Antioxidant lignans and chromone glycosides from Eurya japonica

Article abstract of DOI:10.1021/np3007638

Four new 8,8′,7,2′-lignans, (+)-ovafolinin B-9′-O-β- d-glucopyranoside (1), (-)-ovafolinin B-9′-O-β-d-glucopyranoside (2), (+)-ovafolinin E-9′-O-β-d-glucopyranoside (3), and (-)-ovafolinin E-9′-O-β-d-glucopyranoside (4), two neolignans, eusiderin N (5) an

Full text of DOI:10.1021/np3007638

Article
pubs.acs.org/jnp
Antioxidant Lignans and Chromone Glycosides from Eurya japonica
†
,‡,∇
†,∇
†
†
§
Li-Ming Yang Kuo,
Li-Jie Zhang, Hung-Tse Huang, Zhi-Hu Lin, Chia-Ching Liaw,
†
⊥
⊥
,†,∥
,‡
Hui-Ling Cheng, Kuo-Hsiung Lee, Susan L. Morris-Natschke, Yao-Haur Kuo,* and Hsiu-O Ho*
†
National Research Institute of Chinese Medicine, Taipei 112, Taiwan, Republic of China
‡
School of Pharmacy, Taipei Medical University, Taipei 110, Taiwan, Republic of China
§
StarSci Biotech Co. Ltd, Taipei 112, Taiwan, Republic of China
⊥
Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North
Carolina 27599-7568, United States
∥
Graduate Institute of Integrated Medicine, China Medical University, Taichung 404, Taiwan, Republic of China
*
S Supporting Information
ABSTRACT: Four new 8,8′,7,2′-lignans, (+)-ovafolinin B-9′-
O-β-D-glucopyranoside (1), (−)-ovafolinin B-9′-O-β-D-gluco-
pyranoside (2), (+)-ovafolinin E-9′-O-β-D-glucopyranoside
(
3), and (−)-ovafolinin E-9′-O-β-D-glucopyranoside (4), two
neolignans, eusiderin N (5) and (7S,8R)-3,5,5′-trimethoxy-
′,7-epoxy-8,3′-neolignan-9,9′-diol-4-O-β-D-xylopyranoside
6), and two new chromone glycosides, 5,7-dihydroxy-4H-
4
(
chromen-4-one-3-O-β-D-glucopyranoside (7) and 5,7-dihy-
droxy-4H-chromen-4-one-3-O-β-D-xylopyranoside (8), togeth-
er with 25 known compounds, were isolated from the stems of
Eurya japonica. Structural elucidation of compounds 1−8 was
established by spectroscopic methods, especially 2D NMR
techniques, electronic circular dichroism data, and comparison with reported data. The isolates were evaluated for antioxidant
and anti-NO production activities. Compounds 1, 2, 12−20, and 29 (ED 23.40 μM for 1) demonstrated potent antioxidant
50
activity compared to the positive control α-tocopherol (ED 27.21 μM). On the other hand, compounds 1, 2, 7−9, 12−20, and
50
32 showed only weak anti-NO production activity when compared to the positive control quercetin.
1
8
he fruits and leaves of Eurya japonica Thunberg
(Theaceae) are used in the Chinese traditional medicine
sides A (28), 6′-O-coumaroyl-1′-O-[2-(3,4-dihydroxyphenyl)-
19
T
ethyl]-β-D-glucopyranoside (29), neocalceolarioside D
30), and norbergenin (31); one triterpene, betulinic acid
32); and one steroid, β-sitosterol glucopyranoside (33),
from the ethanol extract of E. japonica. The structures of the
new compounds were elucidated by analysis of spectroscopic
data and comparisons with reported data. Some of these
compounds were also evaluated for antioxidant and anti-NO
production activities.
2
0
21
“
Lingmu” for the treatment of rheumatoid arthritis, tympanites,
(
(
1
2
2
2
23
hemostasis of injuries, etc. The components of the leaves and
3
berries of this plant include halleridone, cornoside, and three
flavonoids; however, phytochemical research on the compo-
nents in the plant stem is rare. We here report the isolation and
characterization of six new lignans (1−6) and two new
chromone glycosides (7, 8) (Figure 1), along with 25 known
compounds, including one chromone, 5,7-dihydroxy-4H-chro-
4
men-4-one-3-O-α-L-arabinopyranoside (9); five lignans, torto-
5
6
RESULTS AND DISCUSSION
side E (10), sakuraresinol (11),7 (−)-2a-O-(β-D-
■
glucopyranosyl)lyoniresinol (12), (+)-3a-O-(β-D-
glucopyranosyl)lyoniresinol (13), and aviculin (14); six
flavonoids, (+)-epitaxifolin 3-O-β-D-xylopyranoside (15),
A 95% aqueous EtOH extract of the stems of E. japonica was
suspended in H O and partitioned successively with n-hexane,
7
8
9
2
EtOAc, and n-BuOH. The EtOAc extract was chromatographed
on a silica gel column and then on a Sephadex LH-20 column.
The bioactive fractions were subjected to semipreparative
HPLC, using a reverse-phase (ODS) column, to yield six new
lignan glycosides (1−6), two new chromone glycosides (7, 8),
and 25 known compounds.
9
(
−)-epitaxifolin 3-O-β-D-xylopyranoside (16), (+)-taxifolin 3-
9
O-β-D-xylopyranoside (17), (−)-taxifolin 3-O-β-D-xylopyrano-
9
10
side (18), (2R,3R)-(+)-glucodistylin (19), and (2S,3S)-(−)-
1
0
glucodistylin (20); 11 phenyl glycosides, di-O-methylcrenatin
1
1
(
21), 2,6-dimethoxy-4-(2-hydroxyethyl)phenol 1-O-β-D-glu-
copyranoside (22), dihydrosyringin (23), 3,4,5-trimethox-
yphenyl-β-D-glucopyranoside (24), 3,4-dimethoxyphenyl-β-D-
glucopyranoside (25), 3-hydroxy-4,5-dimethoxyphenyl-β-D-
1
2
13
14
15
Received: October 31, 2012
16
17
glucopyranoside (26), cremanthodioside (27), junipetriolo-
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
Article
Figure 1. Compounds isolated from E. japonica.
