Phenolic compounds from caesalpinia sappan heartwood and their anti-inflammatory activity

Article abstract of DOI:10.1021/np3003673

Four new phenolic compounds, caesalpiniaphenols A-D (1-4), together with eight known compounds were isolated from Caesalpinia sappan heartwood. The chemical structures were established mainly by NMR, MS, ECD, and Mosher's method. Compounds 4, 5, and 7 sho

Full text of DOI:10.1021/np3003673

Article
pubs.acs.org/jnp
Phenolic Compounds from Caesalpinia sappan Heartwood and
Their Anti-inflammatory Activity
To Dao Cuong,† Tran Manh Hung,‡ Jin Cheol Kim,§ Eun Hee Kim,⊥ Mi Hee Woo,† Jae Sue Choi,∥
Jeong Hyung Lee,∇ and Byung Sun Min*,†
†College of Pharmacy, Catholic University of Daegu, Gyeongsan, Gyeongbuk 712-702, Korea
‡Faculty of Chemistry, University of Natural Science, National University of Hochiminh City, Hochiminh City, Vietnam
§Korea Research Institute of Chemical Technology, Daejeon 305-600, Korea
⊥Korea Basic Science Institute, Daejeon 305-806, Korea
∥Faculty of Food Science and Biotechnology, Pukyung National University, Busan 608-737, Korea
∇College of Natural Science, Kangwon National University, Kangwon 200-701, Korea
S
* Supporting Information
ABSTRACT: Four new phenolic compounds, caesalpiniaphenols
A−D (1−4), together with eight known compounds were iso-
lated from Caesalpinia sappan heartwood. The chemical struc-
tures were established mainly by NMR, MS, ECD, and Mosher’s
method. Compounds 4, 5, and 7 showed weak inhibitory activity
against the LPS-induced NO production in macrophage
RAW264.7 cells with IC50 values of 12.2, 3.5, and 5.7 μM, respectively.
nflammation is a central feature of many pathophysiological
conditions in response to tissue injury and host defenses
Among the protosappanin derivatives, protosappanin B and isopro-
tosappanin B, 10-O-methyprotosappanin B and 10-O-methyliso-
protosappanin B, and protosappanin E1 and protosappanin E2
occur as pairs of epimers. The homoisoflavonoid epimers sappanol
and episappanol, 4-O-methylsappanol and 4-O-methylepisappanol,
and 3′-O-methylsappanol and 3′-O-methylepisappanol have been
successively isolated.9
Choi et al. confirmed that an ethanol extract of C. sappan
ameliorates hypercholesterolemia in C57BL/6 mice and
suppresses inflammatory responses in human umbilical vein
endothelial cells (HUVECs) through an anti-oxidant mecha-
nism.10 Other compounds with a sappanchalcone skeleton show
anti-inflammatory,11,12 antibacterial, and anti-influenza activity.13
In particular, brazilein, one of the main compounds of C. sappan,
shows hepatoprotective,14 anti-oxidant,11 and neuroprotective
activities.15 To further study the inhibitory effect of LPS-induced
NO production in macrophage RAW264.7 cells, fractionation of
the EtOAc-soluble fraction resulted in the isolation of four new
phenolic compounds (1−4), together with eight known com-
pounds (5−12). This study describes the isolation and structural
elucidation of these compounds, as well as the evaluation of their
inhibitory effects on LPS-induced NO production in macro-
phage RAW264.7 cells.
I
against invading microbes. Chronic inflammation and infections
lead to the up-regulation of a series of enzymes and signaling
proteins in affected tissues and cells. Proinflammatory cells,
mainly activated macrophages, mediate most of the cellular
and molecular pathophysiology of inflammation by producing
cytokines and other pro-inflammatory molecules, including
prostaglandins, enzymes, and free radicals such as NO. These
enzymes are known to be involved in the pathogenesis of many
chronic diseases including multiple sclerosis, Parkinson’s and
Alzheimer’s diseases, and colon cancer.1,2 NO is produced by
iNOS in macrophages, hepatocytes, and renal cells, under
stimulation of lipopolysaccharide (LPS), tumor necrosis factor-
alpha (TNF-α), interleukin-1, or interferon-gamma.3 After
exposure to stimulants such as LPS from Gram-negative bacteria
or lipoteichoic acid from Gram-positive bacteria, iNOS can be
induced to trigger several disadvantageous cellular responses,
including inflammation.4 Therefore, NO production induced by
LPS through iNOS can reflect the degree of inflammation, and a
change in NO level through inhibition of iNOS enzyme activity
or iNOS induction provides a means of assessing the effect of
agents on the inflammatory process.
