A concise synthesis of viscolin, and its anti-inflammatory effects through the suppression of iNOS, COX-2, ERK phosphorylation and proinflammatory cytokines expressions

Article abstract of DOI:10.1016/j.ejmech.2011.12.008

In the present report, a concise synthesis of viscolin (1) has been achieved. The anti-inflammatory effect of viscolin was investigated in vitro and in vivo. Viscolin blocked the expression of iNOS and COX-2, and it also inhibited the ERK for the activation of NF-κB in LPS-stimulated RAW 264.7 macrophages. Western blotting and immunohistochemical analysis revealed that viscolin decreased Carr-induced iNOS and COX-2 expressions. These results could help to deduce the anti-inflammatory mechanisms.

Full text of DOI:10.1016/j.ejmech.2011.12.008

European Journal of Medicinal Chemistry 48 (2012) 371e378
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
A concise synthesis of viscolin, and its anti-inflammatory effects through the
suppression of iNOS, COX-2, ERK phosphorylation and proinflammatory cytokines
expressions
,
,
Guan-Jhong Huang a 1, M. Vijaya Bhaskar Reddy b 1, Ping-Chung Kuo c, Chieh-Hung Huang b,
Hung-Cheng Shih b, E-Jian Lee d, Mei-Lin Yang b, Yann-Lii Leu e 1, Tian-Shung Wu b
,
,f,g,
*
a School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan, ROC
b Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan, ROC
c Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan, ROC
d Neurophysiology Laboratory, Neurosurgical Service, Departments of Surgery and Anesthesiology, and Institute of Biomedical Engineering, National Cheng Kung University Medical
Center and Medical School, Tainan 701, Taiwan, ROC
e Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan, ROC
f Department of Pharmacy, China Medical University, Taichung 404, Taiwan, ROC
g Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 404, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present report, a concise synthesis of viscolin (1) has been achieved. The anti-inflammatory effect
of viscolin was investigated in vitro and in vivo. Viscolin blocked the expression of iNOS and COX-2, and it
also inhibited the ERK for the activation of NF-kB in LPS-stimulated RAW 264.7 macrophages. Western
blotting and immunohistochemical analysis revealed that viscolin decreased Carr-induced iNOS and
COX-2 expressions. These results could help to deduce the anti-inflammatory mechanisms.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 9 September 2011
Received in revised form
14 November 2011
Accepted 4 December 2011
Available online 9 December 2011
Keywords:
Viscolin
Anti-Inflammatory
Macrophages
iNOS
COX-2
l-Carrageenin
1. Introduction
coloratum (Loranthaceae) [3], a hemiparasite herb used in tradi-
tional Chinese medicine for the treatment of a number of ailments
Inflammation leads to the signaling proteins in affected cells and
tissues. Inducible nitric oxide synthase (iNOS), catalyzes the
formation of nitric oxide (NO) from L-arginine [1]. Excess produc-
tion of NO has shown to be associated with a number of chronic
diseases, including asthma, rheumatoid arthritis, inflammatory
bowel disease, atherosclerosis, Alzheimer’s disease, and various
human cancers [2]. Thus, inhibition of NO synthesis and PGE2
production stands as an important therapeutic goal. Viscolin 1 is
a naturally occurring 1,3-diarylpropane, identified from Viscum
such as hemorrhage, gout, heart disease, epilepsy, arthritis and
hypertension [4,5]. The structural novelty of viscolin 1, were found
to exhibit potent and selective inhibition of superoxide anion
generation (O2ꢀꢁ) and elastase release induced with N-formyl-
methionyl-leucephenyl alanine combined with cytochalasin B
(fMLP/CB) in human neutrophils with IC50 values of 0.58 ꢂ 0.03 and
4.93 ꢂ 0.54
mg/mL, respectively [6]. Viscolin does not show struc-
tural similarity to any known phosphodiesterase inhibitor and thus
provides a new chemical skeleton in the development of PDE
inhibitors [6]. In addition, viscolin exhibits leukocyte inhibitory
activity by suppressing free radicals, possibly through modulation
of PKC activity and calcium mobilization, and NO production with
moderate free radical-scavenging effects that give viscolin the
potential to be anti-inflammatory agent for the treatment of
oxidative stress-induced diseases [6].