Compounds 1 and 2 were obtained as pale yellow,
amorphous powders and had similar HRESIMS, UV, IR, and
NMR spectra, suggesting that they are stereoisomers. The
elemental formula was C H O from HRESIMS (corre-
2
8
36 13
sponding to 11 degrees of unsaturation). Absorptions for
−1
hydroxy (3382 cm ) and aromatic (1616, 1497, 1457, or 1461
−1
cm ) groups were found in the IR spectra of 1 and 2, and their
1
UV spectra showed absorption maxima at 283 nm. In the H
13
and C NMR spectra, the two compounds possessed the same
resonances for two benzene rings, four aromatic methoxy
groups, three aliphatic methines, three aliphatic methylenes,
and a hexose moiety. The foregoing data accounted for nine of
the 11 required degrees of unsaturation and suggested that both
1
and 2 possessed a tetracyclic skeleton with a sugar moiety.
Their structures and the locations of the attached groups were
1
1
determined from H− H COSY, HMQC, and HMBC data
Figure 2. Key HMBC (→) and COSY (−) correlations of 1 and 2.
1
1
(
Figure 2). In the H− H COSY spectrum, cross-peaks were
found between H-7/H-8/H-9, H-8/H-8′, and H-7′/H-8′/H-9′.
In the HMBC spectrum, long-range correlations were observed
between H-7/C-2, C-6, C-1′, C-2′, and C-3′ and between H-
and the 13C NMR chemical shifts (δ 81.3 C-9′, 153.8 C-6)
C
indicated the presence of an ether bridge between C-9′ and C-
6. Furthermore, a correlation between the anomeric proton (H-
1″) and C-9 established the hexose at C-9. The hexose moiety
7
′/C-1′, C-2′, and C-6′. These facts suggested an 8,8′,7,2′-
lignan skeleton. The positions of the four methoxy groups were
determined at C-2, C-4, C-3′, and C-5′ based on the HMBC
correlations. The long-range correlation between H-9′ and C-6
24
was confirmed as β-glucose, and the D-configuration was
identified by acid hydrolysis of compounds 1 and 2. Thus, the
B
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Journal of Natural Products
Article
25
gross structures of 1 and 2 were determined as shown in Figure
E, and the β-D-glucose moiety was assigned at C-9. The
stereochemical difference between the two compounds was also
determined from the ECD spectra. The ECD spectrum of 3
(Figure 5), which is identical with that of dimethyl (1S,2R)-1,2-
dihydro-1-(3,4-dimethoxyphenyl)-6,7-dimethoxynaphthalene-
2,3-dicarboxylate, showed a positive Cotton effect at 357 nm,
25
1
and are similar to that of ovafolinin B, except for the glucose
moiety.
On the basis of the NOE correlations between H-7 and H-9
and between H-8′ and H-9, the relative configuration was
assigned as 7R*, 8R*, and 8′R* (Figure 3). The stereochemical
28
and that of 4 a negative Cotton effect at 357 nm. Thus, the
absolute configurations of compounds 3 and 4 were confirmed
as 7S, 8R and 7R, 8S, respectively. From the above findings, the
structures of 3 and 4 were identified as (+)-ovafolinin E-9′-O-β-
D-glucopyranoside and (−)-ovafolinin E-9′-O-β-D-glucopyrano-
side, respectively.
Compound 5 was isolated as a white powder. The molecular
formula was established as C H O from the HRESIMS (m/
2
6
34 12
+
z 561.1964 [M + Na] ). The IR spectrum displayed bands for
hydroxy (3407 cm ) and aromatic (1596, 1560, 1505 cm )
moieties. The UV spectrum showed absorption maxima at 273
−1
−1
1
and 230 (sh) nm. The H NMR spectrum showed resonances
at δH 6.49 (d, J = 2.4 Hz) and 6.65 (d J = 2.4 Hz),
corresponding to two meta-coupled aromatic protons, one two-
proton singlet at δH 6.68, and a singlet at δH 3.85 (6H, s)
corresponding to two aromatic methoxy groups. In addition,
resonances were present for a 1,3,4,5-tetrasubstituted aromatic
Figure 3. NOE correlations of 1.
ring: resonances at δ 3.54 (t, J = 6.6 Hz, 2H), 2.56 (m, 2H),
H
and 1.80 (m, 2H) corresponding to a 1-propanol moiety;
difference between the two compounds was further determined
from electronic circular dichroic (ECD) spectra. Compound 1
and ovafolinin B (1a), the acid hydrolysis product of 1, had
similar ECD spectra, indicating that the presence of the glucose
moiety did not influence the Cotton effects (Figure 4).
Comparison of the ECD spectra 1 and 2 showed their Cotton
effects at 284 nm were negative and positive, respectively. The
negative Cotton effect indicates a 7R configuration, while the
resonances at δ 4.56 (d, J = 7.8 Hz), 4.14 (m), and 1.19 (d, J =
H
6
.6 Hz, 3H) corresponding to a 1,2-propylene glycol unit, and a
resonance at δ 4.91 (d, J = 7.8 Hz, 1H) corresponding to the
H
1
3
anomeric proton of a β-D-glucopyranose moiety. The C NMR
spectrum also exhibited the corresponding carbon resonances
for the above structural units, confirming that 5 was a lignan
glucoside. In the HMBC spectrum, a correlation was observed
between H-1″ and C-5′ indicating that the glucose moiety was
linked at C-5′. The lignan portion was assigned as 1,4-
benzodioxane-type, according to the molecular formula and
2
6−28
positive one indicates 7S.
Therefore, the absolute
configurations of compounds 1 and 2 are 7R, 8R, 8′R and
7
S, 8S, 8′S, respectively. Accordingly, the structures of 1 and 2
NMR data [δ 4.56 (d, J = 7.8 Hz, H-7), 4.14 (m, H-8), and
are (+)-ovafolinin B-9′-O-β-D-glucopyranoside and (−)-ovafo-
linin B-9′-O-β-D-glucopyranoside, respectively, as shown in
Figure 1.