Caesalpinia sappan (Leguminosae) is distributed in southeast
Asia, and its heartwood, sappan lignum, is famous as a red
dyestuff. Sappan lignum is also used as an herbal medicine for
inflammation and to improve blood circulation,5 as well as for its
anti-influenza,6 anti-allergic,7 and neuroprotective activities.8
Many reports have shown that the main compounds in sappan
lignum are phenolics, and they are divided into four structural
subtypes: brazilin, chalcone, protosappanin, and homoisoflavonoids.
RESULTS AND DISCUSSION
■
Repeated column chromatography (silica gel, RP-18, and semi-
preparative HPLC) of the EtOAc-soluble fraction from C. sappan
Received: May 27, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Chemical structures of isolated compounds (1−12).
Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1−4 (400 MHz and 100 MHz, methanol-d4, δ values)
1
2
3
4
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
7.10, d (8.4)
131.1
113.5
147.2
115.4
2
4.14, d (11.6)
4.02, d (11.6)
73.5
4.30, d (11.6)
4.19, d (11.6)
74.8
6.70, dd (2.4, 8.4)
7.28, s
3
74.3
195.9
113.4
130.5
112.3
166.8
73.3
194.2
113.4
130.7
112.5
167.0
159.8
108.9
159.2
153.8
116.3
4
6.66, d (2.4)
4.46, s
6.89, d (8.4)
7.29, d (8.4)
5.19, s
4a
5
7.72, d (8.8 )
7.72, d (8.8 )
126.5
130.9
104.9
6
6.55, dd (2.4, 8.8)
6.55, br d (8.8)
78.9
207.9
125.0
117.5
7
7a
8
6.36, d (2.4)
103.7
165.0
41.3
6.35, br s
103.6
164.9
30.9
6.72, s
8a
9
2.91, d (14.0)
2.85, d (14.0)
2.82, d (14.8)
2.71, d (14.8)
145.8
10
145.5
117.6
131.9
127.3
11
6.72, s
11a
11b
1′
2′
3′
4′
5′
6′
127.8
115.5
148.6
146.6
115.9
124.5
56.4
128.3
152.3
72.4
131.2
114.9
146.7
146.6
115.9
119.6
6.81, d (2.0)
7.51, br s
6.84, s
4.98 (2H, overlap)
6.71, d (8.0)
6.66, dd (2.0, 8.0)
3.83, s
177.1
6.71, d (8.0)
6.73, d (8.0)
3′-OCH3
6-CHO
9.67, s
193.2
heartwood resulted in the isolation of 12 phenolic compounds.
The chemical structures of the known compounds were identi-
fied as quercetin-3,7-di-O-methyl ether (5), 3-deoxysappanone
B (6), sappanone A (7), 3′-deoxy-4-O-methylepisappanol (8),
10,11-dihydroxydracaenone C (9), quercetin-3′,4′-di-O-methyl
ether (10), 3′-deoxysappanone B (11), and 3′-deoxysappanone
A (12), by comparing the physicochemical and spectroscopic
data with those previously reported.16−22
B
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Caesalpiniaphenol A (1) was obtained as colorless needles fol-
lowing crystallization from MeOH, with the molecular formula
C17H16O6, as determined by the HRFABMS at m/z 317.1025
[M + H]+. The IR spectrum of 1 suggested the presence of a
hydroxy group (3450 cm−1), a carbonyl group at 1720 cm−1, and
Caesalpiniaphenol B (2) was obtained as a colorless, amor-
phous powder, with the molecular formula C14H12O6, as
determined by the HRFABMS at m/z 277.0712 [M + H]+.
The IR spectrum of 2 suggested the presence of a hydroxy group
(3448 cm−1) and two carbonyl groups (at 1745 and 1740 cm−1).
