 7.6)
3.36 (1H, dd, 8.7, 8.7)
3.30 (1H, dd, 8.7, 8.7)
3.24 (1H, ddd, 8.7, 5.4, 2.3)
3.85 (1H, dd, 11.8, 2.3)
3.65 (1H, dd, 11.8, 5.4)
3.25 (1H, ddd, 8.7, 5.5, 2.2)
3.81 (1H, dd, 11.9, 2.2)
3.63 (1H, dd, 11.9, 5.5)
3.66 (2H, s)
–OCH3
52.1
Numbers of protons, multiplicities and coupling constants in Hz are given in parentheses
Table 4 NMR spectroscopic data for compound 6 (CD3OD, 13C:
1
100 MHz and H: 400 MHz)
C
H
1
139.0
107.5
154.2
134.9
32.1
–
2,6
3,5
4
6.56 (2H, s)
Fig. 2 Results with the modified Mosher’s method (Dd4d–4c
)
–
–
7
2.87 (2H, t, 7.5)
2.64 (2H, t, 7.5)
–
(4e), which was spectroscopically identical with 5. Methyl
ester (5) might be an artifact arising during the extraction
and purification processes.
8
36.6
9
175.1
105.6
75.8
Methyl 3,5-dimethoxy-4-hydroxyphenypropanoate O-b-
D-glucopyranoside (6), [a]D ?10.0, was isolated as an
amorphous powder and its elemental composition was
determined to be C18H26O10 by HR-ESI–MS. The IR
spectrum showed absorption bands assignable to hydroxy
groups (3397 cm-1) and an carbonyl functional group
(1733 cm-1), and the UV spectrum indicated the presence
of an aromatic ring (225 and 332 nm). The 1H NMR
spectrum exhibited one singlet aromatic signal for two
protons, four aliphatic protons, two methoxy signals for
three and six protons, and an anomeric proton, along with
six sugar ring protons. Of the 17 13C NMR peaks observed,
six were attributable to a terminal glucopyranose, and the
remaining 11 peaks comprised four aromatic signals
accounting for six carbon atoms, those of two methylenes,
and one carbonyl carbon, and two methoxy signals (dC 52.1
10
20
30
40
50
60
4.80 (1H, d, 7.5)
3.46 (1H, dd, 7.8, 7.5)
3.42 (1H, dd, 7.8, 7.8)
3.43 (1H, dd, 7.8, 7.8)
3.23 (1H, ddd, 7.8, 5.1, 2.5)
3.78 (1H, dd, 11.8, 2.5)
3.67 (1H, dd, 11.8, 5.1)
3.89 (6H, s)
78.4
71.4
77.9
62.7
–OCH3
–OCH3
57.1
52.1
3.67 (3H, s)
Numbers of protons, multiplicities and coupling constants in Hz are
given in parentheses
and 57.1 9 2) (Table 4). From the above evidence, the
presence of a symmetrically tetrasubstituted aromatic ring
was proposed and the HMBC correlations shown in Fig. 3
established a methyl phenylpropanoate skeleton. Therefore,
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stationary and mobile phases, respectively. Fractions (5 g)
were collected and numbered according to their order of
elution with the mobile phase. HPLC was performed on an
ODS column (Inertsil; GL Science, Tokyo, Japan;
U = 6 mm, L = 25 cm, 1.6 mL/min), and the eluate
was monitored with UV (210 nm) and refractive index
monitors. The (R)-(?)- and (S)-(-)-a-methoxy-a-tri-
fluoromethylphenylacetic acids (MTPA) were purchased
from Nacalai Tesque (Kyoto, Japan).
Fig. 3 Diagnostic HMBC correlations for 6. Arrow tails H, arrow-
heads C. Dual arrow curves indicate HMBC correlations observed in
both directions
Plant material
Whole plants of E. hirta were collected in Kunigami Vil-
lage, Kunigami County, Okinawa in July 2001. A voucher
specimen was deposited in the Herbarium of the Faculty of
Pharmaceutical Sciences, Graduate School of Biomedical
Sciences, Hiroshima University. The plant material was
identified by Emeritus Professor Takakazu Shinzato of the
Faculty of Agriculture, University of Ryukyus.
the structure of compound 6 was elucidated to be as shown
in Fig. 1. Although synapoyl 4-O-b-D-glucopyranoside is
found in some plant sources [22, 23], its reduced form, like
6, is so far not found in nature. However, the methyl ester
might be an artifact, arising during the extraction and
purification processes.