* Corresponding author. Department of Chemistry, National Cheng Kung
University, Graduate Institute of Natural Products, 1, Ta-Shiueh Rd.Tainan 701,
Taiwan, ROC. Tel.: þ886 6 2747538; fax: þ886 2 2740552.
E-mail address: tswu@mail.ncku.edu.tw (T.-S. Wu).
Among authors provided equal contributions to this work.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.008

372
G.-J. Huang et al. / European Journal of Medicinal Chemistry 48 (2012) 371e378
OMe
OMe
OMe
the assistance of the ortho-directing effect of the acyl substituent
[9,10]. The resulting 2-hydroxy-4,6-dimethoxybenzldehyde 6 was
again protected with benzyl group in basic dimethylformamide
solution to afford 7. Baeyer-Villiger oxidation of 7 with m-CPBA in
CH2Cl2 gave the ester [11], which was further hydrolyzed with
aqueous NaOH and methanol to furnish phenol 8. Finally the
phenol group in 8 was methylated with dimethyl sulfate to yield
colorless oil 9. In another pathway, production of the substrate 9
started from the commercially available vanillin 12. Treatment of 12
with Br2 in glacial acetic acid yielded 5-bromovanillin 13 in high
yields [12], which was converted to 5-hydroxyvanillin 14, by reflux
with copper powder and aqueous NaOH solution [13]. Methylation
of 14 with 1.1 equivalent of dimethyl sulfate gave the desired
benzaldehyde 15 in good yields and only trace amount of the
regioisomeric product, syringaldehyde were detected by TLC. This
by-product could be easily removed from the desired product by
over methylation of syringaldehyde to 3,4,5-trimethoxybenzal
dehyde, and alkaline extraction followed by acidification of the
reaction mixture. Compound 15 was protected with benzyl group
in basic acetone solution to afford 16 and further Baeyer-Villiger
oxidation of 16 with m-CPBA in CH2Cl2 gave the ester, which was
hydrolyzed in basic solution to afford phenol 17. This reaction crude
was directly subjected into methylation with dimethyl sulfate to
yield 9 in 30%. Both the routes for the preparation of substrate 9
provided the desired products in good yields ranging from 12 to
27%. Although it offered the substrate 9 with the lower yield in the
second synthetic route, the starting material 12 was cheaper and
more economic for the large scale production of the final products
viscolin (1).
HO
OMe
OH
HO
HO
OMe
OH
OMe
OMe
OMe
OMe
O
1
2
OMe
CHO
OH
+
H3C
OMe
OMe
O
3
4
Fig. 1. Retrosynthetic analysis of viscolin 1.
It has been reported that inflammatory effect induced by Carr
could be associated with free radicals in the previous literature [7].
To explore the in vitro and in vivo anti-inflammatory effects of
viscolin, in the present study we reported a concise synthesis of
viscolin by a strategy distinctly different from that of our previous
report [8]. With the assistance of this concise synthetic method, the
anti-inflammatory effects of viscolin on LPS-induced in RAW 264.7
cells and Carr-induced on paw edema in mice could be examined.
The levels of iNOS and COX-2 in either RAW 264.7 cell or paw
edema were also detected and the activities of CAT, SOD, and GPx at
the 5th hr after Carr injection were also investigated to elucidate
the relationship between the anti-inflammatory mechanism of
viscolin and antioxidant enzymes.
Vilsmeier reaction of 9 with dimethylformamide and phos-
phorus oxychloride gave the mixtures of two isomeric aldehydes 10
and 11 purified by silica gel column chromatography in 51% and 16%
yields, respectively. In addition, compound 18 was protected with
benzyl group in basic dimethylformamide solution to afford 19.
Coupling of aldehydes 10 and 4-O-benzyl-3-methoxyacetophenone
19 was achieved with the aid of Aldol condensation in basic
condition to afford chalcone 20 (Fig. 3). This could be further
reduced by hydrogenation with the catalyst of 10% Pd/C in ethyl
acetate-methanol (9:1) solution to produce viscolin 1 in fewer steps
and better yields (7 steps, 8%), compared with the previous report
(15 steps, 6%) [8].