The 1H NMR spectrum showed the H-2 resonances as a pair of
doublets at δH 4.19 and 4.30 (each 1H, d, J = 11.6 Hz) of ring C
and the methylene signal H-9 at δH 2.71 and 2.82 (each 1H, d, J =
14.8 Hz). The three aromatic protons of ring A resonated at δH
7.72 (d, J = 8.8 Hz, H-5), 6.55 (br d, J = 8.8 Hz, H-6), and 6.35
(br s, H-8) (Table 1). Diagnostic signals for the unsaturated
furanone portion of 2 were the olefinic proton at δH 7.51 (br s,
H-2′), the methylene protons δH 4.98 (2H, overlap), olefinic 13C
NMR signals at δC 128.3 (C-1′) and 152.3 (C-2′), an oxy-
methylene carbon signal at δC 72.4 (C-3′), and a carbonyl at δC
177.1 (C-5′) (Table 1). These conclusions were further sup-
ported by the analysis of the HMQC and HMBC spectroscopic
data. The HMBC spectrum confirmed the correlations between
protons H-9, H-2′, and H-3′ and C-5′ and between H-2′ and
C-1′, C-3′, and C-5′. Diagnostic HMBC correlations were
observed between H-9, H-2, and H-5 and C-4 (Figure 2). The
ECD spectrum of 2 showed a negative Cotton effect at Δε256 −2.64
and a positive Cotton effect at Δε278 +6.84, suggesting a 3R
configuration by comparison with (3R)-3-(3,4-dihydroxybenzyl)-
7-hydroxychroman-4-one.22 On the basis of the above analysis, the
structure of compound 2 was elucidated as (3R)-3,7-dihydroxy-
3-[furan-2′(5H)-one]chroman-4-one.
Caesalpiniaphenol C (3) was obtained as yellow needles fol-
lowing crystallization from MeOH, with the molecular formula
C14H10O5 as determined by the HRFABMS at m/z 259.0612
[M + H]+. The IR spectrum of 3 suggested the presence of a
hydroxy group (3448 cm−1) and a carbonyl group at 1739 cm−1.
The 1H NMR spectrum of 3 showed a singlet methylene signal at
δH 4.46. The 13C NMR spectrum of 3 (Table 1) exhibited one
methylene and one carbonyl carbon signal. The HMBC spec-
trum indicated a correlation between H-6 and the carbonyl
carbon signal (δC 207.9), suggesting the relative position of these
atoms (Figure 2). On the basis of these results, a partial structure
A was proposed for 3.25 The 1H NMR spectrum also showed an
aromatic ABC system at δH 7.10 (1H, d, J = 8.4 Hz, H-1), 6.70
(1H, dd, J = 2.4, 8.4 Hz, H-2), and 6.66 (1H, d, J = 2.4 Hz, H-4)
and a two-proton aromatic singlet at δH 6.72 (2H, s) (Table 1).
The 13C NMR spectrum of 3 exhibited 12 aromatic carbon
signals, and the HMQC revealed these to be methine signals at
δC 131.1, 117.6, 117.5, 113.5, and 108.9, with the remaining
seven signals due to quaternary carbons. The HMBC spectrum
revealed correlations of H-1 and H-11 with C11a (δC 131.9) and
δH 6.70 (H-2) and 6.72 (H-11) with a carbon signal at δC 127.3
(C-11b). In the NOE spectrum, an association was observed
between the protons signals δH 6.72 (H-11) and 7.10 (H-1).
These observations led to a biphenyl-type partial structure for
B.25 The connection of the two partial A and B structures of 3 was
established by HMBC correlations between H-6 and C-4a and
C-7a and also between H-8 and the carbonyl carbon (δC 207.9)
(Figures 2 and 3). The ECD spectrum showed a negative Cotton
effect at Δε256 −8.64, indicating M-helicity (conformation) and
hence an S-configured biphenyl axis.26 Thus, compound 3 was
determined to be (aS)-3,9,10-trihydroxy-7-hydro-6H-dibenz-
[b,d]oxocin-7-one.