As expected from the gallate-containing structures of
hirtionosides A–C (1–3), an antioxidant nature involving
DPPH radical-scavenging activity was observed. The IC50
values of these compounds were 29.4 2.3, 32.7 3.3
and 20.8 0.1 lM, respectively, and that of the positive
control Trolox was 17.5 0.5 lM. Thus, 1–3 showed
nearly comparable activity to that of the positive control.
Extraction and isolation
Whole plants of E. hirta (5.20 kg) were extracted three
times with MeOH (45 L 9 3) at room temperature for
1 week and then concentrated to 3 L in vacuo. The con-
centrated extract was washed with n-hexane (3 L, 93.0 g),
and the MeOH layer was then concentrated to a gummy
mass. The latter was suspended in water (3 L) and then
extracted with EtOAc (3 L) to give 126 g of an EtOAc-
soluble fraction. The aqueous layer was extracted with
1-BuOH (3 L) to give a 1-BuOH-soluble fraction (97.1 g),
and the remaining water layer was concentrated to furnish
302 g of a water-soluble fraction. The 1-BuOH-soluble
fraction (97.0 g) was subjected to Diaion HP-20 CC
(U = 50 mm, L = 45 cm), using H2O–MeOH (4:1, 3 L),
(3:2, 3 L), (2:3, 3 L), and (1:4, 3 L), and MeOH (3 L), 1 L
fractions being collected. The residue (33.5 g) in fractions
3 and 4 was subjected to silica gel (U = 20 mm,
L = 50 cm) CC with increasing amounts of MeOH in
CHCl3 [CHCl3 (6 L), and CHCl3–MeOH (49:1, 3 L),
(24:1, 3 L), (23:2, 3 L), (9:1, 3 L), (17:3, 3 L), (4:1, 3 L),
(3:1, 3 L), and (7:3, 3 L)], 500 mL fractions being col-
lected. The residue (3.68 g out of 7.70 g) in fractions
44–57 was separated by ODC open CC and then the resi-
due (243 mg) in fractions 86–112 was further separated by
DCCC. The residue (25.4 mg) in fractions 38–40 was
finally purified by HPLC (H2O–MeOH, 3:1) to give 3.1 mg
of 11 and 8.8 mg of 10 from the peaks at 15.6 and
25.2 min, respectively.
Experimental
General experimental procedure
Optical rotations were measured on a JASCO P-1030
digital polarimeter. IR and UV spectra were measured on
Horiba FT-710 and JASCO V-520 UV/Vis spectropho-
tometers, respectively. 1H- and 13C-NMR spectra were
taken on a JEOL JNM a-400 at 400 MHz and 100 MHz
with tetramethylsilane as an internal standard. CD spectra
were obtained with a JASCO J-720 spectropolarimeter.
Positive-ion HR-ESI–MS was performed with an Applied
Biosystems QSTAR XL NanoSprayTM System.
A highly porous synthetic resin (Diaion HP-20) was
purchased from Mitsubishi Kagaku (Tokyo, Japan). Silica
gel CC was performed on silica gel 60 (Merck, Darmstadt,
Germany), and ODS open CC on Cosmosil 75C18-OPN
(Nacalai Tesque, Kyoto, Japan) [U = 50 mm, L = 25 cm,
linear gradient: MeOH–H2O (1:9,
1 L) ? (1:1, 1
L) ? (9:1, 1 L), fractions of 10 g being collected]. The
DCCC (Tokyo Rikakikai, Tokyo, Japan) was equipped
with 500 glass columns (U = 2 mm, L = 40 cm), the
lower and upper layers of a solvent mixture of CHCl3–
MeOH–H2O–n-PrOH (9:12:8:2) being used as the
The residue (10.8 g) in fractions 5–9 obtained on Diaion
HP-20 CC was subjected to silica gel (U = 15 mm,
L = 55 cm) CC with increasing amounts of MeOH in
CHCl3 [CHCl3 (3 L), and CHCl3–MeOH (49:1, 1.5 L),
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355
(24:1, 1.5 L), (23:2, 1.5 L), (9:1, 1.5 L), (17:3, 1.5 L),
(4:1, 1.5 L), (3:1, 1.5 L), and (7:3, 1.5 L)], 250 mL