H
1
.19 (d, J = 6.6 Hz, 3H, H-9); δ 82.3 (C-7), 75.5 (C-8), and
C
1
7.4 (C-9)], and the relative configuration of H-7 and H-8 was
29
trans. The ECD spectrum of 5 showed a negative Cotton
effect at 236 nm, indicating an 8R absolute configuration.
Like compounds 1 and 2, compounds 3 and 4 also possessed
similar NMR, UV, IR, and HRESIMS spectra. The HRESIMS
of 3 and 4 suggested an elemental formula of C H O based
29
Thus, the absolute configuration of 5 was determined as 7R, 8R.
On the basis of the above data, the structure of 5, a eusiderin
2
8
34 13
on the quasi-molecular ions at m/z 577.1935 (3) and 577.1932
−
29
(
4) [M − H] . The IR spectra displayed absorption bands for
derivative, was defined and assigned the trivial name eusiderin
hydroxy and aromatic groups, and the UV spectra showed
absorption maxima at 354, 320 (sh), 270 (sh), and 254 nm.
N.
Compound 6, a white powder, has a molecular formula of
1
13
The H and C NMR spectra suggested that 3 and 4 have the
same lignan skeleton. From the HMQC and HMBC spectra,
the aglycone moieties of 3 and 4 were determined as ovafolinin
C H O determined from the HRESIMS spectrum (m/z
2
6
34 11
+
545.1996 [M + Na] ). The IR spectrum displayed bands for
hydroxy (3437 cm ), double-bond (1640 cm ), and aromatic
−1
−1
Figure 4. ECD spectra of compounds 1 (red), 1a (blue), and 2 (black).
C
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Journal of Natural Products
Article
Figure 5. ECD spectra of compounds 3 (blue) and 4 (black).
1
a
Table 1. H NMR Spectroscopic Data (δ) of 1−6 and 1a (J in Hz)
position
1
1a
2
3
4
5
6
2
5
6
7
8
9
6.31, s
6.31, s
6.29, s
6.29, s
6.68, s
6.68, s
6.72, s
6.72, s
6.22, s
4.70, s
6.24, s
4.56, s
6.24, s
4.59, s
4.90, overlapped
3.36, m
4.81, overlapped
3.42, m
4.56, d (7.8)
4.14, m
5.55, d (6.0)
3.45, m
2.29, brt (4.2)
3.86, brd (9.2)
2.15, brt (4.2)
3.61, m
2.35, t (6.8)
3.89, m
3.51, m
3.72, dd (10.2, 4.8) 1.19, d (6.6)
3.20, m
3.86, m
3.58, dd (9.6, 6.8) 3.52, dd (10.8, 7.6) 3.63, m
3.36, m
3.75, dd (11.2, 8.0)
6.74, brs
2′
6′
7′
6.65, d (2.4)
6.42, s
6.44, s
6.45, s
6.98, s
6.98, s
7.51, s
6.49, d (2.4)
2.56, m, 2H
6.71, brs
3.00, dd (17.6, 7.2) 3.10, dd (17.2, 7.2) 3.11, dd (17.6, 7.6) 7.52, s
2.62, m, 2H
2
.80, d (17.6)
2.83, d (17.2)
2.21, brd (6.4)
2.82, d (17.6)
2.24, brd (6.4)
8
′
′
2.22, brd (5.2)
1.80, m, 2H
1.80, m, 2H
3.55, m, 2H
9
4.33, dd (12.0, 2.4) 4.38, dd (12.0, 2.8) 4.36, dd (12.0, 2.8) 9.45, s
.74, m 3.74, dd (12.0, 8.0) 3.56, m
4.26, d (7.6)
9.46, s
3.54, t (6.6), 2H
3
1″
2″
3″
4″
5″
4.24, d (7.6)
3.20, m
4.36, d (7.8)
3.27, t (8.4)
3.37, m
4.23, d (7.8)
3.25, m
4.91, d (7.8)
3.50, t (8.4)
3.46, t (8.4)
3.38, t (8.4)
3.41, m
4.98, d (8.4)
3.54, m
3.20, m
3.33, m
3.27, m
3.25, m
3.34, m
3.33, m
3.45, t (8.0)
3.57, m
3.35, m
3.33, m
3.28, m
3.15, m
3.23, m
3.25, m
3.95, dd (11.6, 4.8)
3
.16, dd (12.0, 8.4)
6″
3.77, m
3.83, m
3.77, dd (12.0, 2.4) 3.82, dd (12.0, 3.6) 3.88, dd (12.0, 2.4)
3.63, dd (12.0, 5.4) 3.64, dd (12.0, 6.0) 3.69, dd (12.6, 5.4)
3.63, m
3.63, m
2
3
4
5
3
5
-OMe
-OMe
-OMe
-OMe
3.89, s, 3H
3.89, s, 3H
3.71, s, 3H
3.90, s, 3H
3.69, s, 3H
3.69, s, 3H
3.85, s, 3H
3.85, s, 3H
3.80, s, 3H
3.67, s, 3H
3.70, s, 3H
3.69, s, 3H
3.54, s, 3H
3.93, s, 3H
3.69, s, 3H
3.59, s, 3H
3.93, s, 3H
3.80, s, 3H
3.87, s, 3H
′-OMe 3.32, s, 3H
′-OMe 3.74, s, 3H
3.36, s, 3H
3.78, s, 3H
3.35, s, 3H
3.77, s, 3H
a
1, 1a, 2, and 6, 400 MHz, 3−5, 600 MHz, all in methanol-d4.
−
1
30
(
796 cm ) groups. The UV spectrum showed absorption
nm, indicating a 7S,8R absolute configuration. Consequently,
compound 6 was determined as (7S,8R)-3,5,5′-trimethoxy-4′,7-
epoxy-8,3′-neolignan-4,9,9′-triol-4-O-β-D-xylopyranoside.
