Isolation, Synthesis, and Antisepsis Effects of a C-Methylcoumarinochromone Isolated from Abronia nana Cell Culture

Article abstract of DOI:10.1021/acs.jnatprod.7b00826

Only a few isoflavones have been isolated from plants of the genus Abronia. The biological properties of compounds isolated from Abronia species have not been well established, and their antisepsis effects have not been reported yet. In the present study, a new C-methylcoumarinochromone, was isolated from Abronia nana suspension cultures. Its structure was deduced as 9,11-dihydroxy-10-methylcoumarinochromone (boeravinone Y, 1) by spectroscopic data analysis and verified by chemical synthesis. The potential inhibitory effects of 1 against high mobility group box 1 (HMGB1)-mediated septic responses were investigated. Results showed that 1 effectively inhibited lipopolysaccharide-induced release of HMGB1 and suppressed HMGB1-mediated septic responses, in terms of reduction of hyperpermeability, leukocyte adhesion and migration, and cell adhesion molecule expression. In addition, 1 increased the phagocytic activity of macrophages and exhibited bacterial clearance effects in the peritoneal fluid and blood of mice with cecal ligation and puncture-induced sepsis. Collectively, these results suggested that 1 might have potential therapeutic activity against various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Full text of DOI:10.1021/acs.jnatprod.7b00826

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Isolation, Synthesis, and Antisepsis Effects of a
C‑Methylcoumarinochromone Isolated from Abronia nana Cell
Culture
Wonhwa Lee,†,‡,§ Doohyun Lee,†,§ Yuri Lee,† Taeho Lee,† Kyung-Sik Song,*,† Eun-Ju Yang,*,†
and Jong-Sup Bae*,†
†College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics Based Creative Drug
Research Team, Kyungpook National University, Daegu 41566, Republic of Korea
‡Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
S
* Supporting Information
ABSTRACT: Only a few isoflavones have been isolated from
plants of the genus Abronia. The biological properties of
compounds isolated from Abronia species have not been well
established, and their antisepsis effects have not been reported
yet. In the present study, a new C-methylcoumarinochromone,
was isolated from Abronia nana suspension cultures. Its structure
was deduced as 9,11-dihydroxy-10-methylcoumarinochromone
(boeravinone Y, 1) by spectroscopic data analysis and verified by
chemical synthesis. The potential inhibitory effects of 1 against
high mobility group box 1 (HMGB1)-mediated septic responses
were investigated. Results showed that 1 effectively inhibited
lipopolysaccharide-induced release of HMGB1 and suppressed
HMGB1-mediated septic responses, in terms of reduction of hyperpermeability, leukocyte adhesion and migration, and cell
adhesion molecule expression. In addition, 1 increased the phagocytic activity of macrophages and exhibited bacterial clearance
effects in the peritoneal fluid and blood of mice with cecal ligation and puncture-induced sepsis. Collectively, these results
suggested that 1 might have potential therapeutic activity against various severe vascular inflammatory diseases via inhibition of
the HMGB1 signaling pathway.
amplify severe vascular inflammatory responses and induce
tissue impairment.12,13 Once released into the extracellular
milieu, HMGB1 can bind to cell surface receptors, such as toll-
like receptors (TLR) 2 and 4, as well as receptor for advanced
glycation end-products (RAGE),9,11,14 to transduce intracellular
signals and elicit cellular responses, including chemotaxis and
proinflammatory cytokine release.12,13 Since HMGB1 is
considered a late mediator of sepsis, in this study, sepsis was
induced by HMGB1 in human endothelial cells.
In a previous study aiming to find antisepsis drug leads from
natural sources, boeravinone X was isolated from Abronia nana
suspension cultures and shown to significantly suppress
HMGB1-mediated septic responses in human umbilical vein
endothelial cells (HUVECs) and C57BL/6 mice.15 Further
investigation of this plant afforded another promising antisepsis
compound. In the current study, we isolated the antisepsis
compound 1 from A. nana suspension cultures and investigated
its potential anti-inflammatory effects in HUVECs and a mouse
model of cecal ligation and puncture (CLP)-induced sepsis.
epsis is a systemic inflammatory response that can lead to
Slethal organ damage. Septic responses result from hyper-
activity of the innate immune system and the ensuing
proinflammatory cascade in response to severe microbial
infections or extensive tissue damage.1,2 Over the last three
decades, considerable progress has been achieved with respect
to understanding the pathophysiology of sepsis. Since tumor
necrosis factor (TNF)-α mimics septic shock in animal models,
several clinical trials have focused on the neutralization of this
proinflammatory mediator.3 However, one trial had very
disappointing results.4,5 Since the early kinetics of systemic
TNF-α accumulation in sepsis makes it a difficult therapeutic
target in clinical settings,6 other late proinflammatory mediators
may offer a wider therapeutic window. High mobility group box
1 (HMGB1), which is secreted in response to bacterial
endotoxins, plays an important role in sepsis.7−9 When
HMGB1 is released from necrotic or activated cells, it triggers
inflammation, immune responses, and tissue regeneration.7−9
In fact, the blockade of HMGB1, even at late stages after the
onset of infection, has been shown to rescue mice from lethal
sepsis.10,11 Therefore, HMGB1 acts as a lethal mediator of
sepsis, in which serum HMGB1 levels substantially increase.9,11
In addition, HMGB1 and its receptor axis have been shown to
Received: September 27, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 1. H (500 MHz) and 13C (125 MHz) NMR
Spectroscopic Data of the Isolated Compound 1
(Boeravinone Y), Synthesized Compound 1, and
Boerharotenoid A (Pyridine-d5, δ in ppm)
a
1
synthesized 1
δH (J in
boerharotenoid A
δH (J in
Hz)
δH (J in
position
1
δC
δC
Hz)
δC
Hz)
122.1 8.25, dd
(7.5, 1.2)
122.2 8.25, dd
127.4 9.18, d
(8)
RESULTS AND DISCUSSION
■
(7.5, 1.4)
Compound 1 was isolated as a gray amorphous powder, and its
molecular formula was determined by high-resolution fast-atom
bombardment mass spectrometry (HRFABMS) to be
C16H10O5 based on the protonated molecular ion peak [M +
H]+ observed at m/z 283.0609 (calcd for C16H11O5, 283.0606).
1a
2
123.5
123.8
100.5
126.1 7.45, dt
126.1 7.42, dt
122.5 7.16, t
(8)
(7.5, 1.2)
(7.5, 1.1)
3
4
126.7 7.38, dt
(7.5, 1.2)
126.1 7.37, dt
(7.5, 1.4)
129.1 7.3, t (8)
1
112.2 7.58, dd
(7.5, 1.2)
112.2 7.57, dd
(7.5, 1.1)
118
7.32, d
(8)
The H NMR spectrum showed a typical methyl signal similar
to that of other C-methyl isoflavones at δH 2.49 (3H, s, CH3-
10).15,16 An intramolecular hydrogen-bonded proton was
observed at δH 13.73 (1H, s, OH-11), indicating the presence
of a hydroxy group attached to C-11. A low-field-shifted
aromatic proton, which resulted from the restricted rotation of
ring A and anisotropic effect of the sterically adjacent carbonyl
double bond, was observed at δH 8.25 (1H, dd, J = 7.5, 1.2 Hz,
H-1). Resonances at δH 7.58 (1H, dd, J = 7.5, 1.2 Hz, H-4),
7.45 (1H, dt, J = 7.5, 1.2 Hz, H-2), and 7.38 (1H, dt, J = 7.5, 1.2
Hz, H-3) were assigned with this proton, which supported the
presence of a 1,2-disubstituted benzene moiety. Acetal and
methylene proton signals around δH 5−6, which are character-
istic of pyran-type rotenoids, were not observed in 1.15−17
Therefore, it was deduced that 1 contains a furan ring, instead
of a pyran ring. Resonances for 16 carbons, including one
carbonyl carbon at δC 179.9, five oxygenated aromatic carbons
at δC 165.7, 164.0, 161.3, 154, and 150.7, and a methyl carbon
at δC 8.6, were observed. All NMR peaks, including the
resonances of two hydroxy groups and one methyl group, were
determined using the heteronuclear multiple-bond correlation
(HMBC) and heteronuclear multiple quantum coherence
(HMQC) spectra of 1. From the HMBC data, it was confirmed
that the hydrogen-bonded hydroxy proton signal at C-11 (δH
13.73, 1H, s, OH-11) correlated with C-11 (δC 161.3) and C-
11a (δC 103.1). A proton at δH 6.83 (1H, s, H-8) correlated
with C-7a (δC 154.0) and C-9 (δC 164). In addition, the methyl
group at δH 2.49 (1H, s, CH3-10) correlated with C-9 (δC
164.0), C-10 (δC 110.1), and C-11 (δC 161.3), indicating that a
methyl group is present at C-10. From these data, the structure
of 1 was deduced as a C-methylcoumarinochromone derivative.