fractions being collected. The residue (860 mg) in frac-
tions 27–35 was separated by ODS open CC and then the
residue (75.0 mg) in fractions 42–72 was purified by
DCCC to give 14.6 mg of 17 in fractions 49–54. The
ODS residue (101 mg) in fractions 81–114 was purified
by DCCC to give 23.9 mg of 9 in fractions 86–99. The
residue (174 mg) in fractions 149–164 was purified by
DCCC to give 16.1 mg of 7 and 4.0 mg of 5 in fractions
87–117 and 124–147, respectively. The ODS residue
(73.0 mg) in fractions 165–204 was separated by DCCC
and then the residue (22.3 mg) in fractions 166–178 was
purified by HPLC (H2O–MeOH, 3:2) to give 6.30 mg of
21 and 5.20 mg of 14 from the peaks at 9.0 and 21.6 min,
respectively. The residue (850 mg) in fractions 36–39
obtained on silica gel CC was separated by ODS open
CC and then the residue (406 mg) in fractions 45–64 was
further separated by DCCC. The residue (85.5 mg) in
fractions 77–88 was finally purified by HPLC (H2O–
MeOH, 4:1) to give 8.0 mg of 12 and 17.5 mg of 16 from
the peaks at 16.8 and 24.0 min, respectively. The residue
(2.49 g) in fractions 40–48 obtained on silica gel CC was
separated by ODS open CC and then the residue
(676 mg) in fractions 33–54 was separated by DCCC.
The residue (122 mg) in fractions 30–39 was purified by
HPLC (H2O–MeOH, 3:1) to give 6.5 mg of 13 from the
peak at 39.6 min. The residue (431 mg) in fractions
55–70 was separated by DCCC and the residue (60.4 mg)
in fractions 87–101 was purified by HPLC (H2O–MeOH,
3:1) to give 17.9 mg of 15 from the peak at 15.6 min.
The residue (318 mg) in fractions 91–110 was separated
by DCCC, and the residue (109 mg) in fractions 49–63
was purified by HPLC (H2O–MeOH, 13:7) to give
26.2 mg of 1 and 38.2 mg of 8 from the peaks at 13.8 and
21.6 min, respectively. The residue (79.9 mg) in frac-
tions 67–83 obtained on DCCC was purified by HPLC
(H2O–MeOH 13:7) to give 38.4 mg of 6 from the peak at
26.4 min.
2, 3.40 mg of 20, and 11.3 mg of 18 from the peaks at 12.0,
31.2, and 36.0 min, respectively. The residue (10.1 mg)
from the peak at 19.2 min was repeatedly purified by
HPLC (H2O–MeOH, 3:2) to give 2.9 mg of 3 from the
peak at 50.4 min. The residue (1.40 g) in fractions 44–50
obtained on silica gel CC was separated by ODS open CC
and then the residue (99.4 mg) in fractions 27–58 was
separated by DCCC to give 14.4 mg of 4 in fractions
20–25.
Hirtionoside A (1)
25
White amorphous powder, ½aꢁD ?11.3 (c 1.74, MeOH); IR
mmax (film) cm-1: 3408, 2959, 2926, 2856, 1730, 1651,
1455, 1376, 1274, 1070, 1047; UV kmax (MeOH) nm (log
1
e): 273 (3.83), 237 (3.96), 218 (4.18); H NMR (CD3OD,
400 MHz): Table 1; 13C NMR (CD3OD, 100 MHz):
Table 2; CD (MeOH) De (nm): ?9.85 (242), -0.57 (314)
(c 3.24 9 10-5 M); HR-ESI–MS (positive-ion mode) m/z:
561.1934 [M?Na]? (calcd for C26H34O12Na: 561.1942).
Hirtionoside B (2)
28
White amorphous powder, ½aꢁD ?17.7 (c 0.12, MeOH); IR
mmax (film) cm-1: 3377, 2962, 2929, 2874, 1635, 1605,
1375, 1224, 1078, 1038, 768; UV kmax (MeOH) nm (log e):
275 (3.84), 240 (4.01), 221 (4.20); 1H NMR (CD3OD,
400 MHz): Table 1; 13C NMR (CD3OD, 100 MHz):
Table 2; CD (MeOH) De (nm): ?7.37 (244), -0.61 (292)
(c 3.38 9 10-5 M); HR-ESI–MS (positive-ion mode) m/z:
547.2148 [M?Na]? (calcd for C26H36O11Na: 547.2150).