Compound 7 was obtained as a white powder. The
HRESIMS indicated a molecular formula of C H O (m/z
1
13
maxima at 280 and 230 (sh) nm. The H and C NMR spectra
showed the presence of two 1,3,4,5-tetrasubstituted benzene
rings, a 1-propanol and a 1,3-propanediol fragment, one
pentose moiety, and three aromatic methoxy groups. These
data also corresponded to a lignan glycoside. The pentose was
determined as β-D-xylose by the acid hydrolysis method. In the
HMBC spectrum, correlations between H-7/C-4′, H-7/C-3′,
H-8/C-2′, H-8/C-3′, H-8/C-4′, and H-1″/C-4 indicated that
the 1-phenylpropane-1,3-diol and 3-phenylpropan-1-ol frag-
ments were linked via 7-O-4′ and 8−3′, which constitutes a
benzofuran neolignan, and the β-D-xylose moiety was
connected at C-4. The relative configuration of H-7 and H-8
was determined as trans, based on association of H-9 and H-7
in the NOE spectrum. The ECD spectrum of 6 showed a
positive Cotton effect at 232 nm and a negative effect at 217
15
16 10
+
379.0656 [M + Na] ). The IR spectrum displayed bands for
−1
−1
hydroxy (3384 cm ), carbonyl (1657 cm ), double-bond
−1
−1
(1618 cm ), and aromatic (1592, 1509, 1451 cm ) groups,
and the UV spectrum showed absorption maxima at 330 (sh),
300, 262 (sh), and 252 nm. In the H NMR spectrum,
resonances were observed for two meta-coupled aromatic
protons at δ 6.34 (d, J = 2.0 Hz) and 6.21 (d, J = 2.0 Hz), one
1
H
double-bond proton at δH 8.23 (s), and a series of proton
1
3
resonances for a β-glucopyranose moiety. In the C NMR
spectrum, the corresponding resonances together with a
conjugated carbonyl carbon at δC 178.4 were found. From
D
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Journal of Natural Products
Article
Table 2. 13C NMR Spectroscopic Data (δ , mult.) for 1−6 and 1a
a
C
C
1
1a
2
3
4
5
6
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
2
3
4
5
3
5
125.5, qC
146.7, qC
136.8, qC
147.8, qC
102.3, CH
153.9, qC
31.3, CH
42.9, CH
73.5, CH2
127.4, qC
124.0, qC
147.3, qC
138.2, qC
148.4, qC
107.4, CH
30.2, CH2
35.6, CH
81.3, CH2
104.6, CH
75.3, CH
78.0, CH
71.2, CH
77.5, CH
62.3, CH2
62.1, CH3
125.6
146.8
136.7
147.8
102.2
153.9
31.3
125.4
146.8
136.7
147.9
102.3
153.9
31.4
136.3, qC
106.2, CH
149.0, qC
135.0, qC
149.0, qC
106.2, CH
38.6, CH
40.9, CH
69.2, CH2
124.2, qC
127.1, qC
147.8, qC
135.7, qC
149.4, qC
109.8, CH
149.5, CH
136.3, qC
194.3, CH
103.9, CH
75.1, CH
78.3, CH
71.5, CH
77.9, CH
62.6, CH2
136.0
106.2
149.0
135.1
149.0
106.2
38.7
129.2, qC
105.9, CH
149.4, qC
137.2, qC
149.4, qC
105.9, CH
82.3, CH
75.5, CH
17.4, CH3
135.7, qC
110.8, CH
147.1, qC
133.3, qC
146.0, qC
112.0, CH
32.7, CH2
35.4, CH2
62.6, CH2
102.8, CH
74.9, CH
77.9, CH
71.4, CH
78.3, CH
62.1, CH2
147.4, qC
103.9, CH
154.5, qC
134.7, qC
154.5, qC
103.9, CH
88.6, CH
55.8, CH
65.1, CH2
137.2, qC
114.2, CH
129.5, qC
140.3, qC
145.3, qC
117.9, CH
32.9, CH2
35.8, CH2
62.2, CH2
104.8, CH
74.3, CH
76.1, CH
71.0, CH
66.3, CH2
45.1
42.5
41.4
65.2
72.5
70.6
′
′
′
′
′
′
′
′
127.5
124.0
147.4
138.2
148.5
107.3
30.0
127.6
123.9
147.3
138.2
148.5
107.5
30.0
124.2
127.1
147.8
136.0
149.4
109.8
149.6
136.2
194.4
105.3
75.2
35.3
35.3
′
81.5
81.4
″
″
″
″
″
″
104.3
75.2
78.2
78.0
71.6
71.7
77.9
78.0
62.8
62.9
-OMe
-OMe
-OMe
-OMe
′-OMe
′-OMe
61.7
56.5
62.0
56.7, CH3
56.7
56.8, CH3
56.8, CH3
56.7, CH3
56.5, CH3
56.5
56.7, CH3
61.0, CH3
56.8, CH3
56.7
61.0
56.8
56.7, CH3
56.8, CH3
60.0, CH3
56.4, CH3
60.0
56.5
60.1
56.5
a
Compounds 1, 1a, 2, and 6, 100 MHz, 3, 4, and 5 150 MHz, all in methanol-d4.
1
13
the HMBC spectrum, correlations of H-6/C-5, C-7, and C-10;
Table 3. H and C NMR Spectroscopic Data (δ in ppm, in
1
13
H-8/C-7, C-9, and C-10; H-2/C-3, C-4, and C-9; and H-1′
methanol-d , 400 MHz for H, 100 MHz for C) for 7 and 8
4
(glucose)/C-3 were observed. Thus, the skeleton is 3,5,7-
7
8
trihydroxy-4H-chromen-4-one, and the glucose moiety is
located at C-3. Accordingly, the structure of 7 was determined
as 5,7-dihydroxy-4H-chromen-4-one-3-O-β-D-glucopyranoside.
