Peroxynitrite-Scavenging Glycosides from the Stem Bark of Catalpa ovata

Article abstract of DOI:10.1021/acs.jnatprod.7b00139

Ten new glycosides, 6,10-O-di-trans-feruloyl catalpol (1), 6,6′-O-di-trans-feruloyl catalpol (2), 3,4-dihydro-6-O-di-trans-feruloyl catalpol (10), (8R,7′S,8′R)-lariciresinol 9′-O-β-d-(6-O-trans-feruloyl)glucopyranoside (17), and ovatosides A-F (18-22, 24), were isolated from the stem bark of Catalpa ovata along with 19 known compounds. All isolates, except 6 (catalposide) and 9 (6-O-veratroyl catalpol), were found to scavenge peroxynitrite (ONOO^-) formed by 3-morpholinosydnonimine. In particular, 12 compounds showed potent activity, with IC₅₀ values in the range 0.14-2.2 μM.

Full text of DOI:10.1021/acs.jnatprod.7b00139

Article
pubs.acs.org/jnp
Peroxynitrite-Scavenging Glycosides from the Stem Bark of Catalpa
ovata
Yun-Seo Kil,† Seong Min Kim,‡ Unwoo Kang,† Hae Young Chung,‡ and Eun Kyoung Seo*,†
†College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Korea
‡College of Pharmacy, Pusan National University, Pusan 46241, Korea
S
* Supporting Information
ABSTRACT: Ten new glycosides, 6,10-O-di-trans-feruloyl
catalpol (1), 6,6′-O-di-trans-feruloyl catalpol (2), 3,4-dihydro-
6-O-di-trans-feruloyl catalpol (10), (8R,7′S,8′R)-lariciresinol
9′-O-β-D-(6-O-trans-feruloyl)glucopyranoside (17), and ovato-
sides A−F (18−22, 24), were isolated from the stem bark of
Catalpa ovata along with 19 known compounds. All isolates,
except 6 (catalposide) and 9 (6-O-veratroyl catalpol), were
found to scavenge peroxynitrite (ONOO−) formed by
3-morpholinosydnonimine. In particular, 12 compounds showed
potent activity, with IC50 values in the range 0.14−2.2 μM.
Catalpa ovata G. Don. is a deciduous tree belonging to the
family Bignoniaceae.1 Its stem bark has been used traditionally
for treating several inflammatory diseases including hepatitis,
cholecystitis, and nephritis.2 Previous phytochemical research
on the stem bark of C. ovata revealed the occurrence of C9-type
iridoids, some of which were shown to inhibit the production
of tumor necrosis factor-α (TNF-α) and nitric oxide (NO),
and showed effects on heat shock protein 90 (HSP90),
respectively.3−5
In the process of inflammation, NO is overproduced by the
expression of inducible NO synthase (iNOS), while reactive
oxygen species (ROS) are released and accumulated by oxygen
uptake of mast cells and leukocytes recruited at the site.6,7
Although regulation of NO production has been targeted for
the treatment of inflammatory diseases, peroxynitrite
(ONOO−) is the ultimate cytotoxic agent, made by combining
NO and ROS and more potent than both.7,8 The reactivity of
ONOO− causes a variety of alterations on DNA, protein, and
lipids.8 Accordingly, the scavenging capacity of ONOO− can
prevent further progression of inflammation and the develop-
ment of chronic disorders. To the best of our knowledge, there
has been no research on C. ovata for the evaluation of its
ONOO−-scavenging effects, even though anti-inflammatory
activities have been studied previously in regard to its tra-
ditional uses.2,9
together with 19 known compounds, 6-O-trans-feruloyl catal-
pol (3),3,10 specioside (4),11 6-O-(4-methoxy-trans-cinnamoyl)
catalpol (5),12 catalposide (6),3 4′-methoxycatalposide (7),13
vanilloyl catalpol (8),14 6-O-veratroyl catalpol (9),15 3,4-dihy-
drocatalposide (11),5 6-O-trans-feruloyl-5,7-bisdeoxycynancho-
side (12),5 6-O-trans-isoferuloyl-5,7-bisdeoxycynanchoside
(13),5 6-O-(4-hydroxybenzoyl)-5,7-bisdeoxycynanchoside
(14),16 6-O-(4-hydroxybenzoyl)ajugol (15),17 6-O-(4-
hydroxybenzoyl)phelipaeside (16),5 grayanoside A (23),18
darendoside A (25),19 2-(4-hydroxyphenyl)ethyl[5-O-(4-hy-
droxybenzoyl)]-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyrano-
side (26),16 phlomisethanoside (27),20 darendoside B (28),19
and 8,5′-diferulic acid (29).21 All isolates were tested for their
ONOO−-scavenging activities.
RESULTS AND DISCUSSION
■
Compound 1 was obtained as a light yellow, amorphous
powder with a molecular formula of C35H38O16, based on its
13C NMR data and the [M + H]+ ion at m/z 715.2242 (calcd
715.2233) in the HRESIMS. The 1H and 13C NMR spectra of 1
showed typical signals for a C9-type iridoid at δH 5.21 (1H, d,
J = 9.6 Hz)/δC 95.4 (C-1), 6.39 (1H, dd, J = 6.0, 2.0 Hz)/142.5
(C-3), 5.00 (1H, dd, J = 6.0, 4.4 Hz)/102.7 (C-4), 2.62 (1H,
m)/36.8 (C-5), 5.09 (1H, dd, J = 8.4, 1.2 Hz)/81.2 (C-6), 3.77
(1H, d, J = 1.2 Hz)/60.6 (C-7), 2.75 (1H, dd, J = 9.6, 8.0 Hz)/
43.4 (C-9), 4.32 and 5.02 (each 1H, d, J = 12.4 Hz)/64.0
(C-10), and δC 64.3 (C-8) (Table 1).5 Besides the iridoid
scaffold, proton signals for two ABX spin systems were observed
at δH 7.22 (1H, d, J = 1.8 Hz, H-2″ exchangeable with H-2‴),
In the present investigation, an EtOAc extract from a MeOH
extract of the stem bark of C. ovata was found to be effective in
scavenging ONOO−. Subsequent fractionation was conducted
using the EtOAc extract, leading to the isolation of 10 new
glycosides, 6,10-O-di-trans-feruloyl catalpol (1), 6,6′-O-di-trans-
feruloyl catalpol (2), 3,4-dihydro-6-O-di-trans-feruloyl catalpol
(10), (8R,7′S,8′R)-lariciresinol 9′-O-β-D-(6-O-trans-feruloyl)-
glucopyranoside (17), and ovatosides A−F (18−22, 24),
Received: February 15, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Chart 1
1
6.815 (1H, d, J = 8.2 Hz, H-5″ exchangeable with H-5‴), 7.10
(1H, dd, J = 8.2, 1.8 Hz, H-6″), 7.21 (1H, d, J = 1.8 Hz, H-2‴
exchangeable with H-2″), 6.805 (1H, d, J = 8.2 Hz, H-5‴
exchangeable with H-5″), and 7.09 (1H, dd, J = 8.2, 1.8 Hz,
H-6‴), together with protons of two trans double bonds
conjugated to carbonyl groups at δH 7.68 (1H, d, J = 15.8 Hz,
H-7″), 6.43 (1H, d, J = 15.8 Hz, H-8″), 7.64 (1H, d, J = 16.0
Hz, H-7‴), and 6.40 (1H, d, J = 16.0 Hz, H-8‴). The 13C NMR
signals of the carbonyl groups resonated at δC 168.91 (C-9″)
and 168.93 (C-9‴). In the H NMR spectrum, two aromatic
methoxy group signals appeared at δH 3.89 (6H, s, OCH3-3″),
as well as the characteristic signals of a β-glucopyranosyl group
at δH 4.78 (1H, d, J = 8.0 Hz), with six protons in the range δH
3.19−3.96.22 The two sets of the 1,3,4-trisubstituted benzene, a
trans double bond conjugated to the carbonyl group, and an
aromatic methoxy group were assignable to two trans-feruloyl
groups on the basis of the HMBC correlations of H-7″/C-2″,
C-6″, C-9″, H-8″/C-1″, C-9″, OCH3-3″/C-3″, H-7‴/C-2‴,
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1
Figure 1. Key H−1H COSY and HMBC correlations of 1, 2, and 10.