While this structure was previously reported as boerharotenoid
A, major differences were found in the NMR data between 1
and the previously reported compound.18 In particular, the
chemical shifts of six carbons of ring A (C-1-C-4, C-1a, and C-
4a) were quite different, despite the use of the same NMR
solvent (Table 1). To verify the proposed structure, 1 was
4a
6a
7a
8
150.7
165.7
154
150.2
165.7
154
157.5
166.2
155.2
94.8
95.1
6.83, s
95.1
6.8, s
6.68, s
9
164
163.8
110.1
161.4
103.9
180
164.1
109
10
11
11a
12
12a
110.1
161.3
103.1
179.9
98.2
160.6
105.3
179.7
123
98.3
CH3-10 8.6
OH-11
2.49, s
8.6
2.49, s
2.45, s
13.73, s
13.73, s
a
The NMR data of boerharotenoid A were obtained from a previous
study.18
HUVECs (Figure 1A). To investigate the effects of 1 on LPS-
mediated release of HMGB1, HUVECs were stimulated with
100 ng/mL LPS for 16 h, followed by treatment with increasing
concentrations of 1 for 6 h. As shown in Figure 1A, 1 inhibited
the release of HMGB1 from HUVECs at optimal effective
concentrations of >0.2 μM. However, in the absence of LPS
pretreatment, 1 did not affect HMGB1 release (Figure 1A). To
confirm these effects in vivo, a CLP-induced sepsis mouse
model was used because it closely resembles human sepsis
more than LPS-induced endotoxemia.3 As shown in Figure 1B,
treatment with 1 resulted in a marked inhibition of CLP-
induced release of HMGB1. The average circulating blood
volume for mice is 72 mL/kg.23 Since the average weight of
mice used in this study was 27 g, and the average blood volume
was 2 mL, the doses of 1 (28 or 56 μg/kg) used were
equivalent to peripheral blood concentrations of 0.5 or 1 μM.
Next, the effects of 1 on the expression of HMGB1 receptors
(TLR2, TLR4, and RAGE) were investigated in HUVECs. As
shown in Figure 1C, treatment with HMGB1 resulted in more
than a 2-fold increase in the expression of TLR2, TLR4, and
RAGE in HUVECs, which was significantly reduced by 1. To
assess the cytotoxicity of compound 1, cell viability assays were
performed using HUVECs treated with 1 for 48 h. At
concentrations up to 5 μM, 1 did not affect cell viability
(Figure 1D). High plasma concentrations of HMGB1 in
patients with inflammatory diseases are associated with poor
prognosis and high mortality. In addition, pharmacological
inhibition of HMGB1 was shown to improve survival in animal
models of acute inflammation in response to endotoxin
challenge.24 Therefore, we suggest that 1 could be a potential
therapy for vascular inflammatory diseases via inhibition of
HMGB1 release.
1
chemically synthesized. The H and 13C NMR data of the
synthetic compound matched very well with those of the
isolated compound 1 (Table 1). Consequently, we concluded
that the reported boerharotenoid A should have a different
structure from that of 1, indicating that 1 is a new compound.
The present compound 1 was given the trivial name
boeravinone Y.
Previous studies have shown that lipopolysaccharides (LPS)
induce HMGB1 release from murine macrophages and human
endothelial cells.19−21 It was shown that 100 ng/mL LPS was
sufficient to induce HMGB1 release.22 Similarly, in the current
study, 100 ng/mL LPS induced the release of HMGB1 from
B
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Effects of boeravinone Y (1) on HMGB1 release and its receptor expression. (A) HUVECs were treated with the indicated concentrations
of 1 for 6 h after stimulation with bacterial LPS (100 ng/mL) for 16 h. HMGB1 release was measured by ELISA. (B) Male C57BL/6 mice
underwent cecal ligation and puncture (CLP) and were treated with 1 (28 or 56 μg/kg) intravenously 12 h after CLP (black bar, n = 5). Mice were
killed 24 h after CLP. Serum HMGB1 levels were measured by ELISA. (C) Confluent HUVECs were activated with HMGB1 (1 μg/mL, 16 h),
followed by incubation with 1 for 6 h. The expression of cell surface receptors TLR2 (white bar), TLR4 (gray bar), and RAGE (black bar) was
determined by cell-based ELISA. (D) The effects of 1 on cell viability were evaluated using the MTT assay. *p < 0.05 vs LPS alone (A), CLP alone
(B), or HMGB1 alone (C). X: 1 μM boeravinone X was used as a positive control.
A permeability assay was performed to investigate the effects
of 1 on barrier integrity in HUVECs. Treatment with 1 μM 1
alone did not alter the barrier integrity (Figure 2A). In contrast,
LPS and HMGB1 have been shown to induce cleavage and
disruption of the endothelial barrier integrity.25−27 Thus,
HUVECs were treated with various concentrations of 1 for 6
h after exposure to LPS (100 ng/mL) or HMGB1 (1 μg/mL).
As shown in Figure 2A and B, treatment with 1 resulted in a
concentration-dependent decrease in LPS- or HMGB1-
mediated disruption of barrier integrity. The effects of 1 on
HMGB1-induced vascular hyperpermeability in mice were
investigated to confirm its vascular barrier protective effects in
vivo. As shown in Figure 2C, treatment with 1 resulted in
marked inhibition of HMGB1-induced peritoneal dye leakage.
HMGB1 has been shown to induce inflammatory responses
by promoting the phosphorylation of p38 mitogen-activated
protein kinase (MAPK).28,29 To determine whether 1 can
inhibit HMGB1-induced activation of p38 MAPK in HUVECs,
cells were activated with HMGB1, followed by incubation with
1 for 6 h. As shown in Figure 2D, HMGB1 increased
phosphorylated p38 expression, which was significantly
inhibited by treatment with 1. These findings indicated that 1
could inhibit HMGB1-induced endothelial disruption and
maintain human endothelial cell barrier integrity.
blood (Figure 3). These results suggested that 1 might increase
macrophage activity and reduce sepsis.