Compound 8 was obtained as a white powder, and its
molecular formula is C H O (HRESIMS m/z 349.0559 [M +
position
δC, type
147.4, CH
141.5, qC
178.4, qC
163.4, qC
100.2, CH
166.5, qC
95.0, CH
159.4, qC
106.1, qC
104.2, CH
74.8, CH
77.4, CH
71.3, CH
78.5, CH
δH (J in Hz)
δC, type
147.4, CH
141.2, qC
178.4, qC
163.4, qC
100.2, CH
166.4, qC
95.0, CH
159.3, qC
106.2, qC
104.6, CH
74.6, CH
77.1, CH
70.9, CH
δH (J in Hz)
2
3
4
5
6
7
8
9
8.23, s
8.13, s
1
4
14
9
+
Na] ). The UV and IR spectra of 8 were similar to those of
compound 7. A comparison of the NMR spectra of 7 and 8
showed that resonances for the glucose moiety in 7 were
replaced by those for a pentose moiety in 8. Furthermore, the
pentose moiety was determined as β-D-xylopyranose. Thus, the
structure of compound 8 was defined as 5,7-dihydroxy-4H-
chromen-4-one-3-O-β-D-xylopyranoside.
6.21, d (2.0)
6.34, d (2.0)
6.22, d (2.0)
6.33, d (2.0)
1
1
2
3
4
5
0
′
′
′
′
4.73, d (7.6)
3.42, m
4.73, d (7.2)
3.40, m
Moreover, 25 known compounds were also isolated from the
3.42, m
3.40, m
95% aqueous EtOH extract of E. japonica. The structures of
3.30, m
3.54, m
isolates 9−33 were identified by comparing their NMR and MS
data with reported analytical data.
′
3.38, m
67.1, CH2 3.93, dd (11.2,
.2)
5
Compounds 1, 2, 7−9, 12−26, and 29−33 were evaluated
for antioxidant activity by using the stable 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH) method. As shown in Table 4,
compounds 1, 2, 12−20, and 29 (ED = 23.40 ± 0.46 to 44.80
3
.30, overlapped
6′
62.6, CH2 3.90, dd (12.4,
2.0)
3.67, dd (12.0,
5
0
6
.4)
±
0.38 μM) had potent antioxidant activities compared with
the positive control α-tocopherol (ED50 = 27.21 ± 0.02 μM).
Fifteen compounds (1, 2, 7−9, 12−20, and 32) were also
evaluated for inhibitory effects on LPS-induced NO production
in RAW 264.7 macrophages, using quercetin as the positive
control (IC50 = 1.30 ± 0.44 μM). As shown in Table 5, the
inhibitory percentages of compounds 7 and 32 exceeded 50%,
while those of the other tested compounds were below 50%
E
dx.doi.org/10.1021/np3007638 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
Table 4. Antioxidant Activity of Compounds 1, 2, 7−9, 12−26, and 29−33
compound
ED50 (μM)
compound
ED50 (μM)
compound
ED50 (μM)
1
2
7
8
9
23.40 ± 0.46
24.70 ± 0.26
−
16
17
18
19
20
21
22
23
24
26.47 ± 0.71
30.39 ± 0.64
25.23 ± 1.26
33.55 ± 0.79
25
26
29
30
31
32
33
−
−
32.95 ± 0.69
−
−
−
−
−
133.34 ± 4.54
37.65 ± 0.09
26.59 ± 1.65
44.80 ± 0.38
26.61 ± 0.96
36.21 ± 0.08
1
1
1
1
2
3
4
5
−
−
−
−
a
“−” means the ED50 (μg/mL) value is >100. Positive control: (±)-α-tocopherol ED50 = 27.21 ± 0.02 μM.
Table 5. Anti-NO Production Activity of Compounds 1, 2, 7−9, 12−20, and 32
a
a
a
a
compound
anti-NO (%)
cell viability (%)
compound
anti-NO (%)
cell viability (%)
1
2
7
8
9
38.99 ± 1.95
40.37 ± 1.94
53.79 ± 1.78
36.24 ± 1.29
38.07 ± 1.94
39.22 ± 1.62
38.76 ± 2.27
39.68 ± 2.27
106.21 ± 4.12
110.30 ± 0.15
105.03 ± 2.53
102.23 ± 0.38
106.42 ± 2.15
100.62 ± 7.29
104.26 ± 0.15
112.41 ± 3.29
15
16
17
18
19
20
32
35.32 ± 5.08
36.70 ± 0.01
33.26 ± 4.22
37.84 ± 6.16
35.55 ± 2.91
35.78 ± 5.18
56.19 ± 2.27
107.73 ± 0.6
102.17 ± 0.08
105.44 ± 5.21
107.31 ± 2.79
101.45 ± 7.60
104.71 ± 12.67
60.31 ± 0.76
−
12
13
14
a
Cells were treated with LPS (1 μg) in combination with test compound (20 μg/mL) for 24 h. Positive control: Quercetin IC50 = 1.30 ± 0.44 μM.
(
20 μg/mL). Yet, the cell viability with compound 32 was
0.31%, measured by the MTT assay, indicating that the anti-
which was subjected to column chromatography (CC) on Sephadex
LH-20, eluting with CHCl /MeOH (1/1), to afford compound 33
6
3
(
65.4 mg, 0.000327%). Fraction E3 (CH Cl /MeOH, 8/2, 52.3 g) was
NO production effect resulted from cell death. Compared with
the positive control, these flavonol glycosides, lignan glycosides,
and chromone glycosides were weak inhibitors of LPS-induced
NO production.