C-6‴, C-9‴, H-8‴/C-1‴, C-9‴, and OCH3-3‴/C-3‴ (Figure 1).
The positions of the aromatic methoxy groups were confirmed
by the NOE correlations with the meta-coupled aromatic
proton signals (H-2″ and H-2‴). The three-bond HMBC
correlations of H-1/C-1′, H-6/C-9″, and H2-10/C-9‴ provided
evidence for the location of the β-glucopyranosyl group and the
trans-feruloyl groups on the iridoid skeleton at C-1, C-6, and
C-10, respectively. The connectivity of the β-glucopyranosyl
group to the iridoid skeleton at C-1 was supported by the NOE
correlation between H-1 and H-1′. Moreover, the NOE
correlations of H-1/H2-10, H-6, H-4/H-6, and H-7/H-10b
together with the coupling constant between H-5 and H-9
(J = 8.0 Hz) supported the stereochemistry of the iridoid
skeleton with a cis ring fusion.23−27 Acid hydrolysis of 1 yielded
a sugar moiety, which was determined as D-glucose by the
HPLC analysis of its thiocarbamoyl-thiazolidine derivative.28−31
The characteristic NMR data for 1 were similar to those of
6-O-trans-feruloyl catalpol (3), which was also identified in the
present study,10 except for the presence of a trans-feruloyl
group at C-10. Thus, the structure of 1 was defined as shown
for the new compound, 6,10-O-di-trans-feruloyl catalpol.
3, which was confirmed by the HMBC correlations of H-3a/
C-1, C-5, H-4a/C-5, and H-4b/C-9 and the COSY correlations
of H-3a with H-4a and H-4b (Figure 1). The sugar moiety in
10 was also identified as D-glucose. Therefore, the structure of
10 was defined as shown for the new substance 3,4-dihydro-6-
O-di-trans-feruloyl catalpol.
Compound 17 was isolated as a light yellow, amorphous gum
with a molecular formula of C36H42O14, based on the 13C NMR
data and the [M + Na]+ ion at m/z 721.2473 (calcd 721.2467)
1
in the HRESIMS. Compound 17 displayed typical H and 13C
NMR signals for lariciresinol,32 a tetrahydrofuran-type lignan, at
δH 6.73 (1H, d, J = 2.0 Hz)/δC 113.4 (C-2), 6.66 (1H, d, J =
8.2 Hz)/116.2 (C-5), 6.56 (1H, d, J = 8.2, 2.0 Hz)/122.3
(C-6), 2.90 (1H, dd, J = 13.8, 4.8 Hz) and 2.46 (1H, dd, J =
13.8, 11.2 Hz)/33.9 (C-7), 2.73 (1H, m)/44.2 (C-8), 3.67
(1H, dd, J = 8.4, 6.4 Hz) and 3.94 (1H, dd, J = 8.4, 6.8 Hz)/
73.8 (C-9), 6.92 (1H, d, J = 1.9 Hz)/110.9 (C-2′), 6.71 (1H,
d, J = 8.2 Hz)/116.0 (C-5′), 6.77 (1H, dd, J = 8.2, 1.9 Hz)/
119.8 (C-6′), 4.82 (1H, d, J = 6.8 Hz)/84.6 (C-7′), 2.48
(1H, m)/51.5 (C-8′), 3.83 (1H, obscured due to signal over-
lapping) and 3.97 (1H, dd, J = 9.6, 6.4)/68.8 (C-9′), 3.78 (3H,
s)/56.43 (OCH3-3), and 3.82 (3H, s)/56.47 (OCH3-3′), along
with those for a trans-feruloyl group and a β-glucopyranosyl
group. The preliminary assignments were supported by the
HMBC correlations of H2-7/C-1, C-2, C-6, C-9, H-7′/C-1′,
C-2′, C-6′, H-8′/C-1′, OCH3-3/C-3, and OCH3-3′/C-3′ and
the COSY correlations of H-8/H2-7, H-8′ and H-8′/H-7′,
H2-9′ (Figure 2). The connectivity of the β-glucopyranosyl
group to the lariciresinol nucleus at C-9′ was established by
the HMBC correlation from H-1″ to C-9′. Moreover, the
HMBC correlation of H2-6″/C-9‴ indicated the C-6″ location
of the trans-feruloyl group. The aromatic methoxy group
proton signals at δH 3.78 (OCH3-3), 3.82 (OCH3-3′), and
3.83 (OCH3-3‴) showed NOE correlations with H-2, H-2′,
and H-2‴, respectively, which confirmed their position at the
aromatic rings. Compound 17 exhibited a specific rotation of
Compound 2 was isolated as a light yellow, amorphous
powder with a molecular formula of C35H38O16, based on
13C NMR data and an [M + H]+ ion at m/z 715.2233 (calcd
715.2233) in the HRESIMS. The 1H and 13C NMR spectra of 2
also showed resonances for a di-trans-feruloyl catalpol unit, as
1
found for 1. However, in the H NMR spectrum of 2, the
methylene group protons at C-10 (δH 3.71 and 4.21) of the
iridoid skeleton were found upfield by 0.61 and 0.81 ppm,
respectively, when compared with those of 1. Downfield shifts
of 0.80 and 0.60 ppm were observed for the CH2-6′ resonances
of the β-glucopyranosyl group (δH 4.47 and 4.54), respectively
(Table 1). The observation suggested that one of the trans-
feruloyl groups was positioned at C-6′ in 2, instead of the C-10
location in 1. The C-6′ location of the trans-feruloyl group was
supported by the important HMBC correlations of H-6′a/C-9‴
and H-6′b/C-9‴ (Figure 1). Acid hydrolysis of 2 afforded
D-glucose. Therefore, the structure of 2 was defined as shown
for the new substance 6,6′-O-di-trans-feruloyl catalpol.