Several previous studies have shown that HMGB1 mediates
inflammatory responses by increasing the expression of cell
adhesion molecules (CAMs), such as vascular cell adhesion
molecule-1 (VCAM-1), intercellular adhesion molecule-1
(ICAM-1), and E-selectin, on the surface of endothelial cells,
thereby promoting the adhesion and migration of leukocytes
across the endothelium to the sites of inflammation.30,31 In the
present study, HMGB1 increased the expression of VCAM-1,
ICAM-1, and E-selectin on the cell surface, which was reduced
by compound 1 (Figure 4A). In addition, the increase in CAM
expression corresponded well with enhanced binding and
migration of human neutrophils to HMGB1-activated endo-
thelial cells (Figure 4B and C). Treatment with 1 reduced
human neutrophil adhesion and migration across the activated
endothelial cells in a concentration-dependent manner (Figure
4B and C). These results suggested that 1 not only inhibited
endotoxin-induced release of HMGB1 from endothelial cells
but also downregulated the proinflammatory signals induced by
HMGB1 release, thereby inhibiting the amplification of
inflammatory responses by nuclear cytokines. To confirm
these effects in vivo, the effects of 1 on HMGB1-induced
migration of leukocytes were examined in mice. HMGB1
induced the migration of leukocytes into the peritoneal cavity
in mice. However, treatment with compound 1 resulted in a
significant reduction of the peritoneal leukocyte counts (Figure
4D).
The bacterial clearance effects of 1 were evaluated by
counting the colony-forming units (CFUs). Twenty-four hours
after CLP surgery, bacterial counts in the peritoneal fluid and
blood significantly increased. However, 1 significantly de-
creased the bacterial counts in both the peritoneal fluid and
Sepsis is a systemic response to serious infections. It has a
poor prognosis when it is associated with organ dysfunction,
C
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 2. In vitro and in vivo effects of boeravinone Y (1) on HMGB1-mediated hyperpermeability. The effects of 1 treatment at different
concentrations for 6 h on barrier disruption induced by bacterial LPS (100 ng/mL, 4 h) or HMGB1 (1 μg/mL, 16 h) were investigated by measuring
the flux of albumin-bound Evans blue across HUVEC monolayers. (C) The effects of 1 (28 or 56 μg/kg) on HMGB1 (2 μg/mouse)-induced
vascular hyperpermeability in mice were examined by measuring the amount of Evans blue in the peritoneal washings (expressed as μg/mouse, n =
5). (D) HUVECs were activated with HMGB1 (1 μg/mL, 16 h), followed by treatment with 1 at different concentrations for 6 h. The effects of 1 on
HMGB1-mediated expression of phospho-p38 were determined by ELISA. *p < 0.05 vs LPS alone (A) or HMGB1 alone (B, C, D). X: 1 μM
boeravinone X was used as a positive control.
Figure 3. Bacterial clearance effects of boeravinone Y (1). Bacterial counts (CFUs) in the peritoneal fluid and blood 24 h after CLP (n = 10). **p <
0.01 vs the sham group; ##p < 0.01 vs the CLP group.
hypoperfusion, or hypotension.12,32 On the bases of our
previous findings, it was hypothesized that 1 might reduce
mortality in a mouse model of CLP-induced sepsis. To test this
hypothesis, 1 was administered to mice after the CLP
procedure. Twenty-four hours after the procedure, mice
showed signs of sepsis, such as shivering, bristled hair, and
weakness. Administration of 1 (56 μg/kg) 12 h after CLP did
not prevent CLP-induced death (data not shown); thus, 1 was
administered twice (12 and 50 h after CLP). Administration of
two doses of 1 resulted in an increase in the survival rate from
40% to 60%, as shown by Kaplan−Meier survival analysis (p <
0.0001; Figure 5A). This marked survival benefit achieved after
administration of two doses of 1 suggested that suppression of
HMGB1 release and HMGB1-mediated inflammatory re-
sponses could be a therapeutic strategy for the management
of sepsis and septic shock.
To confirm the protective effects of 1 against CLP-induced
death, its effects on CLP-induced pulmonary injury were
examined. In the sham group, there were no significant
differences between the lungs of the treated and untreated mice
under a light microscope (data not shown). In the untreated
CLP group, interstitial edema with massive infiltration of
inflammatory cells into the interstitium and alveolar spaces was
observed, and the pulmonary architecture was severely
damaged (Figure 5B). Systemic inflammation during sepsis
commonly causes multiple organ failure, wherein the liver and
D
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 4. Effects of boeravinone Y (1) on HMGB1-mediated inflammatory responses. (A−C) HUVECs were stimulated with HMGB1 (1 μg/mL)
for 16 h, followed by treatment with increasing concentrations of 1 for 6 h. (A) HMGB1-mediated expression of cell adhesion molecules VCAM-1
(white bar), ICAM-1 (gray bar), and E-selectin (black bar) in HUVECs. (B) Adherence of human neutrophils to HUVEC monolayers. (C)
Migration of human neutrophils through HUVEC monolayers was evaluated by ELISA. (D) The effects of 1 (28 or 56 μg/kg) on CLP-induced
leukocyte migration into the peritoneal cavity of mice were analyzed. All results are expressed as the means SD of five independent experiments (n
= 10). *p < 0.05 vs HMGB1 alone or CLP alone. X: 1 μM boeravinone X was used as a positive control.
kidneys are the major target organs.33 CLP resulted in a
significant increase in the plasma levels of alanine transaminase
(ALT) and aspartate transaminase (AST) (markers of hepatic
injury; Figure 5C), as well as creatinine and blood urea nitrogen
(BUN) (markers of renal injury; Figures 5D and E).
Compound 1 was able to reverse all these abnormalities. The
levels of another important marker of tissue injury, lactate
dehydrogenase (LDH), were also reduced by 1 in CLP-
operated mice (Figure 5F). Next, the effects of 1 on the
production of proinflammatory cytokines, such as interleukin
(IL)-1β, IL-6, IL-10, and TNF-α, were examined during sepsis.
IL-10 and TNF-α have been considered essential mediators of
sepsis-induced vascular inflammation;34 in addition, IL-6
blockade with neutralizing antibodies was shown to protect
against CLP-induced septic mortality.35 CLP-induced elevation
of IL-1β, IL-6, IL-10 (Figure 5G), TNF-α (Figure 5H), and
monocyte chemoattractant protein-1 (MCP-1; Figure 5I) levels
was significantly inhibited by compound 1 treatment.
Despite decades of intense research, the basic mechanisms of
sepsis remain elusive.36,37 In both experimental animal models
of sepsis and human patients, substantial pathological changes
have been observed, many of which may result in subsequent
organ injury.36 Variations in age, sex, and medical comorbidities
create additional complexities that influence the outcome in
septic patients.38 Insufficient understanding of the pathophysi-
ology of sepsis has resulted in fundamental problems in the
design of clinical trials conducted to address a better approach
for the management of sepsis. Currently, there are no U.S.
Food and Drug Administration (FDA)- or European Medicines
Evaluation Agency-approved pharmacological therapeutic in-
terventions directed against specific mediators of sepsis. In the
past 30 years, only one FDA-approved therapy was available.
However, it was withdrawn in 2011 by the manufacturer
because follow-up studies failed to show substantial improve-
ment in the survival of patients with septic shock.39 The
complexity of the disease presents substantial challenges to the
understanding of what is aberrant and why the alteration is
deleterious.37,38 Therefore, most antisepsis agents show modest
effects in animal models of the disease, which reflects the
complexity of the underlying pathogenic mechanisms.36,38
Simultaneous disruption of multiple pathways, rather than a
single mediator, likely account for sepsis mortality.37,40
Although several compounds have shown modest in vivo
effects against CLP-induced cell death, the current findings that
HMGB1 (a late sepsis mediator)-mediated septic responses
may be suppressed by 1 might help to discover other antisepsis
agents.