1
aromatic absorption (1430 cm−1). The H NMR spectrum
showed the typical splitting pattern for a 3-hydroxy-3-benzyl-4-
chromanone-type homoisoflavonoid with the H-2 resonances
occurring as a pair of doublets at δH 4.02 and 4.14 (each 1H, d, J =
11.6 Hz), with a benzylic methylene signal H-9 at δH 2.85 and
2.91 (each 1H, d, J = 14.0 Hz). The O-substituted quaternary
carbon at δC 74.3 was assigned to C-3.23 The 1H NMR spectrum
showed one methoxy resonance at δH 3.83 (3H, s). One aromatic
proton singlet at δH 6.81 (d, J = 2.0 Hz, H-2′), and a pair of ortho-
coupled resonances at δH 6.71 (d, J = 8.0 Hz, H-5′) and 6.66
(dd, J = 2.0, 8.0 Hz, H-6′), accounting for the three protons of the
B-ring. Resonances ascribed to H-5 (δH 7.72, d, J = 8.8 Hz), H-6
(δH 6.55 dd, J = 2.4, 8.8 Hz), and H-8 (δH 6.36, d, J = 2.4 Hz) were
located on ring A (Table 1). Correlations in the NOESY spec-
trum between the H-2′ resonances and the methoxy proton
resonances at δH 3.83, respectively, enabled placement of the
methoxy group at C-3′. The 13C NMR spectrum of 1 contained
17 carbon signals. The signal at δC 56.4 belonged to the methoxy
group, and the other 16 signals were consistent with the chroman-
4-one skeleton. The HMQC and 13C NMR spectra indicated the
presence of eight quaternary carbons in 1. The chemical shifts of
these quaternary carbons indicated that of the 12 aromatic
carbons, four were oxygen-bearing carbons. The C-4 carbonyl
functionality was evident at δC 195.9 (Table 1). The complete
NMR assignments and connectivity of 1 were further determined
by analysis of the HMQC and HMBC spectroscopic data. The
HMBC spectrum confirmed the correlations between H-2′(δH 6.81)
and the C-3′ methoxy protons (δH 3.83) and C-3′ (δC 148.6). Diag-
nostic HMBC correlations were observed for H-2 to C-3
(δC 74.3), C-4 (δC 195.9), and C-8a (δC 165.0) and H-9 to
C-3 (δC 74.3), C-4 (δC 195.9), and C-1′ (δC 127.8) (Figure 2).
Figure 2. Selected HMBC correlations (H → C) for new com-
pounds 1−4.
The electronic circular dichroism (ECD) spectrum of 1 showed
a negative Cotton effect at Δε249 −3.11 and a positive Cotton
effect at Δε281 +9.21, suggesting a 3R configuration by com-
parison with (3R)-3-(3,4-dihydroxybenzyl)-7-hydroxychroman-
4-one.22,24 On the basis of the above analysis, the structure of
compound 1 was elucidated as (3R)-3,7-dihydroxy-3-(4′-hydroxy-
3′-methoxybenzyl)chroman-4-one.
Caesalpiniaphenol D (4) was isolated to give a light brown,
amorphous powder with the molecular formula C14H12O6,
as determined by HRFABMS at m/z 277.0714 [M + H]+. The IR
spectrum suggested the presence of a hydroxy group (3440 cm−1), a
C
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Table 2. Inhibition of LPS-Induced NO, TNF-α Production in
Macrophage RAW264.7 Cells, and NO Scavenging Activity of
Compounds 1−12
a
IC50 value (μM)
compound
NO
TNF-α
NO-scavenging
d
1
2
3
4
5
6
7
8
9
>50
>50
>50
N.D.
102.56 25.3
95.62 18.6
86.25 18.8
112.52 27.4
102.95 19.5
65.34 11.2
96.58 23.5
114.56 28.2
156.54 30.4
85.34 12.1
116.82 18.9
95.45 9.5
N.D.
N.D.
12.2 1.5
3.5 0.4
42.4 5.2
5.7 0.6
33.4 0.5
>50
5.8 0.6
N.D.
8.3 0.9
N.D.
>50
35.0 4.1
>50
>50
1.0 0.1
N.D.
10
N.D.
11
N.D.
1
Figure 3. H and 13C chemical shifts and NOE observed in the NOE
12
N.D.
b
e
difference spectrum (↔) in methanol-d4.
celastrol
0.8 0.2
−
−
c
ascorbic acid
−
0.71 0.1
formyl carbonyl group at 1730 cm−1, and aromatic absorption
a
The inhibitory effects are represented as the molar concentration
1
(1421 cm−1). H NMR signals at δH 9.67 (1H, s) and δH 5.19
(μM) giving 50% inhibition (IC50) relative to the vehicle control.