[α]22 +12 (c 0.1, MeOH), and a negative Cotton effect was
D
observed at 239 nm in the electronic circular dichroism (ECD)
curve. The absolute configuration of 17 was determined as
(8R,7′S,8′R) by comparison of the ECD data with those
of alangilignosides C and D.32−34 The configuration was
supported by the NOE correlations of H-7′/H2-9′ and H-9′b/
H-7. Acid hydrolysis of 17 gave D-glucose. Accordingly, the
structure of 17 was defined as shown for the new substance
(8R,7′S,8′R)-lariciresinol 9′-O-β-D-(6-O-trans-feruloyl)-
glucopyranoside.
Compound 10 was isolated as a light yellow, amorphous
powder with a molecular formula of C25H32O13, based on its
13C NMR data and the [M + Na]+ ion at m/z 563.1729 (calcd
563.1735) in the HRESIMS. Compound 10 exhibited closely
comparable 1H and 13C NMR data with 3, except for two meth-
ylene groups replacing the Δ3,4 olefinic group. The methylene
groups were observed at δH 3.59 (1H, td, J = 12.4, 2.0 Hz) and
3.90 (1H, obscured due to signal overlapping)/δC 62.9 (C-3),
and 1.54 (1H, br d, J = 14.4 Hz) and 1.80 (1H, m)/24.0 (C-4).
The observation suggested that 10 is a 3,4-dihydro derivative of
Compound 18 was isolated as a colorless, amorphous gum
with a molecular formula of C36H38O14, based on the 13C NMR
data and an [M + Na]+ ion at m/z 717.2159 (calcd 717.2154)
D
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1
Figure 2. Key H−1H COSY and HMBC correlations of 17−19.
1
in the HRESIMS. The H and 13C NMR spectra of 18 also
determining the absolute configurations at C-7 and C-8 of
the dihydrobenzofuran.36−39 A positive Cotton effect at 231 nm
observed in the ECD curve of 18 was indicative of a (7R,8S)-
configuration. The NOE correlation between H-6 and H-8 was
consistent with the trans configuration in the dihydrobenzofur-
an skeleton.35 Thus, the structure of 18 was defined as shown
for the new compound (2E)-3-[(2R,3S)-2,3-dihydro-2-(4-
hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-1-ben-
zofuran-5-yl]prop-2-enal 9-[6-[(2E)-3-(4-hydroxy-3-methoxy-
phenyl)-1-oxoprop-2-enyl]oxy]-β-D-glucopyranoside, which
was given the trivial name ovatoside A.
showed resonances for a β-(6-O-trans-feruloyl)glucopyranosyl
unit, as found for 17, and the HPLC sugar analysis after
acid hydrolysis indicated the presence of a D-glucose moiety.
Upon comparison of the H and 13C NMR data of these two
1
compounds, differences were observed in the signals of the
aglycones at C-1″. Four methine groups appeared at δH 5.69
(d, J = 6.0 Hz)/δC 90.2 (C-7), 3.81 (obscured due to signal
overlapping)/52.7 (C-8), 7.15 (br s)/114.2 (C-2′), and 7.20
(br s)/120.4 (C-6′) together with four quaternary carbon
signals at δC 129.7 (C-1′), 146.0 (C-3′), 152.8 (C-4′), and
130.9 (C-5′) (Table 2), indicating the presence of a 2,3,5,7-
tetrasubstituted 2,3-dihydrobenzofuran skeleton.35 In the
1H NMR spectrum, signals for a 1,3,4-trisubstituted phenyl
group were observed at δH 6.96 (1H, d, J = 2.0 Hz, H-2), 6.75
(1H, d, J = 8.0 Hz, H-5), and 6.85 (1H, dd, J = 8.0, 2.0 Hz,
H-6), and an extra aromatic methoxy group resonance appeared
Compound 19 was isolated as a colorless, amorphous gum
with a molecular formula of C37H42O14, based on its 13C NMR
data and the [M + Na]+ ion at m/z 733.2471 (calcd 733.2467)
1
in the HRESIMS. The H and 13C NMR spectra of 19 were
closely comparable to those of 18, except for signals for a
methylene and a methoxy group replacing those of a formyl
group. The methylene group appeared at δH 3.98 (2H, br d, J =
6.5 Hz)/δC 74.4 (C-9′) with an additional methoxy group at δH
3.32 (3H, s)/δC 58.0 (OCH3-9′) (Table 2). This observation
suggested the presence of a 3-methoxypropenyl group instead
of the ethylene formyl group in 18, which was supported by the
HMBC correlations of H-2′/C-7′, H-6′/C-7′, H-7′/C-9′, H-8′/
C-1′, and OCH3-9′/C-9′ and the COSY correlations of H-8′
with H-7′ and H2-9′ (Figure 2). The specific rotation was
at δH 3.88 (3H, s, OCH3-3′). The H and 13C NMR data
1
exhibited signals for an olefinic double bond at δH 7.42 (1H, d,
J = 15.9 Hz)/δC 155.9 (C-7′) and 6.58 (1H, dd, J = 15.9, 7.9
Hz)/127.1 (C-8′), which was conjugated to a formyl group
1
at δH 9.49 (1H, d, J = 7.9 Hz)/δC 196.1 (C-9′). These H and
13C NMR assignments were confirmed by further analysis of
2D NMR experiments. Oxygenated methylene protons at δH
4.08 (1H, dd, J = 10.2, 5.2 Hz, H-9a) and 3.91 (1H, dd, J =
10.2, 8.0 Hz, H-9b) showed HMBC correlations with C-7 and
C-5′ of the dihydrobenzofuran and C-1″ of the β-D-
glucopyranoside (Figure 2), suggesting that the β-D-glucopyr-
anosyl group is connected to the dihydrobenzofuran nucleus
through the methylene group. The positions of the 3,4-
disubstituted phenyl group and the ethylene formyl group
were determined as C-7 and C-1′, respectively, due to the
HMBC correlations of H-7/C-2, C-6, H-8/C-1, H-2′/C-7′, H-
6′/C-7′, and H-8′/C-1′. One of the three aromatic methoxy
groups at δH 3.88 was assigned at C-3 based on the HMBC
correlation between the aromatic methoxy protons and C-3′.
The other two aromatic methoxy groups also showed HMBC
correlations of OCH3-3/C-3 and OCH3-3‴/C-3‴, indicating
their C-3 and C-3′ location, respectively. Compound 18 gave a
measured as [α]22 −33 (c 0.1, MeOH). Compound 19 was
D
found to have a (2S,3R)-configuration according to its negative
Cotton effect at 230 nm in the ECD spectrum, opposite that of
18.36,38,39 Like 18, acid hydrolysis of 19 afforded D-glucose.