The molecular mechanisms of the anti-inflammatory effects
of 1 against HMGB1-mediated septic responses might involve
suppression of HMGB1 release (Figure 1A and B), reduction of
the expression of HMGB1 receptors (TLR2, TLR4, and RAGE;
Figure 1C), and decrease in HMGB1-mediated hyperperme-
ability (Figure 2A−C) via suppression of p38 activation (Figure
2D). Furthermore, inhibition of the interaction between
leukocytes and endothelial cells by 1 might be attributable to
the inhibition of the expression of CAMs, such as VCAM,
ICAM, and E-selectin (Figure 4). Compound 1 might mainly
target the interaction of the released HMGB1 with its receptors
in HMGB1-mediated septic responses because binding of
HMGB1 to its receptors mediates severe vascular inflammatory
E
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 5. Effects of boeravinone Y (1) on CLP-induced mortality and pulmonary injury. (A) Male C57BL/6 mice (n = 20) were treated
■
◇
intravenously with 1 (28 μg/kg, or 56 μg/kg, ) at 12 and 50 h after CLP. Animal survival was monitored every 6 h after CLP for 150 h.
●
○
Untreated CLP mice ( ) and untreated sham-operated mice ( ) were administered sterile saline (n = 20). Kaplan−Meier survival analysis was
performed for evaluation of the overall survival rates. (B) Male C57BL/6 mice underwent CLP and were treated with 1 (28 or 56 μg/kg)
intravenously at 12 and 50 h after CLP (n = 5). Mice were killed 96 h after CLP. Photomicrographs of lung tissues (H&E staining, 200×). Untreated
sham group (grade 1), untreated CLP group (grade 3), and CLP group treated with 1 (grade 2). The figure shows representative images from three
independent experiments. (D−G) Similar to (B) and (C) except that mice were bled to death. AST (C), ALT (C), creatinine (D), BUN (E), and
LDH (F) levels in the plasma were measured. Compound 1 decreased the concentrations of IL-1β, IL-6, IL-10, TNF-α, and MCP-1 in the blood of
CLP mice (G−I). Cytokine concentrations were determined by ELISA (n = 10 mice/group/experiment). Results are expressed as the means SD
of five independent experiments (n = 5). *p < 0.05 vs the CLP group. X: 27 μg/kg boeravinone X was used as a positive control.
responses, such as hyperpermeability, adhesion, and migration
of leukocytes to the endothelial cells.9,41−43 Based on the
current findings, 1 might be a potential therapeutic agent for
treatment of severe vascular inflammatory diseases, such as
sepsis and septic shock.
In a previous study, four pyran-type C-methylrotenoids were
isolated from suspension cultures of A. nana. Among them,
boeravinone D (IC50 = 4.8 μM) and mirabijalone D (IC50 = 4.2
μM) were proposed as noncompetitive inhibitors of β-site
amyloid precursor protein cleaving enzyme 1 (BACE1, β-
secretase), which is a key protease involved in Alzheimer’s
disease pathogenesis.17 In addition, a BACE1 inhibitor,
boeravinone E (IC50 = 5.6 μM), was produced by yeast
elicitation of A. nana suspension cultures.16 Furthermore,
abronione, boeravinone C, deguelin,44 and abronisoflavone45
were isolated from the genus Abronia. Although their structures
are similar to those of C-methylrotenoids, the C-methylcou-
marinochromones bearing a furan ring instead of a pyran ring
have been rarely reported. To the best of our knowledge,
boeravinone J,46,47 triquetrumones A, B, and C,48 and
flemingichromone49 are the only reported C-methylcoumar-
inochromones so far. A. nana belongs to the plant family
Nyctaginaceae, which grows in the desert areas of North
America and has not been extensively studied yet. This is the
first study on the antisepsis effects of a C-methylcoumar-
inochromone isolated from the genus Abronia.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Ultraviolet (UV) and
Fourier-transform infrared spectroscopy (FT-IR) spectra were
acquired using Shimadzu UV-1800 (Kyoto, Japan) and Cary 630
FTIR (Agilent, Santa Clara, CA, USA) instruments, respectively. NMR
analysis was performed using a Bruker Avance Digital 500 NMR
spectrometer (Karlsruhe, Germany) with tetramethylsilane (0 ppm) as
F
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 1. Synthesis of Boeravinone Y (1)
an internal standard. HRFABMS was conducted using a JMS700 mass
spectrometer (JEOL, Tokyo, Japan). For plant extraction and
isolation, organic solvents, such as MeOH, EtOAc, and n-hexane,
were purchased from Duksan Chemical (Anseong, Korea).
s), 6.24 (1H, s), 3.84 (3H, s), 3.73 (3H, s), 2.69 (3H, s), and 2.06 (3H,
s).
(E)-3-(Dimethylamino)-1-(6-hydroxy-2,4-dimethoxy-3-
methylphenyl)prop-2-en-1-one (3). Compound 2 (1.45 g, 6.9 mM)
was diluted with dimethylformamide−dimethylacetal (7 mL, 4 equiv),
and the resulting mixture was stirred at 95 °C for 5 h. The volatile
compounds were evaporated in vacuo. The mixture was diluted with
H2O and extracted with diethyl ether. The organic layer was dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The crude
product was washed with a solution of n-hexane−diethyl ether (4:1) to
obtain the desired enamine 3 (1.65 g, 90%) as a yellow solid: 1H NMR
(CDCl3, 500 MHz) δ 14.59 (1H, s), 7.95 (1H, d, J = 12.4 Hz), 6.36
(1H, d, J = 12.4 Hz), 6.23 (1H, s), 3.81 (3H, s), 3.67 (3H, s), 3.17
(3H, s), 2.94 (3H, s), and 2.05 (3H, s).
Plant Material. Suspension cultures of A. nana were prepared as
previously described,16 using the seeds obtained from Alplains (Kiowa,
CO, USA). Briefly, the callus of A. nana was prepared by placing the
seeds onto solid Murashige-Skoog’s (MS) medium (pH 5.8)
supplemented with 30 g/L sucrose, 1 mg/L 2,4-diphenoxyacetic acid
(2,4-D), 3 mg/L 1-naphthaleneacetic acid (NAA), and 2 mg/L kinetin
at 25 °C. The induced callus was transferred into 100 mL of liquid MS
medium supplemented with 30 g/L sucrose, 1 mg/L 2,4-D, and 0.1
mg/L kinetin. The callus was cultured at 25 °C at 100 rpm in the dark
for 25 days. MS medium, sucrose, 2,4-D, NAA, and kinetin were
purchased from Sigma-Aldrich (St. Louis, MO, USA). The callus was
maintained at the Laboratory of Natural Products Medicine, College of
Pharmacy, Kyungpook National University (voucher number
KNUNPM AN-2009-001).
Extraction and Isolation. The 25-day-old suspension culture was
filtered to remove the culture medium (1 kg fresh weight), and fresh A.
nana cells were extracted with 5 L of MeOH twice for 2 h each. The
combined MeOH extract was suspended in 500 mL of deionized water
and partitioned with the same volume of EtOAc three times. The
EtOAc-soluble fraction was combined and evaporated to dryness (5
g). Five fractions (Fr. 1−Fr. 5) were obtained by medium-pressure
silica gel column chromatography (Merck, Art. 9385, 30 × 300 mm; n-
hexane−[EtOAc−MeOH, 1:1], 50:1 → 5:1; 4 mL/min; 254 nm). Fr.
2 was rechromatographed using a high-performance liquid chromatog-
raphy (HPLC) system (Vydac C18 column, 7.8 × 250 mm, 10 μm;
80% MeOH in water; 2.5 mL/min; 254 nm) to purify compound 1
(13 mg, purity >98% by HPLC under UV light at 280 nm).