(1H, s) revealed the presence of formyl and hemiketal methine
groups (Table 1). The spectrum also showed protons at δH
6.71−7.29 ppm (6H), indicating the presence of 1,2,4-
trisubstituted and 1,3,4-trisubstituted benzene rings. The 13C
NMR spectrum showed 14 carbon signals. Those at δC 193.2 and
104.9 belonged to formyl and hemiketal methine groups, and the
remaining were aromatic carbon signals. The full NMR assign-
ments and connectivities of 4 were determined by analysis of
HMQC and HMBC spectroscopic data. The HMBC spectrum
confirmed the correlations between H-7 (δH 5.19, s) and C-1,
C-6′, and C-2′, in agreement with the structure shown. Diag-
nostic HMBC correlations were observed between the formyl
proton (δH 9.67, s) and C-1, C-6, and C-5, suggesting that the
formyl group was located at C-6. The modified Mosher’s method
was used to produce (R)- and (S)-MTPA esters (4a, 4b), and
signals corresponding to H-4, H-5, and 6-CHO were relatively
shielded in 4a compared to 4b. In contrast, signals for H-2′ and
H-6′ were relatively deshielded in 4a compared to 4b (Figure 4),
These data represent the average values of three repeated experiments
b
(mean
SD). Positive control for NO and TNF-α production.
c
d
e
Positive control for NO-scavenging. N.D.: not determined. (−): no
test.
and 7 exhibited an inhibitory effect on the LPS-induced produc-
tion of NO in macrophage RAW264.7 cells, the effects of these
compounds on LPS-induced iNOS expression were investigated.
RAW264.7 cells were stimulated with 1 μg/mL of LPS for 18 h
in the presence of increasing concentrations of 4, 5, and 7, and
the expression levels of iNOS protein were determined by immu-
noblot analyses (Figure 5). Compounds 4, 5, and 7 (0−10 μM)
showed a dose-dependent reduction in LPS-induced iNOS
expression, but did not change the alpha-tubulin expression. The
results showed that 4, 5, and 7 inhibited iNOS activities in
LPS-stimulated RAW264.7 cells, suggesting that these com-
pounds could suppress LPS-induced iNOS expressions at the
transcription level. In addition, these compounds were further
investigated on the LPS-induced TNF-α release. Except for
compound 4, pretreatment of cells with compounds 5 and 7 in
several concentrations (5−50 μM) decreased the TNF-α
production (Table 2). Since degradation of IκB-α protein is an
essential step for NF-κB activation, we examined the effect of
compounds 5 and 7 on the induced degradation and phos-
phorylation of IκB-α protetin by LPS (Figure 6). Cystoplasmic
extracts were analyzed in the presence of IκB-α; it was almost
completely degraded in 15 min after stimulation with LPS and
resynthesized in 30 min. However, preincubation with 5 signifi-
cantly prevented the induced degradation of IκB-α protein at 10
and 15 min; meanwhile, preincubation with 7 prevented the
induced degradation of IκB-α protein at 5 and 15 min. The
resynthesis of IκB-α, which is under control of NF-κB, was also
significantly suppressed by both compounds (Figure 6). In the
NO-scavenging activity assay, all the isolated compounds showed
potential scavenging activity with IC50 values ranging from 65.3
to 156.5 μM.
Figure 4. Results with the modified Mosher’s method (Δ = δS − δR) for
compound 4.
indicating the absolute configuration of 4 as 7S. On the basis
of the above data analyses, compound 4 was established to
be 1-[(7S)-7-hydroxy-1′-(3′,4′-dihydroxyphenyl)methanol]-
3-hydroxybenzaldehyde.
The cytotoxic effects of the isolated compounds (1−12) were
evaluated in the presence or absence of LPS by using the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay. These compounds did not affect the cell viabilities of RAW
264.7 cells in either the presence or absence of LPS, even at a
dose of 50 μM after a period of 24 h (data not shown). As shown
in Table 2, compounds 4, 5, and 7 showed inhibitory effects with
IC50 values of 12.2, 3.5, and 5.7 μM. Compounds 6, 8, and 10 dis-
played moderate effects with IC50 values of 42.4, 33.4, and 35.0 μM,
respectively, but the others were inactive. Because compounds 4, 5,
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation was mea-
sured with a JASCO DIP 370 digital polarimeter. UV spectra were
obtained in MeOH using a Thermo 9423AQA2200E UV spectrometer,
■
D
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Figure 5. Inhibition of LPS-induced iNOS expression in RAW264.7 cells by compounds 4, 5, and 7 (C4, C5, and C7). RAW264.7 cells were pretreated
for 30 min with the indicated concentrations of 4, 5, and 7, followed by stimulation with LPS (1 μg/mL) for 18 h. Total lysates were prepared, and the
expression levels of iNOS were determined by analysis of immunoblot. Histograms show densitometry analyses of relative iNOS expression levels
normalized against α-tubulin.