Hirtionoside C (3)
25
White amorphous powder, ½aꢁD ?25.4 (c 0.35, MeOH); IR
mmax (film) cm-1: 3371, 2926, 1727, 1377, 1272, 1071,
1045; UV kmax (MeOH) nm (log e): 275 (3.78), 240 (3.97),
1
219 (4.17); H NMR (CD3OD, 400 MHz): Table 1; 13C
NMR (CD3OD, 100 MHz): Table 2; CD (MeOH) De (nm):
?9.05 (242), -0.75 (314) (c 2.37 9 10-5 M); HR-ESI–
MS (positive-ion mode) m/z: 545.1986 [M?Na]? (calcd
for C26H34O11Na: 545.1993).
The residue (14.2 g) in fractions 14–19, obtained on
Diaion HP-20 CC was subjected to silica gel (U = 15 mm,
L = 54 cm) CC with increasing amounts of MeOH in
CHCl3 [CHCl3 (6 L), and CHCl3–MeOH (49:1, 3 L),
(24:1, 3 L), (23:2, 3 L), (9:1, 3 L), (17:3, 3 L), (4:1, 3 L),
(3:1, 3 L), and (7:3, 3 L)], 500 mL fractions being col-
lected. The residue (2.40 g) in fractions 31–43 was sepa-
rated by ODS open CC and the precipitate (9.4 mg) in
fractions 153–182 was purified by HPLC (H2O–MeOH,
3:2) to give 3.10 mg of 19 from the peak at 14.4 min. The
residue (793 mg) in fractions 183–221 was separated by
DCCC and the residue (134 mg) in fractions 66–83 was
purified by HPLC (H2O–MeOH, 11:9) to give 5.30 mg of
3-Hydroxyoctanoic acid O-b-D-glucopyranoside (4)
24
Yellow oily material, ½aꢁD -22.4 (c 2.5, MeOH); IR mmax
(film) cm-1: 3405, 2930, 2865, 1716, 1402, 1375, 1164,
1
1077, 1025; H NMR (CD3OD, 400 MHz): Table 3; 13C
NMR (CD3OD, 100 MHz): Table 3; HR-ESI–MS (posi-
tive-ion mode) m/z: 345.1516 [M?Na]? (calcd for
C14H26O8Na: 345.1520).
123

356
J Nat Med (2013) 67:350–358
Methyl 3-hydroxyoctanoate O-b-D-glucopyranoside (5)
Preparation of p-bromophenacyl ester (4a) of 4
26
Compound 4 (11.7 mg) was dissolved in 1 mL EtOH and
then the solution was neutralized with a 0.1 M NaOH
solution and phenolphthalein as a pH indicator. The neutral
solution was made slightly acidic by the addition of a small
amount of 4 (1.2 mg), and then p-bromophenacyl bromide
(15.0 mg) in 1 mL of an EtOH solution was added. After
refluxing for 12 h, the solution was concentrated, and the
resulting residue was subjected to silica gel CC
(U = 20 mm, L = 20 cm) with CHCl3 (100 mL) and
[CHCl3–MeOH (19:1, 100 mL), (9:1, 100 mL) and (17:3,
100 mL)], 10 mL fractions being collected. From fractions
24–29, 11.0 mg of p-bromophenacyl ester (4a) was
Yellow oily material, ½aꢁD -37.6 (c 0.30, MeOH); IR mmax
(film) cm-1: 3395, 2930, 2868, 1729, 1439, 1369, 1268,
1164, 1077, 1037; 1H NMR (CD3OD, 400 MHz): Table 3;
13C NMR (CD3OD, 100 MHz): Table 3; HR-ESI–MS
(positive-ion mode) m/z: 359.1675 [M?Na]? (calcd for
C15H28O8Na: 359.1676).
Methyl 3,5-dimethoxy-4-hydroxyphenypropanoate
O-b-D-glucopyranoside (6)
28
White amorphous powder, ½aꢁD ?10.0 (c 0.21, MeOH); IR
mmax (film) cm-1: 3397, 2922, 2851, 1733, 1681, 1456,
1420, 1121, 1032; UV kmax (MeOH) nm (log e): 332 (3.44),
225 (3.75), 210 (3.95); 1H NMR (CD3OD, 400 MHz):
Table 4; 13C NMR (CD3OD, 100 MHz): Table 4; HR-
ESI–MS (positive-ion mode) m/z: 425.1425 [M?Na]?