3-Iodo-5,7-dimethoxy-6-methyl-4H-chromen-4-one (4). I2 (0.75
g, 3 mM) was added to a stirred suspension of 3 (0.56 g, 2.1 mM) in
20 mL of MeOH at 0 °C. The reaction mixture was stirred at room
temperature (rt) for 4 h. It was quenched with Na2S2O3, extracted with
CH2Cl2, dried over anhydrous MgSO4, and concentrated in vacuo. The
crude product was purified by silica gel column chromatography (n-
hexane−EtOAc, 3:1) to obtain the desired iodochromenone 4 (588
1
mg, 80%) as a white solid: H NMR (CDCl3, 500 MHz) δ 8.12 (1H,
s), 6.62 (1H, s), 3.91 (3H, s), 3.85 (3H, s), and 2.18 (3H, s).
5,7-Dimethoxy-3-(2-methoxyphenyl)-6-methyl-4H-chromen-4-
one (5). Na2CO3 (496 mg, 4.68 mM) and boronic acid (308 mg, 2.03
mM) were added to a solution of 4 (540 mg, 1.56 mM) in
dimethoxyethane−H2O (1:1, 12 mL). Palladium on carbon (5% Pd/C,
166 mg) was then added, and the reaction mixture was stirred at 45 °C
for 5 h. It was then filtered through a short Celite pad and washed
using CH2Cl2. The mixture was diluted with H2O, extracted with
CH2Cl2, dried over MgSO4, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography to yield
the isoflavonone 5 (498 mg, 98%) as a white solid: 1H NMR (CDCl3,
500 MHz) δ 7.79 (1H, s), 7.37−7.3 (2H, m), 7.01 (1H, td, J = 7.5, 1
Hz), 6.97 (1H, d, J = 8.3 Hz), 6.64 (1H, s), 3.92 (3H, s), 3.84 (3H, s),
3.79 (3H, s), and 2.19 (3H, s).
Boeravinone Y (1): gray amorphous powder; UV (MeOH) λmax
(log ε) 327 (3.8), 299 (3.7), and 256 (4.2) nm; IR (KBr) νmax 3411,
1
1618, 1448, 1286, 1117, and 1076 cm−1; H NMR (500 MHz) and
13C NMR (125 MHz), see Table 1; HRFABMS m/z 283.0609 [M +
H]+ (calcd for C16H11O5, 283.0606; Figure S5, Supporting
Information).
5,7-Dihydroxy-3-(2-hydroxyphenyl)-6-methyl-4H-chromen-4-one
(6). BBr3 (0.65 mL, 6.75 mM) was added to a solution of 5 (440 mg,
1.35 mM) in CH2Cl2 (20 mL) at 0 °C. The mixture was heated and
kept under reflux for 10 h. After cooling to rt, the reaction mixture was
quenched with NaHCO3, extracted with EtOAc, dried over anhydrous
MgSO4, and concentrated in vacuo. The crude product was purified by
silica gel column chromatography (n-hexane−EtOAc, 3:1) to obtain
Synthesis of 1. The procedure for the synthesis of 1 is shown in
Scheme 1. The intermediates were prepared as follows.
1-(6-Hydroxy-2,4-dimethoxy-3-methylphenyl)ethanone (2). A
solution of BCl3 (1 M in CH2Cl2; 8.8 mL, 8.8 mM) was added to a
stirred suspension of 3,5-dimethoxy-4-methylphenol (1.34 g, 8 mM) in
50 mL of CH2Cl2 at 0 °C. After 5 min, acetyl chloride (AcCl, 0.68 mL,
9.6 mM) was added dropwise. The mixture was heated and kept under
reflux for 4 h. Next, it was quenched with 1 N HCl (30 mL), extracted
with CH2Cl2, dried over anhydrous MgSO4, and concentrated in vacuo.
The crude product was purified by silica gel column chromatography
(n-hexane−EtOAc, 8:1) to yield the desired acetophenone 2 (1.46 g,
87%) as a light yellow oil: 1H NMR (CDCl3, 500 MHz) δ 13.44 (1H,
1
the desired isoflavonone 6 (340 mg, 89%) as a light yellow solid: H
NMR (DMSO-d6, 500 MHz) δ 13.15 (1H, s), 10.85 (1H, s), 9.42 (1H,
s), 8.21 (1H, s), 7.25−7.16 (2H, m), 6.89 (1H, dd, J = 8, 0.6 Hz), 6.84
(1H, td, J = 7.5, 1 Hz), 6.48 (1H, s), and 2.00 (3H, s).
9,11-Dihydroxy-10-methylcoumaronochromone (boeravinone Y,
1). 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (120 mg, 0.53 mM)
was added to a stirred solution of 6 (100 mg, 0.35 mM) in
G
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
tetrahydrofuran (5 mL), and the reaction mixture was heated at 50 °C
for 4 h. It was then cooled to rt, and the solvent was concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography (n-hexane−EtOAc, 4:1) to obtain the desired 1 (8
compartment chamber model, as previously described.56 Briefly,
HUVECs were plated (5 × 104 cells/well) in Transwells (pore size,
3 μm; diameter, 12 mm) for 3 days. The confluent monolayers were
treated with HMGB1 (1 μg/mL) for 16 h, followed by incubation with
1 for 6 h.
1
mg, 8%) as a white solid: H NMR (DMSO-d6, 500 MHz) δ 12.63
(1H, s), 10.52 (1H, s), 7.52 (1H, m), 7.32 (1H, m), 7.06−6.99 (2H,
In Vivo Permeability and Leukocyte Migration Assays. For
the in vivo study, mice were anesthetized as described above. Then,
they were treated with intravenous HMGB1 (2 μg/mouse) for 16 h,
followed by treatment with 1 (28 or 56 μg/kg) for 6 h. For the in vivo
permeability assay, 1% Evans blue in normal saline was intravenously
injected to each mouse. After 30 min, the mice were killed, and their
peritoneal exudates were collected by washing the peritoneal cavities
with 5 mL of normal saline and centrifuged at 200g for 10 min. The
absorbance of the supernatants was read at 650 nm. Vascular
permeability was determined using a standard curve, as previously
described,57,58 and expressed as μg dye that leaked into the peritoneal
cavity per mouse. For assessment of leukocyte migration, mice were
killed after 6 h, and their peritoneal cavities were washed with 5 mL of
normal saline. Samples (20 μL) of the peritoneal fluid were mixed with
m), 6.2 (1H, s), and 1.58 (3H, s).
Cell Culture, Differentiation, and Transfection. HUVECs were
obtained from Cambrex Bio Science (Charles City, IA, USA) and
maintained as previously described.21 Briefly, the cells were cultured in
endothelial growth basal medium (EBM-2) containing growth
supplements (Cambrex Bio Science) at 37 °C in 5% CO2 until they
reached confluency. All experiments were performed using HUVECs
passaged three or five times. U937 cells (an oncogenic human
monocyte cell line) were maintained at a density of 2 × 105 to 1 × 106
cells/mL in Roswell Park Memorial Institute 1640 medium containing
L-glutamine, 10% heat-inactivated fetal bovine serum, and antibiotics
(penicillin G and streptomycin). These monocytes can differentiate
into macrophages or dendritic cells,50 and their differentiation pattern
depends on the characteristics of the tissue microenvironment.51
Phorbol 12-myristate 13-acetate (PMA) is a phorbol ester that
commits monocytes to the macrophage lineage. Upon PMA treatment,
U937 cells undergo a series of morphological and functional changes.
To induce differentiation, U937 cells were resuspended in a fresh
medium (4 × 105 cells/mL) for 12 h and treated with PMA (10 ng/
mL; Sigma-Aldrich) for up to 24 h, as previously described.52
Animals and Husbandry. Male C57BL/6 mice (6−7 weeks old,
weighing 27 g) were purchased from Orient Bio Co. (Sungnam,
Korea) and acclimatized to the laboratory conditions for 12 days. Five
mice were housed per polycarbonate cage under controlled conditions
of temperature (20−25 °C), humidity (40−45%), and a 12:12 h light−
dark cycle. They were provided with a normal rodent pellet diet and
water ad libitum during acclimatization. All animal experiments were
carried out in accordance with the Guidelines for the Care and Use of
Laboratory Animals of Kyungpook National University (IACUC No.