Figure 6. Inhibition of LPS-induced IκB-α degradation expression in RAW264.7 cells by compounds 5 and 7 (C5 and C7). RAW264.7 cells were
pretreated for 30 min with the indicated concentrations of 5 and 7, followed by stimulation with LPS (1 μg/mL) for 18 h. Total lysates were prepared,
and the expression levels of IκB-α were determined by analysis of immunoblot. Histograms show densitometry analyses of relative IκB-α expression
levels normalized against α-tubulin.
and IR spectra were obtained on a Bruker Equinox 55 FT-IR spec-
trometer. The nuclear magnetic resonance (NMR) spectra were
obtained on a Varian Unity Inova 400 MHz spectrometer. ECD spectra
were recorded on a JASCO J-810 spectropolarimeter. Silica gel (Merck,
63−200 μm particle size) and RP-18 silica gel (Merck, 75 μm particle
size) were used for column chromatography. TLC was carried out using
Merck silica gel 60 F254 and RP-18 F254 plates. HPLC was performed
using a Waters 600 Controller system with a UV detector and a YMC
Pak ODS-A column (20 × 250 mm, 5 μm particle size, YMC Co., Ltd.,
Japan), and HPLC solvents were from Burdick & Jackson, USA.
Plant Material. The C. sappan heartwood was purchased from a folk
medicine market “Yak-ryoung-si” in Daegu, Korea, in May 2010. One of
the authors (B.S.M.) performed botanical identification, and a voucher
specimen (CUD-3174) was deposited at the Herbarium of the College
of Pharmacy, Catholic University of Daegu, Korea.
Extraction and Isolation. The C. sappan heartwood (10 kg) was
extracted with MeOH three times (3 h × 3 L) under reflux. After the
solvent was removed under reduced pressure, the residue was suspended
in H2O and partitioned with n-hexane, EtOAc, and n-BuOH, succes-
sively. The EtOAc-soluble fraction (960 g) was separated on a silica gel
column using a stepwise gradient of CHCl3/MeOH (from 50 mL/1 mL
to 100% MeOH, each 10 L) to yield 14 fractions (Fr.1−Fr.14) according
to their TLC profiles. Fraction 5 (4.4 g) was subjected to reversed-phase
(ODS-A) column chromatography and eluted with MeOH/H2O (from
2 mL/1 mL to 100% MeOH, 2 L for each step) to afford seven
subfractions (Fr.5-1 to Fr.5-7). Further purification of Fr.5-2 (630 mg)
by semipreparative HPLC [using an isocratic solvent system of 30%
MeOH in 0.1% TFA (flow rate 5 mL/min) over 90 min; UV detection at
210 nm; YMC Pak ODS-A column (20 × 250 mm, 5 μm particle size]
resulted in the isolation of 1 (14.1 mg; tR = 38.5 min), 5 (5.8 mg; tR =
44.8 min), and 6 (74.2 mg; tR = 48.2 min). Fraction 5-3 (728 mg) was
purified by semipreparative HPLC [using an isocratic solvent system of
30% MeOH in 0.1% TFA (flow rate 5 mL/min) over 90 min; UV
detection at 210 nm; YMC Pak ODS-A column (20 × 250 mm, 5 μm
particle size], resulting in the isolation of 3 (28.5 mg; tR = 40.8 min), 7
(42.0 mg; tR = 44.9 min), and 10 (15.2 mg; tR = 46.3 min), respectively.
Further purification of Fr.5-5 (510 mg) by semipreparative HPLC
[using an isocratic solvent system of 35% MeOH in 0.1% TFA (flow rate
5 mL/min) over 90 min; UV detection at 210 nm; YMC Pak ODS-A
column (20 × 250 mm, 5 μm particle size] resulted in the isolation of 4
(22.4 mg, tR = 39.2 min), 8 (17.1 mg; tR = 43.8 min), and 12 (16.3 mg,
tR = 46.2 min). Subfraction F.5-7 (354 mg) was further purified by
semipreparative HPLC [using an isocratic solvent system of 40% MeOH
in 0.1% TFA (flow rate 5 mL/min) over 90 min; UV detection at
210 nm; YMC Pak ODS-A column (20 × 250 mm, 5 μm particle size],
resulting in the isolation of 2 (4.8 mg, tR = 42.9 min), 9 (12.7 mg, tR =
46.2 min), and 11 (25.6 mg, tR = 50.2 min).