2. Chemistry
The retrosynthetic analysis of viscolin 1 was illustrated in Fig. 1.
It displayed that hydrogenation of the intermediate 2 which was
prepared through the aldol condensation of the key precursors 3
and 4 could approach the target compound 1. Thus our study
commenced with the preparation of the substrate 9 for the for-
mylation reaction to the analog 10 of the precursors 3 as shown in
Fig. 2. The tetra-oxygenated benzenoid 9 could be synthesized in
two pathways. In the first route, the commercially available 2,4,6-
trimethoxybenzaldehyde
thylated induced by BBr3 as a Lewis acid in ortho-position with
5 was regioselectively mono-deme
OMe
OMe
OMe
R3
R2
R1
R1
R2
OMe
OBn
OHC
MeO
OMe
OBn
+
MeO
MeO
i
OMe
CHO
5 R1 = CHO, R2 = OCH3
6 R1 = CHO, R2 = OH
11
12 R1 = CHO R2 = OH R3 = H
13 R1 = CHO R2 = OH R3 = Br
14 R1 = CHO R2 = OH R3 = OH
15 R1 = CHO R2 = OMe R3 = OH
16 R1 = CHO R2 = OMe R3 = OBn
10
v
ii
vi
7 R1 = CHO, R2 = OBn
iii
vii
8 R1 = OH, R2 = OBn
9 R1 = OMe, R2 = OBn
iv
ii
iii
9
R1 = OMe R2 = OMe R3 = OBn
Reagents and conditions: (i) BBr3 in 1.0M CH2Cl2, -78OC, 1.5 h. (ii) BnCl, DMF, K2CO3,
reflux, 6h. (iii) (1) m-CPBA, CH2Cl2, room temp.(2) NaOH-MeOH, room temp. 3 h. (iv)
(CH3)2SO4, K2CO3, reflux, 4h. (v) Br2/acetic acid, room temp. (vi) NaOH soulution in Cu
powder, reflux. (vii) dimethyl sulphate, Na2CO3, reflux.
Fig. 2. Preparation of the precursors 9 and 10.

G.-J. Huang et al. / European Journal of Medicinal Chemistry 48 (2012) 371e378
373
OMe
3.2. Effects ofviscolin onthe LPS-stimulated activation of iNOS, COX-2,
and mitogen-activated protein kinases (MAPKs)
R
HO
OMe
OH
(i) 10, 30% KOH, Methanol
CH3
MeO
OMe
(ii) 10% Pd/C, EtOAc/MeOH
O
The pathology of inflammation is initiated by complex processes
triggered by microbial pathogens such as LPS, which is a prototyp-
ical endotoxin. LPS can directly activate macrophages, which trigger
OMe
18 R = OH
1
ii
19 R = OBn
the production of inflammatory mediators, such as NO, TNF-
leukotrienes [15]. The pharmacological reduction of LPS-inducible
inflammatory mediators (for example NO and TNF- ) is regarded
a and
Fig. 3. Synthesis of viscolin 1.
a
as one of the essential conditions to alleviate a variety of disorders
caused by activation of macrophages. Thus, RAW 264.7 macro-
phages provide us with an excellent model for anti-inflammatory
drug screening and for subsequently evaluating the inhibitors of
the pathways that lead to the induction of proinflammatory
enzymes and to the production of proinflammatory cytokines [16].
3. Results and discussion
3.1. Effects of viscolin on LPS-induced NO, TNF-
in RAW 264.7 macrophages
a, and PGE2 production
The RAW 264.7 cells were incubated for 24 h with 100 ng/mL of
LPS (lipopolysaccharide) in the absence or presence of viscolin (0, 5,
10, and 20 mM). Viscolin was added 1 h before incubation with LPS.
Cell viability assay was performed using MTT assay. Nitrite
concentration in the medium was determined using Griess reagent.