Therefore, the structure of 19 was defined as shown for the new
compound (2S,3R)-2,3-dihydro-2-(4-hydroxy-3-methoxyphen-
yl)-3-hydroxymethyl-7-methoxy-5-[(E)-3-methoxyprop-1-en-1-
yl]-1-benzofuran 9-[6-[(2E)-3-(4-hydroxy-3-methoxyphenyl)-
1-oxoprop-2-enyl]oxy]-β-D-glucopyranoside (ovatoside B).
Compound 20 was isolated as a light yellow, amorphous
powder with a molecular formula of C23H26O11, based on the
13C NMR data and the [M + Na]+ ion at m/z 501.1367 (calcd
501.1367) in the HRESIMS. Compound 20 exhibited
resonances for a trans-feruloyl group at δH 7.18 (1H, d, J =
1.8 Hz)/δC 111.7 (C-2″), 6.82 (1H, d, J = 8.2 Hz)/116.6
(C-5″), 7.07 (1H, dd, J = 8.2, 1.8 Hz)/124.2 (C-6″), 7.61
specific rotation of [α]22 −102 (c 0.1, MeOH). ECD Cotton
D
effects in the region 220−240 nm provide evidence for
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(1H, d, J = 16.0 Hz)/147.1 (C-7″), 6.36 (1H, d, J = 16.0 Hz)/
115.4 (C-8″), 3.90 (3H, s)/56.5 (OCH3-3″), and δC 169.0
(C-9″), along with a β-glucopyranosyl anomeric proton signal at
δH 4.69 (1H, d, J = 8.0 Hz, H-1′) (Table 3), as found for 17−19.
1
In the H NMR spectrum, an additional ABX spin system was
observed at δH 6.45 (1H, d, J = 2.7 Hz, H-3), 6.22 (1H, dd, J =
8.7, 2.7 Hz, H-5), and 6.96 (1H, d, J = 8.7 Hz, H-6), and an
extra aromatic methoxy group signal appeared at δH 3.79
(3H, s, OCH3-2). The β-glucopyranosyl group and aromatic
methoxy group were assigned at C-1 and C-2, respectively, on
the basis of the HMBC correlations of H-1′/C-1 and OCH3-2/
C-2 (Figure 3). The C-2 position of the aromatic methoxy
group was supported by the NOE correlation between OCH3-2
and H-3. The location of the trans-feruloyl group was
determined as C-6′ of the β-glucopyranosyl group according
to the HMBC connectivity between H2-6′ and C-9″. HPLC
determination after acid hydrolysis showed the presence of a
1
D-glucopyranosyl moiety. The H and 13C NMR data of 20
were also compared with those reported for dunalianoside B,35
which has a hydroxy group at C-2 instead of the methoxy group
in 20. Therefore, the structure for 20 was defined as shown for
the new substance 4-hydroxy-2-methoxybenzene 1-[6-[(2E)-3-
(4-hydroxy-3-methoxyphenyl)-1-oxoprop-2-enyl]oxy]-β-D-glu-
copyranoside (ovatoside C).
Compound 21 was isolated as a white, amorphous powder
with a molecular formula of C24H28O11, based on both the 13C
NMR data and the [M + Na]+ ion at m/z 515.1526 (calcd
515.1524) in the HRESIMS. The 1H NMR spectrum of 21 was
similar to that of 20, except for the presence of a singlet signal
at δH 4.43 (2H, H2-7) (Table 3). The oxygenated methylene
protons showed HMBC correlations with C-3, C-4, and C-5
(Figure 3), as well as the NOE correlations with H-3 and H-5.
The observation indicated the location of the hydroxymethyl
group at C-4. The sugar moiety was determined as D-glucose.
Thus, the structure of 21 was defined as shown for the new
compound 4-hydroxymethyl-2-methoxybenzene 1-[6-[(2E)-3-
(4-hydroxy-3-methoxyphenyl)-1-oxoprop-2-enyl]oxy]-β-D-glu-
copyranoside (ovatoside D).
Compound 22 was isolated as a white, amorphous powder
with a molecular formula of C24H32O11, using the 13C NMR
data and a [M + Na]+ ion at m/z 519.1830 (calcd 519.1837) in
the HRESIMS. The 1H and 13C NMR spectra of 22 also showed
resonances for a trans-feruloyl group and a β-glucopyranosyl
1
group, similar to those of 20. When comparing the H NMR
data of these two compounds, the signals for the 4-hydroxy-2-
methoxyphenyl group at C-1′ of the β-glucopyranosyl group in
20 were absent. Instead, the 1H NMR spectrum of 22 exhibited
resonances for six methylenes at δH 1.98 and 1.83 (each 2H, m,
H2-2 and H2-6), 2.63 and 2.15 (each 2H, m, H2-3 and H2-4),
1.90 (2H, td, J = 6.6, 1.5 Hz, H2-7), and 4.05 and 3.80 (each 1H,
dt, J = 10.4, 6.6 Hz, H2-8) (Table 3). In the 13C NMR spectrum,
quaternary carbon signals appeared at δC 70.4 (C-1) and
1
214.9 (C-4). The observed H and 13C NMR signals were
assignable to a 1-ethyl-1-hydroxycyclohexan-4-one unit,40,41
based on the HMBC correlations of H-2b and H-6b/C-4, H-3a
and H-5a/C-1, C-4, H-3b and H-5b/C-4, H2-7/C-1, C-2,
C-6, and H2-8/C-1 and the COSY correlations of H-3a and
H-5a/H2-2 and H2-6, and H2-7/H2-8 (Figure 3). The HMBC
correlation from H2-8 to C-1′ indicated that the 1-ethyl-1-
hydroxycyclohexan-4-one moiety is linked to the β-glucopyr-
anosyl group at C-1′. The D-glucopyranosyl configuration was
identified based on HPLC analysis following acid hydrolysis.
Therefore, the structure of 22 was defined as shown for the new
G
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H
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1
Figure 3. Key H−1H COSY and HMBC correlations of 20−22 and 24.
a
Table 4. Peroxynitrite (ONOO−)-Scavenging Activities of Compounds 1−29
compound
IC50 (μM)
compound
IC50 (μM)
compound
IC50 (μM)
1
2
0.88 0.05
1.1 0.15
1.6 0.02
13.6 0.47
78.5 2.44
>100
11
12
13
14
15
16
17
18
19
20
58.4 1.95
2.0 0.78
3.9 0.84
39.5 0.67
8.3 1.93
76.7 1.32
0.81 0.09
0.79 0.11
0.34 0.02
0.48 0.07
21
0.17 0.03
1.02 0.06
0.14 0.02
4.5 0.36
15.7 0.39
36.2 1.07
8.6 2.46
7.7 0.91
3.5 1.03
3.4 0.34
22
3
23
4
24
5
25
6
26
7
36.4 1.54
6.0 0.01
>100
27
8
28
29
9
10
2.2 0.10
DL-penicillamine
a
Inhibitory activities of ONOO− from decomposition of SIN-1 are expressed as IC50 values. Data were described as mean SD (obtained from
three different experiments). DL-Penicillamine was used as a positive control.
compound 1-ethyl-1-hydroxycyclohexan-4-one 8-[6-[(2E)-3-
(4-hydroxy-3-methoxyphenyl)-1-oxoprop-2-enyl]oxy]-β-D-glu-
copyranoside (ovatoside E).