KNU 2017-102).
Cecal Ligation and Puncture. Mice were anesthetized using 2%
isoflurane (Forane, JW Pharmaceutical, Seoul, Korea) in oxygen
delivered via a small rodent gas anesthesia machine (RC2, Vetequip,
Pleasanton, CA, USA) first in a breathing chamber and then via a
facemask. They were allowed to breathe spontaneously during the
procedure. A CLP-induced sepsis model was used as previously
described.53,54 Briefly, a 2 cm midline incision was made to expose the
cecum and adjoining intestine. The cecum was then tightly ligated with
a 3-0 silk suture at 5 mm from the cecal tip and punctured once using a
22-gauge needle for induction of high-grade sepsis.55 This was gently
squeezed to extrude a small amount of feces from the perforation site
and then returned to the peritoneal cavity. The laparotomy site was
then sutured with a 4-0 silk suture. In the sham control mice, the
cecum was exposed, but neither ligated nor punctured, and returned to
the abdominal cavity. This protocol was approved by the Animal Care
Committee at Kyungpook National University prior to the study (IRB
No. KNU 2017-102).
0.38 mL of Turk’s solution (0.01% crystal violet in 3% acetic acid), and
̈
the number of leukocytes was counted under a light microscope.
Determination of Phosphorylated p38 MAPK Levels. The
levels of phosphorylated p38 MAPK were determined in accordance
with the manufacturer’s instructions, using a commercially available
enzyme-linked immunosorbent assay (ELISA) kit (Cell Signaling
Technology, Danvers, MA, USA). The absorbance was measured using
a microplate reader.
Bacterial Count. Peritoneal lavage fluid and blood were collected
24 h after CLP. Samples were centrifuged at 13 000 rpm for 10 min.
Diluted samples were cultured in 5% sheep blood agar (Teknova,
Hollister, CA, USA) at 37 °C for 24 h. Bacterial count was determined
by counting the CFUs.
Expression of CAMs. The expression of VCAM-1, ICAM-1, and
E-selectin in HUVECs was determined by whole-cell ELISA, as
previously described.59,60 Briefly, confluent HUVEC monolayers were
treated with HMGB1 (1 μg/mL) for 16 h followed by treatment with
1 (0−1 μM) for 6 h. The medium was then removed, and the cells
were washed with phosphate-buffered saline (PBS) and fixed with 50
μL of 1% paraformaldehyde for 15 min at rt. After washing, 100 μL of
mouse anti-human monoclonal antibody (anti-VCAM-1, anti-ICAM-1,
and anti-E-selectin; Millipore, Temecula, CA, USA; 1:50 dilution
each) was added. After 1 h (37 °C, 5% CO2), the cells were washed
three times, and 100 μL of peroxidase-conjugated anti-mouse IgG
antibody (Sigma-Aldrich; 1:2000 dilution) was added for 1 h. The cells
were washed three times and developed using O-phenylenediamene
(Sigma-Aldrich) as a substrate. The absorbance was measured at 490
nm.
Cell−Cell Adhesion Assay. Adhesion of the monocytes to
endothelial cells was evaluated by fluorescent labeling of the
monocytes, as previously described.61−63 Briefly, human neutrophils
(1.5 × 106 cells/mL, 200 μL/well) were labeled with vybrant DiD dye.
Then, they were added to washed and stimulated HUVECs. HUVEC
monolayers were treated with HMGB1 (1 μg/mL) for 16 h, followed
by treatment with 1 (0−1 μM) for 6 h.
In Vitro Migration Assay. The migration assay was performed in
Transwell plates (pore size, 8 μm; diameter, 6.5 mm). HUVECs (6 ×
104 cells/well) were cultured for 3 days to obtain confluent endothelial
monolayers. Before addition of the monocytes to the upper
compartment, cell monolayers were treated with HMGB1 (1 μg/
mL) for 16 h, followed by treatment with 1 for 6 h. Cells in the upper
chamber of the filter were aspirated, and nonmigrating cells on the top
of the filter were removed using a cotton swab. Monocytes on the
lower chamber of the filter were fixed with 8% glutaraldehyde and
stained with 0.25% crystal violet in 20% MeOH (w/v). Each
experiment was carried out in duplicate, and within each well, nine
randomly selected high-power microscopic fields (200×) were
counted and expressed as the migration index.
Expression of Receptors. The same experimental procedures
were used for measuring the expression of the cell surface receptors
TLR2, TLR4, and RAGE using specific antibodies (A-9, H-80, and A-
9, respectively) obtained from Santa Cruz Biotechnology Inc. (Santa
Cruz, CA, USA).
Cell Viability Assay. Microculture tetrazolium (MTT) was used as
an indicator of cell viability. Cells were grown in 96-well plates at a
density of 5 × 103 cells/well. After 24 h, the cells were washed with
fresh medium and treated with compound 1. After 48 h of incubation,
the cells were washed, and 100 μL of MTT (1 mg/mL) was added,
followed by incubation for 4 h. Finally, 150 μL of dimethyl sulfoxide
(DMSO) was added to solubilize the formazan salt. The amount of
formazan salt was determined by measuring its optical density at 540
nm using a microplate reader (Tecan Austria GmbH, Grodig, Austria).
̈
In Vitro Permeability Assay. Permeability was quantitated by
spectrophotometric measurement of the flux of albumin-bound Evans
blue across functional HUVEC monolayers using a modified two-
Hematoxylin and Eosin Staining and Histopathological
Examination. Male C57BL/6 mice underwent CLP and were treated
with compound 1 (28 or 56 μg/kg) intravenously 12 and 50 h after
H
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
CLP (n = 5). They were sacrificed 96 h after CLP. To investigate the
changes in lung morphology, lung samples were removed from each
mouse, washed three times with PBS (pH 7.4) to remove any
remaining blood, and fixed in 4% formaldehyde (Junsei, Tokyo, Japan)
in PBS (pH 7.4) for 20 h at 4 °C. After fixation, the samples were
dehydrated using serial dilutions of ethanol, embedded in paraffin,
sectioned at 4 μm, and placed on a slide. The slides were
deparaffinized in an oven at 60 °C, rehydrated, and stained with
hematoxylin (Sigma-Aldrich). To remove the excess stain, the slides
were quickly dipped three times in 0.3% acid alcohol and
counterstained with eosin (Sigma-Aldrich). The excess stain was
removed using serial dilutions of ethanol and xylene, and then the
slides were coverslipped. Examination of the lung specimens was
conducted by a blinded observer using a light microscope to evaluate
the pulmonary architecture, tissue edema, and inflammatory cells’
infiltration, as previously defined.64
Measurement of Organ Injury Markers. The levels of AST,
ALT, BUN, creatinine, and LDH in fresh serum isolated from the
plasma of septic mice were measured using biochemical kits
(Mybiosource, San Diego, CA, USA). To determine the concen-
trations of IL-1β, IL-6, IL-10, MCP-1, and TNF-α, commercially
available ELISA kits were used according to the manufacturer’s
protocols (R&D Systems, Minneapolis, MN, USA). The absorbance
was measured using a microplate reader.
REFERENCES
■
(1) Weis, S.; Rubio, I.; Ludwig, K.; Weigel, C.; Jentho, E. Int. J. Mol.
Sci. 2017, 18, 1273.