TNF-a and PGE2 levels in the medium were determined using ELISA
kit. Cells cultured with or without viscolin did not change cell
viability significantly (Fig. S1A). NO plays a role as neurotrans-
mitter, vasodilator, and immune regulator in a variety of tissues at
physiological concentration. High levels of NO produced by iNOS
have been defined as a cytotoxic molecule in inflammation [1]. In
the present study, effects of viscolin on LPS-induced NO production
in RAW 264.7 macrophages were investigated. Nitrite accumulated
in the culture medium was estimated by the Griess reaction as an
index for NO release from the cells. Viscolin did not interfere with
In order to investigate whether the inhibitions of NO, TNF-a, and
PGE2 production were due to the decreased iNOS and COX-2 protein
levels, the effects of viscolin on iNOS and COX-2 protein expression
were studied by immunoblot. The results exhibited that incubation
with viscolin (0, 5, 10, and 20
mL) for 24 h inhibit iNOS and COX-2 proteins expression in mouse
macrophage RAW 264.7 cells in dose-dependent manner
(Fig. S2A). The detection of -actin was also performed in the same
mM) in the presence of LPS (100 ng/
a
b
blot as an internal control. The intensity of protein bands were
analyzed using Kodak Quantity software in three independent
experiments and showed an average of 83.8 and 70.6% down-
regulation of iNOS and COX-2 proteins, respectively, after treat-
ment with viscolin at 20
(Fig. S2B). These in vitro data showed that viscolin suppressed
LPS-induced productions of NO, TNF- , and PGE2, which are the
mM compared with the LPS-alone
a
the reaction between nitrite and Griess reagents at 20
m
M (data not
expression products of inflammatory protein such as iNOS and
COX-2.
MAPKs play critical roles in the regulation of cell growth and
differentiation, and control cellular responses to cytokines and
stresses. In particular, ERK, p38, and JNK are known to be impor-
shown). Unstimulated macrophages after 24 h of incubation in
culture medium produced background levels of nitrite. When RAW
264.7 macrophages were treated with different concentrations of
viscolin (0, 5, 10, and 20 mM) together with LPS (100 ng/mL) for
24 h, a significant concentration-dependent inhibition of nitrite
production was detected (Fig. S1B). There was either a significant
tant for the activation of NF-
kB [17]. To explore whether the
inhibition of NF- B activation by viscolin is mediated through the
k
decrease in the nitrite production of group treated with 5
colin (p < 0.05), or highly significant decrease of groups treated
respectively with 10 and 20 M of viscolin when compared with the
LPS-alone group (p < 0.01 or p < 0.001). The IC50 value for inhibi-
tion of nitrite production of viscolin was 17.80 ꢂ 1.52 M.
TNF- plays an important role in the promotion of the inflam-
matory response, which in turn causes many clinical problems
associated with autoimmune disorders, such as rheumatoid
arthritis, Crohn’s disease, psoriasis, and asthma [14]. After treat-
m
M vis-
MAPK pathway, MAPK phosphorylation was examined by Western
blot in RAW 264.7 cells pretreated with viscolin and then with LPS.
As shown in Fig. S3, we showed that ERK, JNK, and p38 were
phosphorylated with LPS stimulation. Furthermore, phosphoryla-
tion of ERK was inhibited by viscolin at 30 min of LPS stimulation,
whereas there was no effect of viscolin on p-p38 or p-JNK. MAPKs
are also likely targets for the development of novel anti-
inflammatory drugs; however, signaling from MAPKs to tran-
scription factors mediating iNOS and COX-2 expression is not fully
understood.
m
m
a
ment with LPS (100 ng/mL) for 24 h, the TNF-a concentration
increased in the medium. When RAW 264.7 macrophages were
treated with different concentrations of viscolin (0, 5, 10, and
3.3. Effects of viscolin on Carr-induced mice paw edema
20
concentration-dependent inhibition of TNF-
(Fig. S1C). There was either a significant decrease in the TNF-
of group treated with 5 M viscolin (p < 0.05), or highly significant
decrease of groups treated respectively with 10 and 20 M of vis-
colin when compared with the LPS-alone group (p < 0.01 or
p < 0.001). The IC50 value for inhibition of TNF- level of viscolin
M.