2 2
separated into five subfractions (E3.1 to E3.5) by CC on Sephadex
LH-20, eluting with 100% MeOH. Fraction E3.2 (12.78 g) was then
separated into four subfractions (E3.2.1 to E3.2.4) by CC on Sephadex
LH-20, eluting with CHCl /MeOH (1/1). Finally, fraction E3.2.2
3
(
3.24 g) was separated into four subfractions (E3.2.2.1 to E3.2.2.4) by
Sephadex LH-20 CC, eluting with 100% MeOH. Fraction E3.2.2.2
1.79 g) was applied to an ODS open column, eluting with gradient
EXPERIMENTAL SECTION
■
(
General Experimental Procedures. Optical rotations were
obtained on a JASCO P-2000 polarimeter. ECD spectra were
measured with a JASCO J-715 spectropolarimeter. IR spectra were
recorded neat, using an IR-FT Mattson Genesis II spectrometer. NMR
spectra were recorded using Bruker UltraShield 400 MHz and Varian
Inova-600 MHz spectrometers. HRESIMS spectra were recorded on a
Shimadzu IT-TOF HR mass spectrometer. For column chromatog-
raphy, silica gel 60 (70−230, 230−400 mesh, Merck) and Sephadex
LH-20 (GE Healthcare) were used. Precoated silica gel (Merck 60 F-
mobile phase (10% to 100% MeOH in H O, v/v), to give eight
2
subfractions, E3.2.2.2.1 to E3.2.2.2.8. Fraction E3.2.2.2.1 (0.1296 g)
was subjected to semipreparative HPLC [250 × 10 mm i.d., Cosmosil
5
C18 AR-II column (also used for all HPLC experiments), MeCN/
H O, using the concentration gradient 10−20% for 20 min, 20−30%
2
for 10 min, 100% for 10 min, flow rate 2 mL/min] to afford
compounds 21 (t : 11.9 min, 1.3 mg, 0.0000065%), 22 (t : 15.1 min,
R
R
3
.9 mg, 0.0000185%), and 23 (t : 19.0 min, 0.9 mg, 0.0000045%).
R
Fraction E3.2.2.2.2 (0.2722 g) was purified by semipreparative HPLC
2
54) plates were used for TLC. The spots on TLC were detected by
with MeCN/H O (17−30% for 30 min, 100% for 10 min, flow rate 2
spraying with 5% H SO followed by heating at 110 °C. MPLC was
2
2
4
mL/min) to give 11 subfractions (E3.2.2.2.2.1 to E3.2.2.2.2.11).
performed on a system equipped with a Buchi B-688 pump, Buchi B-
84 fraction collector, and Buchi columns. HPLC separations were
Fraction E3.2.2.2.2.5 was purified by semipreparative HPLC (MeOH/
6
H O, 4/1, flow rate 2.5 mL/min) to afford compounds 4 (t : 14.0 min,
performed on a Hitachi L-2130 pump with an L-2450 diode array
detector, equipped with a 250 × 10 mm i.d. preparative Cosmosil 5C18
AR-II column (Nacalai Tesque, Inc.).
Plant Material. Stems of E. japonica (20 kg) were collected at
Yangming Mountain, Taiwan, in April 2008, and identified by one of
the authors (Y.-H.K.). A voucher specimen (No. NRICM20080410B)
has been deposited in the National Research Institute of Chinese
Medicine, Taipei, Taiwan.
2
R
0
.7 mg, 0.0000035%) and 3 (t : 14.6 min, 1.0 mg, 0.0000050%).
R
Fraction E3.2.2.2.3 (0.4222 g) was purified by semipreparative HPLC
MeCN/H O, 21% for 30 min, 100% for 10 min, flow rate 2 mL/
(
2
min), to give 11 subfractions (E3.2.2.2.3.1 to E.2.2.2.3.11). By using
preparative TLC (CHCl /MeOH/EtOAc/acetone, 4.5/1/1/1), com-
3
pound 6 (4.6 mg, 0.000023%) was obtained from fraction E3.2.2.2.3.7,
and compounds 10 (0.8 mg, 0.000004%) and 5 (0.6 mg, 0.000003%)
were obtained from fraction E3.2.2.2.3.8. Preparative TLC with a
Extraction and Isolation. The stems of E. japonica (20 kg) were
extracted with 95% aqueous EtOH at 55 °C (3 × 80 L) in an airtight
heating extraction tank, and the extract was concentrated under
different mobile solvent system (CHCl /MeOH/EtOAc/acetone, 5/
3
1/1/1) was used for the purification of compound 2 (23 mg,
0.000115%) from fraction E3.2.2.2.3.9 and compounds 1 (75.5 mg,
0.000378%) and 11 (2.3 mg, 0.000012%) from fraction E3.2.2.2.3.10.
Fraction E3.2.3 (5.15 g) was separated into five subfractions (E3.2.3.1
to E3.2.3.5) by Sephadex LH-20 CC, eluting with 100% MeOH.
Fraction E3.2.3.3 (3.45 g) was applied to an ODS open column,
reduced pressure. The EtOH extract (1500 g) was suspended in H O,
2
and the suspension was extracted successively with n-hexane, EtOAc,
and n-BuOH. The EtOAc layer gave 247.3 g of extract that was
separated on an MPLC silica gel column, eluted with CH Cl /MeOH
2
2
(
(
10/0, 9/2, 8/2, 7/3), to yield four fractions (E1 to E4). Fraction E2
CH Cl /MeOH, 9/2, 90 g) gave an insoluble precipitate, which was
eluting with gradient mobile phase (10−100% MeOH in H O, v/v), to
2
2
2
identified as compound 32 (about 40 g, 0.2%), and a soluble part,
afford nine subfractions (E3.2.3.3.1 to E3.2.3.3.9). Fraction E3.2.3.3.2
F
dx.doi.org/10.1021/np3007638 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
0.6815 g) was subjected to semipreparative HPLC (MeCN/H O,
(7S,8R)-3,5,5′ -Trimethoxy-4′,7-epoxy-8,3′-neolignan-4,9,9′-triol-
2
2
5
1
0% for 5 min, 10−20% for 15 min, 20−30% for 10 min, 100% for 5
min, flow rate 2 mL/min) to afford 11 subfractions (E3.2.3.3.2.1 to
.2.3.3.2.11). Fraction E3.2.3.3.2.2 was purified by semipreparative
HPLC (MeOH/H O, 3/7, flow rate 2 mL/min) to give 28 (t : 32.0
4-O-β-D-xylopyranoside (6): white powder, mp 218 °C; [α]
D
−19.1
(c 0.2, MeOH); UV λmax (MeOH) nm (log ε) 280 (2.98), 230 (3.70,
sh) nm; ECD [θ]232 +846.8, [θ]217 −3021.2; IR νmax (KBr) 3437,
3
−
1
1
13
2966, 1640, 1025, 796 cm ; H NMR and C NMR data, Tables 1
2
R
+
min, 0.9 mg, 0.0000045%) and 27 (t : 33.8 min, 1.5 mg, 0.0000075%).
and 2; HRESIMS m/z 545.1996 [M + Na] (calcd for C26H O11Na,
34
R
Compounds 24 (42.0 mg, 0.00021%) and 13 (27.8 mg, 0.00014%)
were obtained from fractions E3.2.3.3.3 and E3.2.3.3.4, respectively, by
recrystallization with MeOH. Fractions E3.2.3.3.5 and E3.2.3.3.6 were
545.2001).