D-apiose based on HPLC analysis of the thiocarbamoyl-thiazo-
1
lidine derivatives. The H and 13C NMR data for 24 were also
compared with those for darendoside A (25),19 in which the
trans-4-coumaroyl group at C-5″ was absent. Thus, the
structure of 24 was defined as shown for the new substance
1-ethyl-4-hydroxybenzene 8-[5-[(2E)-3-(4-hydroxyphenyl)-1-
oxoprop-2-enyl]oxy]-β-D-apiofuranosyl-(1→2)-β-D-glucopyra-
noside (ovatoside F).
Compound 24 was isolated as a white, amorphous gum with
a molecular formula of C28H34O13, based on its 13C NMR data
and a [M + Na]+ ion at m/z 601.1897 (calcd 601.1892) in the
1
HRESIMS. In the H NMR spectrum of 24, two AA′BB′ spin
systems appeared at δH 7.02 (2H, d, J = 8.6 Hz, H-2 and H-6),
6.64 (2H, d, J = 8.6 Hz, H-3 and H-5), 7.41 (2H, d, J = 8.6 Hz,
H-2‴ and H-6‴), and 6.79 (2H, d, J = 8.6 Hz, H-3‴ and H-5‴)
(Table 2). Two coupled doublet signals at δH 7.64 (1H, d,
J = 16.0 Hz, H-7‴) and 6.34 (1H, d, J = 16.0 Hz, H-8‴) were
attributed to an olefinic double bond conjugated to a carbonyl
group (δC 169.1). The presence of a β-glucopyranosyl group
and a β-apiofuranosyl group was deduced from their character-
istic anomeric proton signals at δH 4.328 (1H, d, J = 7.6 Hz,
H-1′) and 5.43 (1H, d, J = 1.2 Hz, H-1″), respectively.16,19
Additionally, two methylene proton signals resonated at δH 2.79
(2H, br t, J = 7.6 Hz, H2-7) and 4.00 and 3.65 (each 1H, m, H-
8a and H-8b) and were coupled to each other in the COSY
spectrum of 24 (Figure 3). The HMBC correlations of
H2-7/C-2 and C-6 and H2-8/C-1, C-1′ suggested the presence
of a 4-hydroxyphenethyl group attached to C-1′ of the
β-glucopyranosyl group. The anomeric proton signal of the
β-apiofuranosyl group showed the HMBC correlation with C-2′
of the β-glucopyranosyl group, indicating the connectivity of
two sugar moieties. Moreover, a trans-4-coumaroyl group was
assignable at C-5″ of the β-apiofuranosyl group according to
the HMBC correlations of H2-5″/C-9‴, H-7‴/C-2‴ and C-6‴,
C-9‴, and H-8‴/C-1‴, C-9‴. Acid hydrolysis of 24 gave
a mixture of sugars, which were identified as D-glucose and
ONOO−-scavenging activity was evaluated for all iso-
lates produced by 3-morpholinosydnonimine (SIN-1),8 using
DL-penicillamine as a positive control (Table 4).42 Of the
compounds tested, 27 had the effect of scavenging ONOO−.
Two compounds were inactive, namely, 6 and 9. In particular,
12 compounds, 1−3, 10, 12, and 17−23 (IC50 0.88, 1.1, 1.6,
2.2, 2.0, 0.81, 0.79, 0.34, 0.48, 0.17, 1.0, and 0.14 μM,
respectively), showed more potent activity than DL-penicill-
amine (IC50 3.4 μM). Compounds 13 and 29 also exhibited
ONOO−-scavenging effects similar to the positive control
with IC50 values of 3.9 and 3.5 μM, respectively. In the present
study, the feruloyl group was commonly found in the com-
pounds with significant activity (1−3, 10, 12, 17−23, and 29),
suggesting that this functionality is important for ONOO−-
scavenging activity.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a P-1010 polarimeter. UV spectra were recorded on
a U-3000 spectrophotometer. ECD measurements were performed
using a JASCO J-810 spectropolarimeter. 1D and 2D NMR spectra
were recorded on a Varian Unity Inova 400 MHz FT-NMR
instrument with tetramethylsilane (TMS) as internal standard. Mass
spectrometry was performed on a Waters Acquity UPLC system
I
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coupled to a Micromass Q-Tof Micro mass spectrometer and Agilent
6220 Accurate-Mass TOF LC/MS system. Silica gel (230−400 mesh,
Merck, Germany), RP-18 (YMC gel ODS-A, 120 Å, S-150 μm, YMC
Co., Japan), and Sephadex LH-20 (GE Healthcare Bio-Science AB,
Uppsala, Sweden) were used for column chromatography (CC). Thin-
layer chromatographic (TLC) analysis was performed on Kieselgel
60 F254 (silica gel, 0.25 mm layer thickness, Merck, Germany) and
RP-18 F254s (Merck, Germany) plates, with visualization under UV
light (254 and 365 nm) and 10% (v/v) sulfuric acid spray followed by
heating (120 °C, 5 min). A YMC-Pack Pro C18 column (5 μm,
250 mm × 20 mm i.d.) was used for preparative HPLC, as performed
on an Acme 9000 system (Young Lin, Korea). Analytical HPLC was
conducted on a Phenomenex Luna C18 column (5 μm, 250 mm ×
4.6 mm i.d.) equipped with a Waters 1525 binary pump and a Waters
996 photodiode array detector (Milford, MA, USA). D-Glucose,
L-glucose, and D-apiose were purchased from Sigma-Aldrich (St. Louis,
MO, USA). L-Cysteine methyl ester hydrochloride and o-tolylisothio-
cyanate were purchased from TCI Chemicals (Tokyo, Japan).
Plant Material. The stem bark of C. ovata was collected at the
Medicinal Plant Garden, College of Pharmacy, Ewha Womans
University, in December 2013 and identified by Professor Je-hyun
Lee (College of Oriental Medicine, Dongguk University). A voucher
specimen (no. EA343) has been deposited at the Natural Product
Chemistry Laboratory, College of Pharmacy, Ewha Womans
University.
Extraction and Isolation. The dried and chopped stem bark of
C. ovata (8 kg) was extracted with MeOH (4 × 18 L) at room
temperature overnight. After removing the solvent under reduced
pressure, the concentrated MeOH extract (2.6 kg) was suspended in
distilled H2O (2 L) and then partitioned with hexanes (5 × 3 L),
EtOAc (10 × 3 L), and BuOH (5 × 3 L), sequentially, to afford
hexanes- (30 g), EtOAc- (250 g), and BuOH (1.3 kg)-soluble extracts
with a residual aqueous extract (1 kg). The EtOAc extract was
subjected to CC over silica gel by elution with gradient mixtures of
CH2Cl2−MeOH (999:1 → 1:4) to afford eight fractions (F1−F8).