m
M) together with LPS (100 ng/mL) for 24 h, a significant
level was detected
level
a
Since viscolin effectively inhibited iNOS and COX-2 expressions
in macrophages, studies were extended to determine whether
viscolin affected acute phase inflammation in animal models. In the
present study, the Carr-induced edema model was performed due
to its widely adaptation for screening the effects of anti-
inflammatory drugs. Carr-induced paw edema is shown in
Fig. S4A. Viscolin (8 mg/kg) significantly inhibited (p < 0.001) the
development of paw edema-induced by Carr after 4th and 5th hr of
treatment, comparable with the reference compound Indo (10 mg/
kg). Similarly, the MDA level increased significantly in the edema
paw at 5th hr after Carr injection (p < 0.001). However, the MDA
level was decreased significantly by treatment with viscolin (8 mg/
kg) (p < 0.001) as well as 10 mg/kg Indo (Fig. S4B). In addition, the
a
m
m
a
was 17.32 ꢂ 0.08
m
An increase of PGE2 production has been demonstrated by LPS
treatment. After treatment with LPS (100 ng/mL) for 24 h, the
amount of PGE2 elevated clearly in the medium, and viscolin at 10
or 20 mM in the presence of LPS was able to significantly suppress
the LPS-induced production of PGE2 in RAW 264.7 macrophages
when compared with the LPS-alone group (p < 0.01 or p < 0.001)
(Fig. S1D). The IC50 value for inhibition of PGE2 level of viscolin was
NO and TNF-
a levels increased significantly in the edema serum at
about 14.37 ꢂ 0.12
mM.
5th hr after Carr injection (p < 0.001). Viscolin (8 mg/kg)

374
G.-J. Huang et al. / European Journal of Medicinal Chemistry 48 (2012) 371e378
Table 1
significantly decreased the serum NO and TNF-a levels (p < 0.001).
Effects of viscolin and indomethacin (Indo) on changes in CAT, SOD, and GPx
activities in Carr-induced paw edema (5th hr) in mice.
The inhibitory potency was similar to that of Indo (10 mg/kg) at 5th
hr after induction (Fig. S4C and D).
Groups
CAT
SOD
GPx
The Carr-induced rat paw edema is a suitable test for evaluating
anti-inflammatory drugs and has frequently been used to assess the
anti-edematous effect of natural products [18]. The degree of
swelling of the Carr-injected paws was maximal 3rd hr after
injection. Statistical analysis revealed that viscolin and Indo
significantly inhibited the development of edema 4 h after treat-
ment (p < 0.001). They both showed anti-inflammatory effects in
Carr-induced mice edema paw. It is well known that the third phase
of the edema-induced by Carr, in which the edema reaches its
highest volume, is characterized by the presence of prostaglandins
and other compounds of slow reaction found that the injection of
Carr into the rat paw induces the liberation of bradykinin, which
later induces the biosynthesis of prostaglandin and other autacoids,
which are responsible for the formation of the inflammatory
(U/mg protein) (U/mg protein) (U/mg protein)
Control
Carr
Carr þ Indo (10 mg/kg)
5.26 ꢂ 0.14
3.54 ꢂ 0.15a
4.65 ꢂ 0.17c
4.31 ꢂ 0.16
1.65 ꢂ 0.13a
3.85 ꢂ 0.24c
2.45 ꢂ 0.12b
3.46 ꢂ 0.11c
4.08 ꢂ 0.19a
12.96 ꢂ 0.12
7.21 ꢂ 0.07a
10.45 ꢂ 0.09c
8.64 ꢂ 0.05b
10.97 ꢂ 0.11c
11.54 ꢂ 0.16a
Carr þ viscolin (2 mg/kg) 3.92 ꢂ 0.13b
Carr þ viscolin (4 mg/kg) 4.28 ꢂ 0.11c
Carr þ viscolin (8 mg/kg) 4.98 ꢂ 0.23a
Each value represents as mean ꢂ S.E.M.
a
p < 0.001 as compared with the control group.
p < 0.05 and.
b
c
p < 0.01 as compared with the Carr (l-carrageenan) group (one-way ANOVA
followed by Scheffe’s multiple range test).