5,7-Dihydroxy-4H-chromen-4-one-3-O-β-D-glucopyranoside (7):
2
5
white powder, mp 202 °C; [α]
−68.7 (c 0.2, MeOH); UV λmax
D
purified by preparative TLC (CHCl /MeOH/EtOAc/acetone, 4.5/1/
(MeOH) nm (log ε) 330 (3.46, sh), 300 (3.60), 262 (3.98, sh), and
252 (3.99) nm; IR νmax (KBr) 3384, 1657, 1618, 1592, 1509, 1451,
1206, 1074, 800 cm ; H NMR and C NMR data, Table 3;
3
1
/1) to afford 26 (14.6 mg, 0.000073%) and 25 (20 mg, 0.0001%),
respectively. Fraction E3.2.3.4 was further purified by preparative TLC
CHCl /MeOH, 4/1) to give 29 (28.3 mg, 0.000142%) and 30 (20.1
−1
1
13
+
(
HRESIMS m/z 379.0656 [M + Na] (calcd for C15H O10Na,
16
3
mg, 0.000101%). Fraction E3.3 (5 g, total 22.64 g) was subjected to
379.0643).
Sephadex LH-20 CC, eluting with 100% MeOH, to yield five fractions
5,7-Dihydroxy-4H-chromen-4-one-3-O-β-D-xylopyranoside (8):
25
(
E3.3.1 to E3.3.5). Fraction E3.3.2 (1.5 g) was purified by
white powder, mp 208 °C; [α]
D
−70.6 (c 0.2, MeOH); UV λmax
semipreparative HPLC (MeCN/H O, 13.5/86.5, flow rate 3 mL/
(MeOH) nm (log ε) 330 (3.63, sh), 300 (3.84), 262 (4.20, sh), and
252 (4.22) nm; IR νmax (KBr) 3368, 1655, 1616, 1588, 1460, 1204,
2
min) to give 31 (t : 9.4 min, 28.3 mg, 0.000642%) and 7 (t : 15.3 min,
R
R
−
1
1
13
3
81 mg, 0.00861%). Fraction E3.3.3 (1.2 g) was purified by
1038, 803 cm ; H NMR and C NMR data, Table 3; HRESIMS m/
+
semipreparative HPLC (MeCN/H O, 15−17% for 20 min, 17−45%
z 349.0559 [M + Na] (calcd for C H O Na, 349.0538).
2
14 14
9
for 20 min, 100% for 10 min, 3 mL/min) to yield compounds 8 (t :
Ovafolinin B (1a): pale yellow, amorphous powder, mp 125 °C;
R
[α]25 +176.8 (c 0.2, MeOH); UV λmax (MeOH) nm (log ε) 284
1
0
3.9 min, 449 mg, 0.010147%) and 9 (t : 18.6 min, 44.0 mg,
.000994%). Fraction E3.3.4 (0.6 g) was purified by semipreparative
R
D
(3.66) nm; ECD [θ]2 −13 892, and [θ] +14 826.2 (c 2.5 × 10−
5
85
246
1
13
HPLC (MeCN/H O, 17% for 20 min, 25% for 20 min, 100% for 10
M, MeOH); H NMR and C NMR data, see Tables 1 and 2.
Scavenging Activity of 1,1-Diphenyl-2-picrylhydrazyl Radi-
cal. The radical scavenging activities of the 25 compounds on the
DPPH free radical were measured using the method of Rangkadilok et
2
min, flow rate 3 mL/min) to yield 17 (t : 18.1 min, 120 mg, 0.0027%),
R
1
8 (t : 19.7 min, 50 mg, 0.00112%), 15 (t : 28.6 min, 18.8 mg,
R
R
0
.000424%), and 16 (t : 30.6 min, 45.3 mg, 0.001021%). Fraction E4
R
3
1
32
(
eluted with CH Cl /MeOH, 7/3, 57 g) was subjected to Sephadex
al. and Chung et al. with minor modifications. An aliquot (120 μL)
of each compound (100−10 μg/mL) or (±)-α-tocopherol (40−10
μg/mL, Fluca Biochemika, ≥97.0% HPLC) was mixed with 30 μL of
0.75 mM DPPH methanol solution in a 96-well microplate. The
mixture was shaken vigorously with an orbital shaker in the dark at
room temperature for 30 min, and then the absorbance was measured
at 517 nm with an ELISA reader. (±)-α-Tocopherol was used as the
positive control. The negative control contained no test compound.
The final results were reported as ED50, the concentration of
compound that scavenged 50% of DPPH radicals in the reaction
solution.
2
2
LH-20, eluting with 100% MeOH, to afford five fractions (E4.1 to
E4.5). Fraction E4.4 (0.5 g, total 15.9 g) was purified by
semipreparative HPLC (MeCN/H O, 10% for 25 min, 25% for 20
2
min, flow rate 3 mL/min) to afford compounds 19 (t : 22.1 min, 53.4
R
mg, 0.00849%), 20 (t : 26.5 min, 20.5 mg, 0.00327%), 14 (t : 34.1
R
R
min, 57.2 mg, 0.00909%), and 12 (t : 36.1 min, 23.4 mg, 0.00372%).