Fraction F4 (14 g), eluted with CH2Cl2−MeOH (23:1), was subjected
to RP-18 CC using a MeOH−H2O gradient solvent system (1:4 →
7:3) to yield 15 subfractions (F4.01−F4.15). Subfraction F4.12
(500 mg) was further purified by RP-18 CC (MeOH−H2O, 3:2) and
Sephadex LH-20 CC (MeOH 100%), sequentially, to afford 29
(1 mg). Fraction F5 (30 g), eluted with CH2Cl2−MeOH (9:1) from
the first separation, was chromatographed over silica gel using gradient
mixtures of CHCl3−MeOH (49:1 → 1:1) to obtain 15 subfractions
(F5.01−F5.15). Subfraction F5.08 (500 mg) was subjected to RP-18
CC, eluted with a MeOH−H2O gradient solvent system (2:3 → 11:9),
to yield 19 subfractions (F5.08.01−F5.08.19). Compound 19 (3 mg)
was purified from subfraction F5.08.17 (4 mg) by preparative HPLC
with MeOH−H2O (4:1, 2 mL/min, tR 36.8 min). Subfraction F5.09
(300 mg) was fractionated by RP-18 CC using gradient mixtures
of MeOH−H2O (2:3 → 3:2) as eluent, affording 15 subfractions
(F5.09.01−F5.09.15). Subfraction F5.09.11 (12 mg) was further puri-
fied using preparative HPLC with MeOH−H2O (3:2, 2 mL/min) to
obtain 18 (4 mg, tR 45.8 min). Subfraction F5.10 (2 g) was subjected
to RP-18 CC, eluted by a MeOH−H2O gradient solvent system
(2:3 → 9:1), to yield 1 (100 mg) and 2 (180 mg) with 24 subfractions
(F5.10.01−F5.10.24). Subfraction F5.10.06 (400 mg) was chromato-
graphed over Sephadex LH-20 using MeOH (100%) and then purified
by separation on preparative HPLC with MeOH−H2O (11:9, 2 mL/min)
to give 22 (17 mg, tR 34.5 min). Subfraction F5.10.08 (100 mg) was
chromatographed using Sephadex LH-20 with MeOH (100%) to yield
20 (30 mg). Subfraction F5.10.13 (50 mg) was separated by Sephadex
LH-20 CC (MeOH 100%) and then further purified by preparative
HPLC with MeOH−H2O (3:2, 2 mL/min) to afford 23 (10 mg,
tR 42.8 min). A part of subfraction F5.10.16 (70 mg) was subjected
to preparative HPLC with MeOH−H2O (3:2, 2 mL/min) to afford
17 (5 mg, tR 53.9 min). Subfraction F5.12 (4 g) was separated by
RP-18 CC with a MeOH−H2O gradient solvent system (2:3 → 1:1)
as eluent to give 5 (240 mg), 9 (50 mg), and 21 (9 mg) with 15 sub-
fractions (F5.12.01−F5.12.15). Subfraction F5.12.13 (1.4 g) was
purified by separation over RP-18 CC with MeOH−H2O (3:2) to
yield 7 (80 mg). Fraction F6 (130 g), eluted with CH2Cl2−MeOH
(4:1) from the first separation, was subjected to silica gel CC using a
CHCl3−MeOH−H2O gradient solvent system (90:10:1 → 15:10:2)
for elution to obtain 13 subfractions (F6.01−F6.13). A part (5 g) of
subfraction F6.08 (40 g) was fractionated by RP-18 CC with gradient
mixtures of MeOH−H2O (1:4 → 1:1) as eluent to give 3 (2.4 g) and 8
(2 mg) with 17 subfractions (F6.08.01−F6.08.17). Subfraction
F6.08.08 (5 mg) was further purified by separation on preparative
HPLC with MeOH−H2O (11:9, 2 mL/min) to yield 10 (2 mg, tR 39.6
min). Subfraction F6.11 (5 g) was chromatographed over an RP-18
column by elution with a MeOH−H2O gradient solvent system
(1:9 → 1:1), affording 6 (120 mg) with 12 subfractions (F6.11.01−
F6.11.12). Subfraction F6.08.11.06 (200 mg) was further separated by
RP-18 CC using MeOH−H2O (11:9) as eluent to give 12 (60 mg)
and 15 (1 mg). Subfraction F6.08.11.08 (160 mg), eluted with
MeOH−H2O (1:1), was subjected to RP-18 CC by elution with
MeOH−H2O (1:1) to afford 4 (18 mg) and two additional
subfractions, which were further purified using preparative HPLC to
yield 13 (2 mg, MeOH−H2O, 11:9; 2 mL/min, tR 29.5 min) and 27
(20 mg, MeOH−H2O, 2:3; 2 mL/min, tR 37.1 min), respectively.
Subfraction F6.12 (1.4 g) was separated by RP-18 CC using a
MeOH−H2O gradient solvent system (1:9 → 1:1) as eluent, affording
18 subfractions (F6.12.01−F6.12.18). Subfractions F6.12.04 (13 mg)
and F6.12.05 (12 mg) were chromatographed over Sephadex LH-20
with MeOH (100%) to give 25 (6 mg) and 28 (5 mg), respectively.
Compounds 11 (1 mg) and 14 (3 mg) were separated from sub-
fraction F6.12.07 (17 mg) by preparative HPLC with MeOH−H2O
(1:1, 2 mL/min, tR 11: 49.9, 14: 52.8 min). Subfractions F6.12.08
(30 mg), F6.12.12 (100 mg), and F6.12.15 (80 mg) were further puri-
fied by preparative HPLC using MeOH−H2O (11:9, 2 mL/min) to
afford 16 (5 mg, tR 36.9 min), 26 (70 mg, tR 32.3 min), and 24 (20 mg,
tR 40.9 min), respectively.
6,10-O-Di-trans-feruloyl catalpol (1): light yellow, amorphous
powder; [α]22 −135 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218
D
1
(4.48), 236 (4.40), 298 (4.49), 327 (4.64) nm; H NMR (400 MHz,
methanol-d4) and 13C NMR (100 MHz, methanol-d4) data, see Table 1;
HRESIMS m/z 715.2242 [M + H]+ (calcd for C35H39O16, 715.2233).
6,6′-O-Di-trans-feruloyl catalpol (2): light yellow, amorphous
powder; [α]22 −132 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
D
217 (4.47), 237 (4.39), 298 (4.46), 327 (4.63) nm; 1H NMR
(400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4)
data, see Table 1; HRESIMS m/z 715.2233 [M + H]+ (calcd for
C35H39O16 715.2233).
3,4-Dihydro-6-O-trans-feruloyl catalpol (10): light yellow, amor-
phous powder; [α]22D −90 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
218 (4.24), 234 (4.14), 297 (4.17), 327 (4.33) nm; 1H NMR
(400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4)
data, see Table 1; HRESIMS m/z 563.1729 [M + Na]+ (calcd for
C25H32O13Na, 563.1735).