(calcd for C18H26O10Na: 425.1418).
24
obtained. Compound 4a: Amorphous powder, ½aꢁD -19.9
(c 0.73, MeOH); IR mmax (film) cm-1: 3395, 2928, 2857,
1740, 1704, 1587, 1398, 1167, 1073, 972, 817. UV kmax
(MeOH) nm (log e): 255 (4.29), 210 (4.11); 1H NMR
(CD3OD, 400 MHz) d: 7.87 (2H, d, J = 8.6 Hz, C-200 and
600), 7.69 (2H, d, J = 8.6 Hz, C-300 and 500), 5.42 (1H d,
J = 16.7 Hz, H-800a), 5.36 (1H, d, J = 16.8 Hz, H-800b),
H-4.36 (1H, d, J = 7.3 Hz, H-10), 4.14 (1H, tt, J = 6.0,
5.8 Hz, H-3), 3.83 (1H, dd, J = 11.8, 2.3 Hz, H-60a), 3.66
(1H, dd, J = 11.8, 5.4 Hz, H-60b), 3.35 (1H, t, J = 8.7 Hz,
H-30), 3.27 (1H, t, J = 8.7 Hz, H-40), 3.24 (1H, ddd,
J = 8.7, 5.4, 2.3 Hz, H-50), 3.15 (1H, dd, J = 8.7, 7.6 Hz,
H-20), 2.91 (1H, dd, J = 15.5, 5.8 Hz, H-2a), 2.67 (1H, dd,
J = 15.5, 6.0 Hz, H-2b), 1.64 (2H, m, H2-4), 1.34 (2H, m,
H2-5), 1.33 (2H, m, H2-6), 1.32 (2H, m, H2-7), 0.91 (3H, t,
J = 7.1 Hz, H3-8); 13C NMR (CD3OD, 100 MHz) d: 193.8
(C-200), 172.8 (C-1), 134.5 (C-300), 133.3 (C-300 and 500),
130.6 (C-200 and 600), 129.8 (C-60), 104.4 (C-10), 78.07
(C-3), 78.05 (C-30), 77.7 (C-50), 75.2 (C-20), 71.6 (C-40),
67.4 (C-100), 62.8 (C-10), 41.7 (C-2), 35.8 (C-4), 32.9 (C-6),
25.7 (C-5), 23.6 (C-7), 14.3 (C-8); HR-ESI–MS (positive-
ion mode) m/z: 541.1048 [M?Na]? (calcd for
C22H31O9Na79Br: 541.1054) and 543.1003 [M?Na]?
(calcd for C22H31O9Na81Br: 543.1009).
Sugar analysis
About 500 lg of each compound was hydrolyzed with 1 M
HCl (0.1 mL) at 90 °C for 2 h. The reaction mixtures were
partitioned with an equal amount of EtOAc (0.1 mL), and
the water layers were analyzed with a chiral detector
(JASCO OR-2090 plus) on an amino column [Asahipak
NH2P-50 4E, U = 4.6 mm, L = 25 cm, CH3CN–H2O
(3:1), 1 mL/min]. The hydrolyzates of 1–6 each gave a
peak for D-glucose at 13.9 min with a positive optical
rotation sign. The peaks were identified by co-chroma-
tography with authentic D-glucose.
Mild alkaline hydrolysis of hirtionoside A (1)
Hirtionoside A (4.1 mg) in 1 mL of 0.1 M NaOMe in
MeOH was stirred for 1 h under a N2 atmosphere. The
reaction mixture was neutralized with Amberlite [IR-120B
(H?)] and then concentrated to leave a residue, which was
purified by silica gel CC (U = 10 mm, L = 16 cm) with
CHCl3–MeOH (10:1, 100 mL) and CHCl3–MeOH–H2O
(15:6:1, 200 mL), 8 mL fractions being collected to give
1.0 mg methyl gallate (1a) and 1.5 mg cochoionoside C
(1b) in fractions 24–28 and 34–39, respectively. Methyl
gallate (1a): HR-ESI–MS (positive-ion mode) m/z:
207.0265 [M?Na]? (calcd for C8H8O5Na: 207.0264).