(2) Stearns-Kurosawa, D. J.; Osuchowski, M. F.; Valentine, C.;
Kurosawa, S.; Remick, D. G. Annu. Rev. Pathol.: Mech. Dis. 2011, 6,
19−48.
(3) Buras, J. A.; Holzmann, B.; Sitkovsky, M. Nat. Rev. Drug Discovery
2005, 4, 854−865.
(4) Abraham, E.; Anzueto, A.; Gutierrez, G.; Tessler, S.; San Pedro,
G.; Wunderink, R.; Dal Nogare, A.; Nasraway, S.; Berman, S.; Cooney,
R.; Levy, H.; Baughman, R.; Rumbak, M.; Light, R. B.; Poole, L.;
Allred, R.; Constant, J.; Pennington, J.; Porter, S. Lancet 1998, 351,
929−933.
(5) Abraham, E.; Wunderink, R.; Silverman, H.; Perl, T. M.;
Nasraway, S.; Levy, H.; Bone, R.; Wenzel, R. P.; Balk, R.; Allred, R. J.
Am. Med. Assoc. 1995, 273, 934−941.
(6) Tracey, K. J.; Fong, Y.; Hesse, D. G.; Manogue, K. R.; Lee, A. T.;
Kuo, G. C.; Lowry, S. F.; Cerami, A. Nature 1987, 330, 662−664.
(7) Nightingale, K.; Dimitrov, S.; Reeves, R.; Wolffe, A. P. EMBO J.
1996, 15, 548−561.
(8) Scaffidi, P.; Misteli, T.; Bianchi, M. E. Nature 2002, 418, 191−
195.
(9) Wang, H.; Bloom, O.; Zhang, M.; Vishnubhakat, J. M.;
Ombrellino, M.; Che, J.; Frazier, A.; Yang, H.; Ivanova, S.;
Borovikova, L.; Manogue, K. R.; Faist, E.; Abraham, E.; Andersson,
J.; Andersson, U.; Molina, P. E.; Abumrad, N. N.; Sama, A.; Tracey, K.
J. Science 1999, 285, 248−251.
(10) Wang, H.; Ward, M. F.; Sama, A. E. Shock 2009, 32, 348−357.
(11) Yang, H.; Ochani, M.; Li, J.; Qiang, X.; Tanovic, M.; Harris, H.
E.; Susarla, S. M.; Ulloa, L.; Wang, H.; DiRaimo, R.; Czura, C. J.; Roth,
J.; Warren, H. S.; Fink, M. P.; Fenton, M. J.; Andersson, U.; Tracey, K.
J. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 296−301.
(12) Bae, J. S. Arch. Pharmacal Res. 2012, 35, 1511−1523.
(13) Park, J. S.; Svetkauskaite, D.; He, Q.; Kim, J. Y.; Strassheim, D.;
Ishizaka, A.; Abraham, E. J. Biol. Chem. 2004, 279, 7370−7377.
(14) Neeper, M.; Schmidt, A. M.; Brett, J.; Yan, S. D.; Wang, F.; Pan,
Y. C.; Elliston, K.; Stern, D.; Shaw, A. J. Biol. Chem. 1992, 267, 14998−
15004.
Statistical Analysis. All experiments were performed at least five
independent times. Student’s t-test was used for comparison. Values
were expressed as the means
standard deviation (SD). Kaplan−
Meier survival analysis was performed to determine the overall survival
rates. SPSS for Windows, version 16.0 (SPSS; Chicago, IL, USA) was
used to perform all statistical analyses. P values of <0.05 were
considered statistically significant.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00826.
1H and 13C NMR, HMQC and HMBC, HRFABMS, IR
spectra, and HPLC chromatogram of 1 (PDF)
(15) Yang, E. J.; Lee, W.; Song, K. S.; Bae, J. S. Biochem. Pharmacol.
2016, 110−111, 58−70.
(16) Kim, S. I.; Yang, E. J.; Park, S. H.; Kim, C. K.; Song, K. S. Appl.
Biochem. Biotechnol. 2014, 172, 3529−3537.
AUTHOR INFORMATION
Corresponding Authors
*Tel: 82-53-950-8565. Fax: 82-53-950-8557. E-mail: kssong@
knu.ac.kr (K.-S. Song).
■
(17) Park, S. H.; Yang, E. J.; Kim, S. I.; Song, K. S. Bioorg. Med. Chem.
Lett. 2014, 24, 2945−2948.
(18) Nazir, M.; Saleem, M.; Riaz, N.; Hafeez, M.; Sultan, M.; Jabbar,
A.; Ali, M. S. Nat. Prod. Commun. 2011, 6, 1651−1652.
(19) El Gazzar, M. Inflammation Res. 2007, 56, 162−167.
(20) Mullins, G. E.; Sunden-Cullberg, J.; Johansson, A. S.;
Rouhiainen, A.; Erlandsson-Harris, H.; Yang, H.; Tracey, K. J.;
Rauvala, H.; Palmblad, J.; Andersson, J.; Treutiger, C. J. Scand. J.
Immunol. 2004, 60, 566−573.
*Tel: 82-53-950-8565. Fax: 82-53-950-8557. E-mail: ejy125@
gmail.com (E.-J. Yang).
*Tel: 82-53-950-8570. Fax: 82-53-950-8557. E-mail: baejs@
knu.ac.kr (J.-S. Bae).
Author Contributions
§W. Lee and D. Lee contributed equally to this work.
(21) Bae, J. S.; Rezaie, A. R. Blood 2011, 118, 3952−3959.
(22) Chen, G.; Li, J.; Ochani, M.; Rendon-Mitchell, B.; Qiang, X.;
Susarla, S.; Ulloa, L.; Yang, H.; Fan, S.; Goyert, S. M.; Wang, P.;
Tracey, K. J.; Sama, A. E.; Wang, H. J. Leukocyte Biol. 2004, 76, 994−
1001.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(23) Diehl, K. H.; Hull, R.; Morton, D.; Pfister, R.; Rabemampianina,
Y.; Smith, D.; Vidal, J. M.; van de Vorstenbosch, C. J. Appl. Toxicol.
2001, 21, 15−23.
This study was supported by a grant from the National
Research Foundation of Korea, funded by the Korean
Government (NRF-2015R1A2A1A15055871, NRF-
2016R1A6A3A11933576, and NRF-2015M3A9D9067478), by
the Bio and Medical Technology Development Program of the
National Research Foundation (NRF) funded by the Ministry
of Science and ICT (2017M3A9G8083382), and by a grant of
the Korea Health Technology R and D Project through the
Korea Health Industry Development Institute (KHIDI),
funded by the Ministry of Health and Welfare, Republic of
Korea (grant number HI15C0001).
(24) Sama, A. E.; D’Amore, J.; Ward, M. F.; Chen, G.; Wang, H.
Acad. Emerg. Med. 2004, 11, 867−873.
(25) Wolfson, R. K.; Chiang, E. T.; Garcia, J. G. Microvasc. Res. 2011,
81, 189−197.
(26) Yang, H.; Wang, H.; Czura, C. J.; Tracey, K. J. J. Leukocyte Biol.
2005, 78, 1−8.
(27) Lee, W.; Ku, S. K.; Lee, Y. M.; Bae, J. S. Food Chem. Toxicol.
2014, 63, 1−8.
(28) Qin, Y. H.; Dai, S. M.; Tang, G. S.; Zhang, J.; Ren, D.; Wang, Z.
W.; Shen, Q. J. Immunol. 2009, 183, 6244−6250.
I
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(29) Sun, C.; Liang, C.; Ren, Y.; Zhen, Y.; He, Z.; Wang, H.; Tan, H.;
Pan, X.; Wu, Z. Basic Res. Cardiol. 2009, 104, 42−49.