Caesalpiniaphenol A (1): colorless needles (MeOH); mp 181−
183 °C; [α]2D5 49.2 (c 0.13, MeOH); IR νmax (KBr) 3450, 2942, 1720,
1430, 1260, 1166 cm−1; UV (MeOH) λmax (log ε) 232 (4.12), 278
(3.87) nm; ECD (c 0.032 mM, MeOH) Δε249 −3.11, Δε281 +9.21; 1H
NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4)
spectroscopic data, see Table 1; HRFABMS m/z 317.1025 [M + H]+
(calcd for C17H17O6, 317.1025).
Caesalpiniaphenol B (2): colorless, amorphous powder; [α]2D5 56.2
(c 0.10, MeOH); IR νmax (KBr) 3448, 2941, 1745, 1740, 1427, 1257,
1169 cm−1; UV (MeOH) λmax (log ε) 226 (4.16), 270 (3.81), 284 (3.52)
nm; ECD (c 0.036 mM, MeOH) Δε256 −2.64, Δε278 +6.84; 1H NMR
(400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4)
spectroscopic data, see Table 1; HRFABMS m/z 277.0712 [M + H]+
(calcd for C14H13O6, 277.0712).
Caesalpiniaphenol C (3): yellow needles (MeOH); mp 262−
264 °C; [α]2D5 −22.7 (c 0.40, MeOH); IR νmax (KBr) 3448, 2926, 1739,
1260, 1158, 1031 cm−1; UV (MeOH) λmax (log ε) 252 (4.46), 285
(4.13) nm; ECD (c 0.046 mM, MeOH) Δε256 −8.64; HRFABMS m/z
1
259.0612 [M + H]+ (calcd for C14H11O5, 259.0606); H NMR (400
MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4) spec-
troscopic data, see Table 1; 1H NMR (400 MHz, pyridine-d5) δH 7.43
(1H, d, J = 8.4 Hz, H-1), 7.13 (1H, dd, J = 2.4, 8.4 Hz, H-2), 7.21 (1H, d,
J = 2.4 Hz, H-4), 4.73 (2H, s, H-6), 7.38 (1H, s, H-8), 7.40 (1H, s, H-11);
13C NMR (100 MHz, pyridine-d5) δC 131.2 (C-1), 113.9 (C-2), 160.7
(C-3), 109.7 (C-4), 159.2 (C-4a), 78.8 (C-6), 206.4 (C-7), 124.7
(C-7a), 118.5 (C-8), 147.4 (C-9), 147.1 (C-10), 118.6 (C-11), 131.5
(C-11a), 126.9 (C-11b).
Caesalpiniaphenol D (4): light brown, amorphous powder; [α]D25
−24.4 (c 0.17, MeOH); IR νmax (KBr) 3440, 2924, 1730, 1421, 1264
1165, 1031 cm−1; UV (MeOH) λmax (log ε) 233 (4.30), 279 (4.16),
1
312 (4.09) nm; H NMR (400 MHz, methanol-d4) and 13C NMR
(100 MHz, methanol-d4) spectroscopic data, see Table 1; HRFABMS
m/z 277.0714 [M + H]+ (calcd for C14H13O6, 277.0712).
Preparation of (R)- and (S)-MTPA Esters 4a and 4b from 4. A
solution of 4 (1.0 mg) in dry pyridine (50 μL) was reacted with 5 μL
of (S)-(+)-MTPA-Cl and 2 mg of 4-DMAP at room temperature for
30 min. The reaction mixture was dried by N2 gas. The dried product
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dx.doi.org/10.1021/np3003673 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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was partitioned with CH2Cl2 and H2O. The organic layer was dried
(Na2SO4), filtered, and concentrated under reduced pressure. The resi-
due was purified by preparative TLC silica gel (0.25 mm thickness)
developed with CH2Cl2−MeOH (5 mL/1 mL), and the product
was eluted with CH2Cl2−MeOH (100 mL/10 mL) to furnish the ester,
4a (1.3 mg). In a similar manner, 4b (1.7 mg) was prepared from 4
(1.0 mg) using (R)-MTPA-Cl (5 μL) and 4-DMAP (2.0 mg).27
(1-[(7S)-7-Hydroxy-1′-(3′,4′-dihydroxyphenyl)methanol]-3-hy-
droxybenzaldehyde (R)-MTPA ester (4a): colorless oil; 1H NMR
(pyridine-d5, 400 MHz) δ 9.984 (1H, s, 6-CHO), 8.111 (1H, d, J =
8.4 Hz, H-5′), 7.985 (1H, d, J = 2.0Hz, H-2′), 7.818 (1H, dd, J = 2.0, 8.4Hz,
H-6′), 7.520 (1H, s, H-2), 7.291 (1H, d, J = 8.0 Hz, H-5), 8.100 (1H, d, J =
7.6 Hz, H-4), 7.46−7.53 (5H, m, aromatic protons).
ASSOCIATED CONTENT
■
S
* Supporting Information
Copies of 1H, 13C NMR and 2D spectra for 1−4 can be accessed
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +82-53-850-3613. Fax: +82-53-850-3602. E-mail: bsmin@
cu.ac.kr.