3.5. Effects of viscolin on Carr-induced iNOS and COX-2 protein
expressions in mice paw edema
exudates [19]. The proinflammatory cytokines such as TNF-
small secreted proteins, which mediate and regulate immunity and
inflammation. The production of TNF- is crucial for the synergistic
induction of NO synthesis in IFN- and/or LPS-stimulated macro-
phages. TNF- induces a number of physiological effects including
septic shock, inflammation, and cytotoxicity [20]. Also, TNF- is
a are
a
To investigate whether the inhibition of NO production was due
to a decreased iNOS and COX-2 protein levels, the effects of viscolin
on iNOS and COX-2 proteins expression were studied by Western
blot. The results showed that injection of viscolin (8 mg/kg) on
Carr-induced for 5th hr inhibited iNOS and COX-2 proteins
expression in mouse paw edema (Fig. S5A). The intensity of protein
bands were analyzed using Kodak Quantity software in three
independent experiments and showed an average of 63.6% and
76.2% down-regulation of iNOS and COX-2 proteins, respectively,
after the treatment with viscolin compared with the Carr-induced
alone (Fig. S5B). In addition, the protein expressions displayed an
average of 69.1 and 73.1% down-regulation of iNOS and COX-2
protein after the treatment with Indo at 10.0 mg/kg compared
with the Carr-induced alone. The down-regulation of iNOS and
COX-2 activity of viscolin (8 mg/kg) exhibited as well as Indo
(10.0 mg/kg) did.
g
a
a
a mediator of Carr-induced inflammatory incapacitation, and is able
to induce the further release of kinins and leukotrienes, which is
suggested to be an important role in the maintenance of long-
lasting nociceptive response [21]. In the above results, viscolin
significantly decreased the TNF-a level in serum after Carr injection
by treatment with 2, 4, and 8 mg/kg, respectively. It suggested that
anti-inflammatory effects of viscolin were resulted from the inhi-
bition of the proinflammatory cytokines in the Carr-induced paw
edema.
3.4. Effects of viscolin on activities of antioxidant enzymes
The acute inflammatory response is associated with the
production of reactive oxygen species (ROS), which have been
proposed to mediate cell damage in the paw tissue. At 5th hr after
the intrapaw injection of Carr, paw tissues were analyzed for the
biochemical parameters, such as CAT, SOD, and GPx activities. CAT,
SOD, and GPx activities in paw tissue were decreased significantly
by Carr administration. CAT, SOD, and GPx activity were increased
significantly after treated with 8 mg/kg viscolin and 10 mg/kg Indo
(p < 0.01) (Table 1). This local acute inflammation model induces
a biphasic edema consisting of an early phase (up to 2 h) followed
by a more sustained late phase (2e6 h). The early phase of Carr
edema is related to the production of immediate inflammation
mediators such as histamine, bradykinin, leukotrienes, platelet-
activating factor and cyclooxygenase products in the inflamed
tissue. The late phase is related to neutrophil infiltration and the
production of ROS. In a number of pathophysiological conditions
associated with inflammation or oxidant stress, these ROS have
been proposed to mediate cell damage via a number of indepen-
dent mechanisms including the initiation of lipid peroxidation, the
inactivation of a variety of antioxidant enzymes and depletion of
glutathione. Giving the importance of the oxidative status in the
formation of edema, the anti-inflammatory effect exhibited by
drug in this model might be related to its antioxidant properties
[22]. In our study, there is a significantly increment in CAT, SOD,
and GPx activities with viscolin treatment. Furthermore, signifi-
cant decreases in MDA level with viscolin treatment were also
found. These results indicated that the suppression of MDA
production is probably due to the enhancements of CAT, SOD, and
GPx activities.