Enzymatic hydrolysis of 4a to 4b
A solution of 4a in 50 lL DMSO was poured into 2 mL
H2O, and then 19.8 mg b-glucosidase was added. The
reaction mixture was stood at 37 °C for 16 h and then
concentrated. The residue was purified by silica gel CC
(U = 20 mm, L = 15 cm) with CHCl3 (100 mL) and
CHCl3–MeOH [(19:1, 100 mL), (9:1, 100 mL), (17:3,
100 mL) and (4:1, 100 mL)], 10 mL fractions being col-
lected. From fractions 29–35 and 42–46, 4.4 mg 4b and
2.0 mg D-glucose were obtained, respectively. Compound
24
Corchoionoside C (1b = 16): amorphous powder, ½aꢁD
?36.0 (c 0.10, MeOH); UV kmax (MeOH) nm (log e): 274
(3.26), 231 (3.76); NMR: identical with those of 16; CD
(MeOH) De (nm): -0.44 (326), ?7.52 (241) (c
2.58 9 10-5 M); HR-ESI–MS (positive-ion mode) m/z:
409.1835 [M?Na]? (calcd for C19H30O8Na: 409.1833).
24
4b: Amorphous powder, ½aꢁD -16.3 (c 0.29, MeOH); IR
mmax (film) cm-1: 3436, 2922, 2849, 1697, 1647, 1171,
123

J Nat Med (2013) 67:350–358
357
1014, 817. UV kmax (MeOH) nm (log e): 252 (4.27), 210
(4.04). 1H NMR (CD3OD, 400 MHz) d: 7.88 (2H, d,
J = 8.4 Hz, H-200 and 600), 7.70 (2H, d, J = 8.4 Hz, H-300
and 500), 5.43 (1H, d, J = 16.8 Hz, H-800a), 5.37 (1H, d,
J = 16.8 Hz, H-800b), 4.03 (1H, tt, J = 7.6, 5.1 Hz, C-3),
2.65 (1H, dd, J = 15.0, 5.1 Hz, H-2a), 2.65 (1H, dd,
J = 15.0, 7.6 Hz, H-2b), 1.52 (2H, m, H2-4), 1.34 (2H, m,
H2-5), 1.33 (2H, m, H2-6), 1.32 (2H, m, H-7), 0.90 (3H, t,
J = 7.1 Hz, H-8); 13C NMR (CD3OD, 100 MHz) d: 193.9
(C-700), 172.7 (C-1), 134.5 (C-100), 133.3 (C-300 and 500),
130.6 (C-200 and 600), 129.8 (C-400), 69.3 (C-3), 67.3 (C-700),
43.1 (C-2), 37.9 (C-4), 32.8 (C-6), 26.2 (C-5), 23.6 (C-7),
14.3 (C-8); HR-ESI–MS (positive-ion mode) m/z: 379.0526
[M?Na]? (calcd for C16H21O4Na79Br: 379.0515) and
381.0488 [M?Na]? (calcd for C16H21O4Na81Br: 381.0494).
J = 9.0 Hz, H-200 and 600), 7.63 (2H, d, J = 9.0 Hz, H-300
and 500), 7.53–7.56 (2H, m, MTPA aromatic protons), 7.37–
7.39 (3H, m, MTPA aromatic protons), 5.52 (1H, tt,
J = 7.8, 5.4 Hz, H-3), 5.19 (1H, d, J = 16.4 Hz, H-800a),
5.13 (1H, d, J = 16.4 Hz, H-800b), 3.55 (3H, q, J = 1 Hz,
–OCH3), 2.83 (1H, dd, J = 16.2, 7.8 Hz, H-2a), 2.76 (1H,
dd, J = 16.2, 5.4 Hz, H-2b), 1.74–1.79 (2H, m, H-4), 1.34
(2H, m, H-6), 1.32 (2H, m, H-5), 1.30 (2H, m, H-7), 0.89
(3H, t, J = 6.7 Hz, H-8); HR-ESI–MS (positive-ion mode)
m/z: 595.0914 [M?Na]? (calcd for C26H28O6F3Na79Br:
595.0913) and 597.0882 [M?Na]? (calcd for C26H28O6F3-
Na81Br: 597.0893).
Methylation of 4
23
D-glucose: ½aꢁD ?34.1 (c 0.25, H2O, 24 h after being dis-
Five milligrams of 4 was dissolved in 1 mL MeOH and
methylated with an excess amount of trimethylsilyl
solved in H2O).
diazomethane. On removal of the solvent, 5.1 mg 4e (= 5)
26
was obtained. Compound 4e: Amorphous powder, ½aꢁD
-
Preparation of (R)-ester (4c) and (S)-MTPA ester (4d)
from 4b
36.9 (c 0.34, MeOH); NMR: identical with those of 5; HR-
ESI–MS (positive-ion mode) m/z: 359.1675 [M?Na]?