Notes
The authors declare no competing financial interest.
(1-[(7S)-7-Hydroxy-1′-(3′,4′-dihydroxyphenyl)methanol]-3-hy-
droxybenzaldehyde (S)-MTPA ester (4b): colorless oil; 1H NMR
(pyridine-d5, 400 MHz) δ 10.014 (1H, s, 6-CHO), 8.110 (1H, d, J =
8.4 Hz, H-5′), 7.910 (1H, d, J = 2.0Hz, H-2′), 7.523 (1H, dd, J = 2.0, 8.4Hz,
H-6′), 7.522 (1H, s, H-2), 7.317 (1H, d, J = 8.0 Hz, H-5), 8.260 (1H, d, J =
7.6 Hz, H-4), 7.46−7.53 (5H, m, aromatic protons).
ACKNOWLEDGMENTS
■
This research was supported by the National Research Foundation
of Korea Grant funded by the Korean Government (MEST)
(KRF-2012R1A2A06046921 and KRF-2012R1A1A2003547).
We are grateful to Korea Basic Science Institute (KBSI) for MS
and NMR spectral measurements.
Determination of NO Production and the Cell Viability Assay.
The level of NO production was determined by measuring the amount
of nitrite present in cell culture supernatants as described previously.28
Briefly, the RAW264.7 cells (1 × 105 cells/well) were stimulated with or
without 1 μg/mL of LPS (Sigma Chemical Co., St. Louis, MO, USA) for
24 h in the presence or absence of the test compounds (0.5−25 μM).
The cell culture supernatant (100 μL) was then reacted with 100 μL
of Griess reagent (1% sulfanilamide in 5% phosphoric acid and
0.1% naphthylethylenediamine dihydrochloride in distilled H2O). The
absorbance at 540 nm was determined with a microplate reader (Molec-
ular Devices, Emax, Sunnyvale, CA, USA), and the absorption coef-
ficient was calibrated using a NaNO2 solution standard. The amount of
TNF-α in the culture supernatant was measured using the ELISA kit
(R&D systems, Minneapolis, MN, USA). Cell viability was measured
with the MTT-based colorimetric assay. For this experiment, celastrol
was used as a positive control.
Immunoblot Analysis. Proteins were extracted from cells in ice-
cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM
EDTA, 1 mM phenylmethyl sulfonyl fluoride, 1 μg/mL leupeptin, 1 mM
sodium vanadate, 150 mM NaCl). A 50 μg amount of protein (for iNOS)
per lane was separated by sodium dodecyl sulfate−polyacrylamide gel
electrophoresis and transferred to a polyvinylidene difluoride membrane
(Millipore, Bedford, MA, USA). The membrane was blocked with 5%
skim milk and then incubated with the corresponding antibody. The
antibody for iNOS was obtained from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). The antibody for α-tubulin was obtained from Sigma.
Antibody for IκB-α was obtained from Cell Signaling Technology
(Danvers, MA, USA). After binding of an appropriate secondary antibody
coupled to horseradish peroxidase, proteins were visualized by enhanced
chemiluminescence according to the instructions of the manufacturer
(Amersham Pharmacia Biotec, Buckinghamshire, UK).29
Nitric Oxide Free Radical Scavenging Activity. A 50 μL amount
of each of the concentrations of compounds previously dissolved in
DMSO, as well as ascorbic acid (standard compound), was taken in
separate tubes, and the volume was uniformly made up to 150 μL
with MeOH. To each tube was added 2.0 mL of sodium nitroprusside
(10 mM) in phosphate buffer saline. The solutions were incubated at
room temperature for 150 min. A similar procedure was repeated with
MeOH as blank, which served as a control. After the incubation, 5 mL of
Griess reagent was added to each tube including the control. The absor-
bance of chromophore formed was measured at 546 nm on an Infinite
F200Pro UV−visible spectrometer, Tecan, Switzerland. Ascorbic acid
was used as a positive control. The IC50 value for each test compound as
well as standard preparation was calculated.30
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