(30) Sager, H. B.; Dutta, P.; Dahlman, J. E.; Hulsmans, M.; Courties,
G.; Sun, Y.; Heidt, T.; Vinegoni, C.; Borodovsky, A.; Fitzgerald, K.;
Wojtkiewicz, G. R.; Iwamoto, Y.; Tricot, B.; Khan, O. F.; Kauffman, K.
J.; Xing, Y.; Shaw, T. E.; Libby, P.; Langer, R.; Weissleder, R.; Swirski,
F. K.; Anderson, D. G.; Nahrendorf, M. Sci. Transl. Med. 2016, 8,
342ra380.
(58) Lee, J. D.; Huh, J. E.; Jeon, G.; Yang, H. R.; Woo, H. S.; Choi, D.
Y.; Park, D. S. Int. Immunopharmacol. 2009, 9, 268−276.
(59) Che, W.; Lerner-Marmarosh, N.; Huang, Q.; Osawa, M.; Ohta,
S.; Yoshizumi, M.; Glassman, M.; Lee, J. D.; Yan, C.; Berk, B. C.; Abe,
J. Circ. Res. 2002, 90, 1222−1230.
(60) Kim, T. H.; Ku, S. K.; Lee, I. C.; Bae, J. S. BMB Rep. 2012, 45,
200−205.
(61) Akeson, A. L.; Woods, C. W. J. Immunol. Methods 1993, 163,
181−185.
(31) Wang, Y. H. W.; Meyer, R. D.; Bondzie, P. A.; Jiang, Y.; Rahimi,
I.; Rezazadeh, K.; Mehta, M.; Laver, N. M. V.; Costello, C. E.; Rahimi,
N. J. Mol. Biol. 2016, 428, 5019−5033.
(62) Kim, I.; Moon, S. O.; Kim, S. H.; Kim, H. J.; Koh, Y. S.; Koh, G.
Y. J. Biol. Chem. 2001, 276, 7614−7620.
(63) Bae, J. W.; Bae, J. S. Inflammation Res. 2011, 60, 751−758.
(64) Ozdulger, A.; Cinel, I.; Koksel, O.; Cinel, L.; Avlan, D.; Unlu, A.;
Okcu, H.; Dikmengil, M.; Oral, U. Shock 2003, 19, 366−372.
(32) Cohen, J. Nature 2002, 420, 885−891.
(33) Astiz, M. E.; Rackow, E. C. Lancet 1998, 351, 1501−1505.
(34) Stenvinkel, P.; Ketteler, M.; Johnson, R. J.; Lindholm, B.;
Pecoits-Filho, R.; Riella, M.; Heimburger, O.; Cederholm, T.; Girndt,
M. Kidney Int. 2005, 67, 1216−1233.
(35) Riedemann, N. C.; Neff, T. A.; Guo, R. F.; Bernacki, K. D.;
Laudes, I. J.; Sarma, J. V.; Lambris, J. D.; Ward, P. A. J. Immunol. 2003,
170, 503−507.
(36) Gustot, T. Curr. Opin. Crit. Care 2011, 17, 153−159.
(37) Remick, D. G. Am. J. Pathol. 2007, 170, 1435−1444.
(38) Wynn, J.; Cornell, T. T.; Wong, H. R.; Shanley, T. P.; Wheeler,
D. S. Pediatrics 2010, 125, 1031−1041.
(39) Ranieri, V. M.; Thompson, B. T.; Barie, P. S.; Dhainaut, J. F.;
Douglas, I. S.; Finfer, S.; Gardlund, B.; Marshall, J. C.; Rhodes, A.;
Artigas, A.; Payen, D.; Tenhunen, J.; Al-Khalidi, H. R.; Thompson, V.;
Janes, J.; Macias, W. L.; Vangerow, B.; Williams, M. D. N. Engl. J. Med.
2012, 366, 2055−2064.
(40) Surbatovic, M.; Veljovic, M.; Jevdjic, J.; Popovic, N.; Djordjevic,
D.; Radakovic, S. Mediators Inflammation 2013, 2013, 362793.
(41) Abraham, E.; Arcaroli, J.; Carmody, A.; Wang, H.; Tracey, K. J. J.
Immunol. 2000, 165, 2950−2954.
(42) Andersson, U.; Wang, H.; Palmblad, K.; Aveberger, A. C.;
Bloom, O.; Erlandsson-Harris, H.; Janson, A.; Kokkola, R.; Zhang, M.;
Yang, H.; Tracey, K. J. J. Exp. Med. 2000, 192, 565−570.
(43) Wang, H.; Yang, H.; Czura, C. J.; Sama, A. E.; Tracey, K. J. Am.
J. Respir. Crit. Care Med. 2001, 164, 1768−1773.
(44) Starks, C. M.; Williams, R. B.; Norman, V. L.; Lawrence, J. A.;
Goering, M. G.; O’Neil-Johnson, M.; Hu, J. F.; Rice, S. M.; Eldridge,
G. R. Phytochem. Lett. 2011, 4, 72−74.
(45) Wollenweber, E.; Papendieck, S.; Schilling, G. Nat. Prod. Lett.
1993, 3, 119−122.
(46) Do, T. M.; Truong, A. V.; Pinnock, T. G.; Pratt, L. M.;
Yamamoto, S.; Watarai, H.; Guillaume, D.; Nguyen, K. P. Chem.
Pharm. Bull. 2013, 61, 624−630.
(47) Ahmed-Belkacem, A.; Macalou, S.; Borrelli, F.; Capasso, R.;
Fattorusso, E.; Taglialatela-Scafati, O.; Di Pietro, A. J. Med. Chem.
2007, 50, 1933−1938.
(48) Xiang, W.; Li, R. T.; Mao, Y. L.; Zhang, H. J.; Li, S. H.; Song, Q.
S.; Sun, H. D. J. Agric. Food Chem. 2005, 53, 267−271.
(49) Shiao, Y. J.; Wang, C. N.; Wang, W. Y.; Lin, Y. L. Planta Med.
2005, 71, 835−840.
(50) Lawrence, T.; Natoli, G. Nat. Rev. Immunol. 2011, 11, 750−761.
(51) Kigerl, K. A.; Gensel, J. C.; Ankeny, D. P.; Alexander, J. K.;
Donnelly, D. J.; Popovich, P. G. J. Neurosci. 2009, 29, 13435−13444.
(52) Garcia, A.; Serrano, A.; Abril, E.; Jimenez, P.; Real, L. M.;
Canton, J.; Garrido, F.; Ruiz-Cabello, F. Exp. Hematol. 1999, 27, 353−
364.
(53) Bae, J. S.; Lee, W.; Nam, J. O.; Kim, J. E.; Kim, S. W.; Kim, I. S.
Am. J. Respir. Crit. Care Med. 2014, 189, 779−786.
(54) Wang, H.; Liao, H.; Ochani, M.; Justiniani, M.; Lin, X.; Yang, L.;
Al-Abed, Y.; Metz, C.; Miller, E. J.; Tracey, K. J.; Ulloa, L. Nat. Med.
2004, 10, 1216−1221.
(55) Rittirsch, D.; Huber-Lang, M. S.; Flierl, M. A.; Ward, P. A. Nat.
Protoc. 2009, 4, 31−36.
(56) Qureshi, S. H.; Manithody, C.; Bae, J. S.; Yang, L.; Rezaie, A. R.
Biophys. Chem. 2008, 134, 239−245.
(57) Bae, J. S.; Lee, W.; Rezaie, A. R. J. Thromb. Haemostasis 2012,
10, 1145−1151.
J
DOI: 10.1021/acs.jnatprod.7b00826
J. Nat. Prod. XXXX, XXX, XXX−XXX