(8R,7′S,8′R)-Lariciresinol 9′-O-β-D-(6-O-trans-feruloyl)-
glucopyranoside (17): light yellow, amorphous gum; [α]22 +12
D
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.33), 230 (4.33), 287
(4.13), 327 (4.21) nm; ECD (MeOH) λmax (Δε) 239 (−3.25), 289
(−2.86) nm; 1H NMR (400 MHz, methanol-d4) and 13C NMR
(100 MHz, methanol-d4) data, see Table 2; HRESIMS m/z 721.2473
[M + Na]+ (calcd for C36H42O14Na, 721.2467).
Ovatoside A (18): colorless, amorphous gum; [α]22 −102 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 230 (4.45), 290 (4.26), 329
(4.49) nm; ECD (MeOH) λmax (Δε) 231 (+1.50), 242 (−2.36), 288
(+2.58) nm; 1H NMR (400 MHz, methanol-d4) and 13C NMR
(100 MHz, methanol-d4) data, see Table 2; HRESIMS m/z 717.2159
[M + Na]+ (calcd for C36H38O14Na, 717.2154).
Ovatoside B (19): colorless, amorphous gum; [α]22 −33 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 218 (4.15), 281 (3.91), 320
(3.79) nm; ECD (MeOH) λmax (Δε) 230 (−1.80), 239 (+1.53), 290
(−1.89) nm; 1H NMR (400 MHz, methanol-d4) and 13C NMR
(100 MHz, methanol-d4) data, see Table 2; HRESIMS m/z 733.2471
[M + Na]+ (calcd for C37H42O14Na, 733.2467).
Ovatoside C (20): light yellow, amorphous powder; [α]22 −58
D
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.34), 231 (4.26), 295
J
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Article
(4.18), 326 (4.31) nm; 1H NMR (400 MHz, methanol-d4) and
13C NMR (100 MHz, methanol-d4) data, see Table 3; HRESIMS
m/z 501.1367 [M + Na]+ (calcd for C23H26O11Na, 501.1367).
Notes
The authors declare no competing financial interest.
Ovatoside D (21): white, amorphous powder; [α]22 −38 (c 0.1,
D
ACKNOWLEDGMENTS
■
MeOH); UV (MeOH) λmax (log ε) 219 (4.22), 231 (4.16), 285
(3.98), 298 (4.03), 326 (4.19) nm; 1H NMR (400 MHz, methanol-d4)
and 13C NMR (100 MHz, methanol-d4) data, see Table 3; HRESIMS
m/z 515.1526 [M + Na]+ (calcd for C24H28O11Na, 515.1524).
This research was supported by Ewha Womans University.
REFERENCES
Ovatoside E (22): white, amorphous powder; [α]22 −15 (c 0.1,
■
D
(1) Inouye, H.; Okuda, T.; Hirata, Y.; Nagakura, N.; Yoshizaki, M.
Chem. Pharm. Bull. 1967, 15, 782−792.
MeOH); UV (MeOH) λmax (log ε) 218 (4.12), 234 (4.04), 298
(4.06), 325 (4.20) nm; 1H NMR (400 MHz, methanol-d4) and
13C NMR (100 MHz, methanol-d4) data, see Table 3; HRESIMS
m/z 519.1830 [M + Na]+ (calcd for C24H32O11Na, 519.1837).
(2) Pae, H. O.; Oh, G. S.; Choi, B. M.; Shin, S.; Chai, K. Y.; Oh, H.;
Kim, J. M.; Kim, H. J.; Jang, S. I.; Chung, H. T. J. Ethnopharmacol.
2003, 88, 287−291.
Ovatoside F (24): white, amorphous gum; [α]22 −56 (c 0.1,
D
(3) Young, H. S.; Kim, M. S.; Park, H. J.; Chung, H. Y.; Choi, J. S.
Arch. Pharmacal Res. 1992, 15, 322−327.
MeOH); UV (MeOH) λmax (log ε) 212 (4.39), 224 (4.43), 314
(4.50) nm; 1H NMR (400 MHz, methanol-d4) and 13C NMR
(100 MHz, methanol-d4) data, see Table 2; HRESIMS m/z 601.1897
[M + Na]+ (calcd for C28H34O13Na, 601.1892).
(4) Oh, H.; Pae, H.-O.; Oh, G.-S.; Lee, S. Y.; Chai, K.-Y.; Song, C. E.;
Kwon, T.-O.; Chung, H.-T.; Lee, H.-S. Planta Med. 2002, 68, 685−
689.
Acid Hydrolysis and Absolute Configuration Determination
of Sugar Moieties. The absolute configurations of sugar moieties
were determined using a HPLC-UV-based method.28−31 Compounds
1, 2, 10, 17−22, and 24 (each 1−2 mg) were hydrolyzed in the
presence of 2 M HCl (dioxane−H2O, 1:1) at 80 °C for 2 h.
The dioxane was removed from each reaction mixture under vacuum,
and then EtOAc was used for extraction. The aqueous layer was
neutralized with Amberlite IRA-67 (OH− form), dried under a vacuum
evaporator, and dissolved in anhydrous pyridine (0.5 mL) with
addition of L-cysteine methyl ester hydrochloride (2 mg). After the
reaction mixture was heated at 60 °C for 1.5 h, o-tolylisothiocyanate
(50 μL) was added and the mixture was kept at 60 °C for 1 h. The
reaction product was directly analyzed by RP-18 HPLC (MeCN−
H2O, 1:3; 1 mL/min). The monosaccharides D-glucose and D-apiose in
1, 2, 10, 17−22, and 24 were identified based on comparison of the
retention times with those of authentic samples (tR: D-glucose 20.9
min, L-glucose 19.0 min, and D-apiose 36.7 min).
(5) Piaz, F. D.; Vassallo, A.; Temraz, A.; Cotugno, R.; Belisario, M.
A.; Bifulco, G.; Chini, M. G.; Pisano, C.; De Tommasi, N.; Braca, A. J.
Med. Chem. 2013, 56, 1583−1595.
(6) Aktan, F. Life Sci. 2004, 75, 639−653.
(7) Reuter, S.; Gupta, S. C.; Chaturvedi, M. M.; Aggarwal, B. B. Free
Radical Biol. Med. 2010, 49, 1603−1616.
(8) Szabo, C. Shock 1996, 6, 79−88.
(9) Yang, G.; Choi, C.-H.; Lee, K.; Lee, M.; Ham, I.; Choi, H.-Y. J.
Ethnopharmacol. 2013, 145, 416−423.
(10) Stuppner, H.; Wagner, H. Planta Med. 1989, 55, 467−469.
(11) El-Naggar, S. A.; Doskotch, R. W. J. Nat. Prod. 1980, 43, 524−
526.
(12) Houghton, P. J.; Hikino, H. Planta Med. 1989, 55, 123−126.
(13) Bobbitt, J. M.; Spiggle, D. W.; Mahboob, S.; von Philipsborn,
W.; Schmid, H. Tetrahedron Lett. 1962, 3, 321−329.
(14) Han, X. H.; Lee, C.; Lee, J. W.; Jin, Q.; Jang, H.; Lee, H. J.; Lee,
D.; Lee, S.-J.; Hong, J. T.; Lee, M. K.; Hwang, B. Y. Helv. Chim. Acta
2015, 98, 381−385.