3.6. Histological examination
Paw biopsies of the control mice displayed marked cellular
infiltration in the connective tissue. The infiltrates accumulated in
collagen fibers and intercellular spaces. Paw biopsies of mice
treated with viscolin (8 mg/kg) showed a reduction in inflamma-
tory responses induced by Carr. Histologically, inflammatory cells
were reduced in number and confined to the surroundings of the
vascular areas. Intercellular spaces did not show any cellular infil-
trations. Collagen fibers were regular in shape and exhibited
a reduction in intercellular spaces. Moreover, the hypodermis
connective tissues were not damaged (Fig. S6A). Neutrophils were
increased with Carr treatment (Fig. S6B). Indomethacin and viscolin
(8 mg/kg) could decrease the neutrophils numbers as compared to
the Carr-treated group (Fig. S6C and D, respectively). At 5th h after
intraplantar Carr injection, numerous iNOS and COX-2 immuno-
reactive cells were observed in the brown site of paw tissue
(Fig. S6EeH). Administration of indomethacin and viscolin (8 mg/
kg) 30 min prior to the Carr injection markedly reduced the
increase in iNOS and COX-2 immunoreactive cells in paws
(Fig. S6IeL).
4. Conclusion
In the present study, a concise synthesis of viscolin has been
achieved to construct the 1,3-diarylpropane skeleton and main-
tain the hydroxyl groups at para position in both rings. In addi-
tion, we demonstrated anti-inflammatory activities of viscolin in

G.-J. Huang et al. / European Journal of Medicinal Chemistry 48 (2012) 371e378
375
both in vitro and in vivo experimental systems, using LPS-
stimulated RAW 264.7 macrophages and a mouse model of
topical inflammation respectively. The anti-inflammatory mech-
anism of viscolin may be related to iNOS, COX-2, and it is asso-
ciated with the increase in the activities of antioxidant enzymes
(CAT, SOD, and GPx). Dual inhibitory activities against iNOS as
shown in in vitro assays appear to confer on viscolin a potent
in vivo efficacy in mouse, Carr-induced, paw edema, comparable
with a potent and well known COX inhibitor, Indo, suggesting
that viscolin may be used as a pharmacological agent in the
prevention or treatment of disease in which free radical forma-
tion in a pathogenic factor.
5.2.3. 2-(Benzyloxy)-4,6-dimethoxyphenol (8)
To a solution of 7 (10.0 g, 36.8 mmol) in CH2Cl2 at room
temperature, the m-CPBA (9.48 g, 55.1 mmol) was added slowly in
portions. The reaction mixture was stirred for 4 h at room
temperature and then the CH2Cl2 was removed under reduced
pressure to obtain crude ester. This was hydrolyzed by NaOH (10%,
30 mL) in MeOH (30 mL) at room temperature stirring for 3 h. The
reaction mixture was neutralized with 2 M HCl and extracted with
EtOAc. The crude was further purified by column chromatography
over a silica gel using n-hexane:EtOAc (8:2) to obtain brown red oil
(5.2 g, 54%); IR (neat) nmax 3460, 2943, 1697, 1600, 1512, 1458, 1319,
1149, 1103, 802 cmꢁ1
; d 7.43e7.31 (5H,
1HNMR (300 MHz, CDCl3)
m), 6.21 (1H, s), 6.20 (1H, s), 5.09 (2H, s), 3.85 (3H, s), 3.71 (3H, s);
13CNMR (75 MHz, CDCl3)
d 152.8, 147.5, 146.2, 136.6, 129.5, 128.5,
5. Experimental section
128.1, 127.6, 93.4, 92.4, 71.4, 56.1, 55.6; EIMS m/z (rel. int.): 260 [M]þ
(14), 169 (100), 141 (39), 91 (66), 69 (12), 65 (14); HREIMS calcd for
C15H16O4 [M]þ 260.1049, found 260.1052.