(calcd for C15H28O8Na: 359.1676).
A solution of 4b (1.0 mg) in 1 mL dehydrated CH2Cl2 was
reacted with (R)-MTPA (10.2 mg) in the presence
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (EDC) (9.5 mg) and N,N-dimethyl-4-aminopyri-
dine (4-DMAP) (5.1 mg), and the mixture was then stirred
occasionally at 40 °C for 18 h. After the addition of 1 mL
CH2Cl2, the solution was washed with H2O (1 mL), 5 %
HCl (1 mL), NaHCO3-saturated H2O, and brine (1 mL),
successively. The organic layer was dried over Na2SO4 and
then evaporated under reduced pressure. The residue was
purified by preparative TLC [silica gel (0.25 mm thick-
ness); the residue being applied for 18 cm, developed with
CHCl3-(CH3)2CO (20:1) for 9 cm, and then eluted with
CHCl3–MeOH (5:1)] to furnish an ester, 4b (0.5 mg).
Through a similar procedure, 4c (0.5 mg) was prepared
from 4a (1.0 mg) using (S)-MTPA (17.4 mg), EDC
(9.4 mg), and 4-DMAP (6.6 mg). Compound 4c: Amor-
phous powder, 1H NMR (CDCl3, 400 MHz) d: 7.75 (2H, d,
J = 9.0 Hz, H-200 and 600), 7.64 (2H, d, J = 9.0 Hz, H-300
and 500), 7.53–7.56 (2H, m, MTPA aromatic protons), 7.37–
7.39 (3H, m, MTPA aromatic protons), 5.53 (1H, tt,
J = 7.8, 5.4 Hz, H-3), 5.26 (1H, d, J = 16.4 Hz, H-800b),
5.31 (1H, d, J = 16.4 Hz, H-800a), 3.55 (3H, q, J = 1 Hz,
–OCH3), 2.89 (1H, dd, J = 16.2, 7.8 Hz, H-2a), 2.79 (1H,
dd, J = 16.2, 5.4 Hz, H-2b), 1.67–1.73 (2H, m, H2-4), 1.31
(2H, m, H2-6), 1.28 (2H, m, H2-5), 1.27 (2H, m, H2-7),
0.86 (3H, t, J = 6.7 Hz, H-8); HR-ESI–MS (positive-ion
mode) m/z: 595.0935 [M?Na]? (calcd for C26H28O6F3-
Na79Br: 595.0913) and 597.0876 [M?Na]? (calcd for
C26H28O6F3Na81Br: 597.0893). Compound 4d: Amor-
phous powder, 1H NMR (CDCl3, 400 MHz) d: 7.73 (2H, d,
Antioxidant activity with a DPPH radical-scavenging
system
The antioxidant activity was evaluated using a DPPH
radical-scavenging system. In a 96-well plate, 2 lL-ali-
quots of the DMSO solution of the compounds were diluted
with 98 lL MeOH in triplicate. A 100 lL-aliquot of a
methanolic solution of DPPH was added to each well to
give a final concentration of 100 lM. The compounds were
tested at final concentrations of 50, 30, 10 and 5 lM. Each
mixture was incubated in the dark for 30 min at room
temperature, followed by measurement of the absorbance
at 515 nm using a Molecular Devices Versamax tunable
microplate reader. DMSO was used as a negative control
and Trolox as a positive control. Radical-scavenging
activity was expressed as the inhibition percentage and was
calculated using the following formula:
% Inhibition ¼ ½ðAcontrol ꢂ AtestÞ = Acontrolꢁ ꢃ 100
where Acontrol is the absorbance of the control (DMSO) and
Atest the absorbance of a test compounds.
Acknowledgments The authors are grateful for access to the
superconducting NMR instrument (JEOL JNM a-400) at the Ana-
lytical Center of Molecular Medicine of the Hiroshima University
Faculty of Medicine, and an Applied Biosystem QSTAR XL system
ESI (Nano Spray)-MS at the Analysis Center of Life Science of the
Graduate School of Biomedical Sciences, 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
123

358
J Nat Med (2013) 67:350–358
the Research Foundation for Pharmaceutical Sciences and the Takeda
Science Foundation for financial support.
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