R
(
+)-Ovafolinin B-9′-O-β-D-glucopyranoside (1): pale yellow,
25
amorphous solid; [α]
12869.6, [θ]246 +11727, and [θ]237 −13010.5 (c 2.5 × 10 M,
MeOH); UV λ (MeOH) nm (log ε) 283 (3.76) nm; IR ν (neat)
+193.1 (c 0.2, MeOH); ECD [θ]
285
D
−5
−
max
max
−1 1
3
382, 2931, 1616, 1497, 1457, 1121, 1085, 807 cm ; H NMR and
Cell Culture and NO Measurement. The macrophage cell line
RAW 264.7 was obtained from ATCC (Rockville, MD, USA), cultured
in DMEM containing 5% heat-inactivated fetal calf serum, 100 U/mL
1
3
C NMR data, see Tables 1 and 2; HRESIMS m/z 579.2099 [M −
−
H] (calcd for C H O , 579.2077).
28
35 13
(
−)-Ovafolinin B-9′-O-β-D-glucopyranoside (2): pale yellow,
penicillin, and 100 μg/mL streptomycin, and grown at 37 °C with 5%
25
5
amorphous solid; [α]
nm (log ε): 283 (3.92) nm; ECD [θ] +19052.5, [θ] −12866.8,
and [θ]237 +15282.2 (c 2.5 × 10 M, MeOH); IR ν (neat) 3382,
927, 1616, 1497, 1461, 1125, 1077, 800 cm ; H NMR and
NMR data, see Tables 1 and 2; HRESIMS m/z 579.2090 [M − H]
calcd for C H O , 579.2077).
−127 (c 0.2, MeOH); UV λ (MeOH)
CO in fully humidified air. Cells were plated at a density of 2 × 10
2
D
max
cells/well in 96-well culture plates and stimulated with LPS (1000 ng/
284
246
−5
mL) in the presence (20 μg/mL) or absence of test compound for 24
max
−1
1
13
2
C
h. All compounds were dissolved in DMSO and further diluted with
−
−
sterile PBS. Nitrite (NO
) accumulation in the medium was used as
2
(
an indicator of NO production, which was measured by adding the
Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine in 5%
28
35 13
(
+)-Ovafolinin E-9′-O-β-D-glucopyranoside (3): yellow, amorphous
25
solid; [α] +64.9 (c 0.2, MeOH); UV λ (MeOH) nm (log ε) 354
phosphoric acid). NaNO was used to generate a standard curve, and
2
D
max
(
+
1
1
2
5
3.66), 320 (3.56, sh), 270 (3.51, sh), and 254 (3.87) nm; ECD [θ]357
6446.9, [θ] −3559.6, [θ]276 +2323.68, and [θ]261−938.1 (c 5.0 ×
nitrite production was determined by measuring optical density at 550
nm. All experiments were performed in triplicate. NO production by
LPS stimulation was designated as 100% for each experiment.
Quercetin (Sigma, 98.0% HPLC) was employed as a positive
3
23
−
5
0
M, MeOH); IR ν (neat) 3422, 2966, 2930, 1683, 1572, 1461,
max
−1
1
13
350, 1085, 796 cm ; H NMR and C NMR data, see Tables 1 and
; HRESIMS m/z 577.1935 [M − H] (calcd for C H O ,
−
33
control.
28
33 13
77.1920).
Acid Hydrolysis of Glycosides. A methanol solution of
compound 1 (3.0 mg) was placed on a TLC plate (20 × 10 cm),
and this plate was placed in an atmosphere of concentrated HCl for 30
(
−)-Ovafolinin E-9′-O-β-D-glucopyranoside (4): yellow, amor-
25
D
phous solid; [α]
−5.9 (c 0.2, MeOH); UV λ
(MeOH) nm
max
(
log ε) 354 (3.61), 320 (3.53, sh), 270 (3.54, sh), and 253 (3.84) nm;
min. The HCl and H
plate was developed in CHCl
2
O were then evaporated in a vacuum oven. The
/MeOH (3/1) (saturated with H O).
ECD [θ]357 −4589.6, [θ] +5289.7, [θ]276 −1080.0, and [θ]
+
3
2
324
261
−5
1838.2 (c 5.0 × 10 M, MeOH); IR ν
918, 1686, 1556, 1259, 1089, 800 cm ; H NMR and C NMR
(neat) 3399, 2961,
The aglycone band was detected by UV absorption at 254 nm. To
detect the glycone band, part of the TLC plate was cut off, sprayed
max
−1
1
13
2
−
data, see Tables 1 and 2; HRESIMS m/z 577.1932 [M − H] (calcd
for C H O , 577.1920).
with 5% H SO in EtOH, and then heated at 110 °C. The aglycone
2 4
and glycone bands were removed from the TLC plate. The glycone
was dissolved in EtOH and subjected to HPLC (Shimadzu HPLC
equipped with LC-20AT pump, RID-10A detector, and SCL-10AVP
system controller; column: Chiralpak AD-H column (Daicel), 4.6 ×
250 mm, 5 μm; mobile phase: EtOH/n-hexane:TFA, 3/7/0.05 (v/v);
2
8
33 13
25
Eusiderin N (5): white powder, mp 195 °C; [α]
−5.0 (c 0.2,
D
MeOH); UV λmax (MeOH) nm (log ε) 273 (3.48), 230 (4.25, sh) nm;
ECD [θ]236 −15 926; IR ν (KBr) 3407, 2928, 1596, 1560, 1505,
1
max
−1
1
13
081, 800 cm ; H NMR and C NMR data, Tables 1 and 2;
+
34
HRESIMS m/z 561.1964 [M + Na] (calcd for C H O Na,
flow rate: 0.5 mL/min). The sugar was identified by comparison
26
34 12
5
61.1950).
with authentic samples: t (min) 14.23 (D-glucose), 14.69 (L-glucose);
R
G
dx.doi.org/10.1021/np3007638 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
8.96 (D-xylose), 18.35 (L-xylose). Compounds 2, 6, 7, and 8 were
(17) Wang, A.-X.; Zhang, Q.; Jia, Zh.-J. Pharmazie 2004, 59, 889−
treated in the same manner. All compounds produced either D-glucose
or D-xylose. Therefore, although compounds 3, 4, and 5 were not
obtained in sufficient quantity to perform the acid hydrolysis
experiment, we postulated they would also contain D-glucose.
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