Measurement of ONOO−-Scavenging Activity. The ONOO−-
scavenging ability was measured by monitoring the oxidation of
dihydrorhodamine 123 (DHR 123). The method reported by Kooy
et al. was slightly modified.43 A stock solution of DHR 123 (5 mM) in
dimethylformamide was purged with N2 and stored at −80 °C.
A working solution of DHR 123 (5 μM) was diluted from the stock
solution and immediately placed on ice and kept in the dark before
use. The buffer was composed of 90 mM NaCl, 50 mM Na3PO4,
5 mM KCl (pH 7.4), and 100 μM diethylenetriaminepentaacetic acid.
All materials for the buffer were prepared with high-quality deionized
H2O, purged with N2, and kept on ice. Oxidation of DHR 123 was
measured on an FL 500 microplate fluorescence reader (Bio-Tek
Instruments Inc., Winooski, VT, USA) with excitation and emission
wavelengths of 480 and 530 nm, respectively. DHR 123 was oxidized
gradually by decomposition of SIN-1. The background and final
fluorescent intensities were measured 5 min after treatment with and
without SIN-1 (10 μM). The results are expressed as means SD of
three independent experiments for the final fluorescence intensity
minus background fluorescence. The effects were described by the
percent inhibition of DHR 123 oxidation, and DL-penicillamine was
used as a positive control.
(15) Joshi, K. C.; Prakash, L.; Singh, L. B. Phytochemistry 1975, 14,
1441−1442.
(16) Iwagawa, T.; Hamada, T.; Kurogi, S.; Hase, T.; Okubo, T.; Kim,
M. Phytochemistry 1991, 30, 4057−4060.
(17) Nishimura, H.; Sasaki, H.; Morota, T.; Chin, M.; Mitsuhashi, H.
Phytochemistry 1989, 28, 2705−2709.
(18) Shimomura, H.; Sashida, Y.; Adachi, T. Phytochemistry 1987, 26,
2363−2366.
(19) Calis, I.; Saracoglu, I.; Basaran, A. A.; Sticher, O. Phytochemistry
1993, 32, 1621−1623.
(20) Takeda, Y.; Kinugawa, M.; Masuda, T.; Honda, G.; Otsuka, H.;
Sezik, E.; Yesilada, E. Phytochemistry 1999, 51, 323−325.
(21) Ralph, J.; Quideau, S.; Grabber, J. H.; Hatfield, R. D. J. Chem.
Soc., Perkin Trans. 1 1994, 3485−3498.
(22) Machida, K.; Ando, M.; Yaoita, Y.; Kakuda, R.; Kikuchi, M.
Chem. Pharm. Bull. 2001, 49, 732−736.
(23) Bobbitt, J. M.; Spiggle, D. W.; Mahboob, S.; Schmid, H.; von
Philipsborn, W. J. Org. Chem. 1966, 31, 500−506.
(24) Bobbitt, J. M.; Kiely, D. E.; Lam, A. Y.; Snyder, E. I. J. Org.
Chem. 1967, 32, 1459−1461.
(25) Jia, Q.; Hong, M. F.; Minter, D. J. Nat. Prod. 1999, 62, 901−903.
(26) Huang, S. X.; Zhou, Y.; Nie, Q. J.; Ding, L. S.; Peng, S. L. J.
Asian Nat. Prod. Res. 2006, 8, 259−263.
(27) Fukuyama, Y.; Minoshima, Y.; Kishimoto, Y.; Chen, I.-S.;
Takahashi, H.; Esumi, T. J. Nat. Prod. 2004, 67, 1833−1838.
(28) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I.
Chem. Pharm. Bull. 2007, 55, 899−901.
(29) Muhit, M. A.; Umehara, K.; Mori-Yasumoto, K.; Noguchi, H. J.
Nat. Prod. 2016, 79, 1298−1307.
(30) Ge, Y.-W.; Tohda, C.; Zhu, S.; He, Y.-M.; Yoshimatsu, K.;
Komatsu, K. J. Nat. Prod. 2016, 79, 1834−1841.
(31) Odonbayar, B.; Murata, T.; Batkhuu, J.; Yasunaga, K.; Goto, R.;
Sasaki, K. J. Nat. Prod. 2016, 79, 3065−3071.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.7b00139.
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1D and 2D NMR and HRESIMS spectra of compounds
1, 2, 10, 17−22, and 24 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel (E. K. Seo): 82-2-3277-3047. Fax: +82-2-3277-3051.
E-mail: yuny@ewha.ac.kr.
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DOI: 10.1021/acs.jnatprod.7b00139
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Article
(32) Warashina, T.; Shikata, K.; Miyase, T.; Fujii, S.; Noro, T. Chem.
Pharm. Bull. 2008, 56, 1159−1163.
(33) Yuasa, K.; Ide, T.; Otsuka, H.; Ogimi, C.; Hirata, E.; Takushi, A.;
Takeda, Y. Phytochemistry 1997, 45, 611−615.
(34) Achenbach, H.; Benirschke, M.; Torrenegra, R. Phytochemistry
1997, 45, 325−335.
(35) Zhao, P.; Tanaka, T.; Hirabayashi, K.; Zhang, Y.-J.; Yang, C.-R.;
Kouno, I. Phytochemistry 2008, 69, 3087−3094.
(36) Matsuda, N.; Sato, H.; Yaoita, Y.; Kikuchi, M. Chem. Pharm.
Bull. 1996, 44, 1122−1123.
(37) Machida, K.; Takano, M.; Kakuda, R.; Yaoita, Y.; Kikuchi, M.
Chem. Pharm. Bull. 2002, 50, 669−671.
(38) Feng, W.-S.; Zang, X.-Y.; Zheng, X.-K.; Wang, Y.-Z.; Chen, H.;
Li, Z. J. Asian Nat. Prod. Res. 2010, 12, 557−561.
(39) Lemiere, G.; Gao, M.; De Groot, A.; Dommisse, R.; Lepoivre, J.;
Pieters, L.; Buss, V. J. Chem. Soc., Perkin Trans. 1 1995, 1775−1779.
(40) Liu, S.-S.; Zhou, T.; Zhang, S.-W.; Xuan, L.-J. Helv. Chim. Acta
2009, 92, 1070−1079.
(41) Li, C.; Dai, Y.; Zhang, S.-X.; Duan, Y.-H.; Liu, M.-L.; Chen, L.-
Y.; Yao, X.-S. Phytochemistry 2014, 104, 105−113.
(42) Althaus, J. S.; Oien, T. T.; Fici, G. J.; Scherch, H. M.; Sethy, V.
H.; VonVoigtlander, P. F. Res. Commun. Chem. Pathol. Pharmacol.
1994, 83, 243−254.
(43) Kooy, N. W.; Royall, J. A.; Ischiropoulos, H.; Beckman, J. S. Free
Radical Biol. Med. 1994, 16, 149−156.
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