5.1. General
Unless stated otherwise, the chemicals were acquired from
commercial sources and used without further purification. Melting
points were measured on a Yanaco MP-S3 micro melting point
apparatus and are uncorrected. IR spectra were determined on
a Shimadzu FT-IR Prestige 21 spectrophotometer. 1H and 13CNMR
spectra were recorded on a Bruker Avance 300 spectrometer, using
tetramethylsilane (TMS) as internal standard; all chemical shifts are
5.2.4. 1-(Benzyloxy)-2,3,5-trimethoxybenzene (9) from 8
To a solution of 8 (10.0 g, 38.5 mmol), anhydrous K2CO3 (2.7 g,
19.5 mmol) and dimethyl sulfate (5.0 g, 39.6 mmol) in DMF (50 mL)
was stirred at 80 ꢃC for 6 h, cooled to room temperature, and then
poured into H2O (150 mL) and extracted with EtOAc. The crude
product was further purified by silica gel column chromatography
(n-hexane/EtOAc) to give 9 (6.2 g, 59%) as colorless oil; IR (neat)
nmax 2939, 2835, 1600, 1504, 1458, 1431, 1381, 1230, 1060, 948, 813,
reported in parts per million (ppm, d). EIMS and HREIMS spectra
were obtained on a VG 70-250S mass spectrometer. Column
chromatography was performed on silica gel (70e230 mesh,
230e400 mesh). TLC was conducted on pre-coated Kieselgel 60
F254 plates (Merck), and the spots were examined under UV light
and revealed by a sulfuric acid-anisaldehyde spray.
744, 698 cm1; 1HNMR (300 MHz, CDCl3)
d 7.38 (5H, m), 6.16 (2H, d,
J ¼ 3.0 Hz), 5.09 (2H, s), 3.80 (6H, s), 3.69 (3H, s); 13CNMR (75 MHz,
CDCl3)
d 155.8, 153.6, 152.5, 136.9, 132.7, 128.2, 127.6, 127.0, 93.4,
92.1, 70.8, 60.7, 55.7, 55.1; EIMS m/z (rel. int.): 274 [M]þ (59), 183
(22), 155 (98), 125 (19), 91 (100); HREIMS calcd for C16H18O4 [M]þ
274.1205, found 274.1208.
5.2. Chemistry
5.2.5. 3-Bromo-4-hydroxy-5-methoxybenzaldehyde (13)
5.2.1. 2-Hydroxy-4,6-dimethoxybenzaldehyde (6)
To a solution of vanillin 12 (15.2 g, 100 mmol) in glacial acetic
acid (75 mL) was added bromine (17.6 g, 110 mmol). After stirring
for 1.5 h, the reaction mixture was diluted with ice water (200 mL).
Precipitates were formed and filtered, washed with H2O, and dried
to give 5-bromvanillin 13 (21.5 g, 94%) as colorless solid, m.p.
162e164 ꢃC; IR (neat) nmax 3278, 1674, 1585, 1496, 1423, 1350, 1284,
To a solution of 2,4,6-trimethoxybenzaldehyde (5) (10.0 g,
51.0 mmol) in dry CH2Cl2 (80 mL) was added drop wise BBr3 (30 mL,
1.0 M in CH2Cl2) at ꢁ78 ꢃC. The reaction mixture was allowed to
warm up to room temperature, poured into ice, and the aqueous
portion was extracted with EtOAc and dried. It was further purified
by column chromatography eluted with n-hexane:EtOAc (8:2)
afforded 6 (8.25 g, 89%) as colorless solid, mp 67e69 ꢃC; IR (neat)
1157, 1041, 968, 852, 786 cmꢁ1
;
1HNMR (300 MHz, DMSO-d6)
d
10.72 (1H, s, OH), 9.75 (1H, s, CHO), 7.69 (1H, s), 7.39 (1H, s), 3.89
nmax 2978, 1643, 1423, 1303, 1219, 1157, 1045, 937, 798 cmꢁ1
1HNMR (300 MHz, DMSO-d6)
12.36 (1H, s, OH), 9.98 (1H, s, CHO),
6.11 (1H, d, J ¼ 2.1 Hz), 6.06 (1H, d, J ¼ 2.1 Hz), 3.84 (3H, s), 3.82 (3H,
;
(3H, s); 13CNMR (75 MHz, DMSO-d6)
d 190.3, 149.7, 148.6, 128.9,
d
128.7, 109.5, 109.2, 56.3; EIMS m/z (rel. int.): 231 [M]þ (82), 230
(100), 229 (74), 187 (12); HREIMS calcd for C8H7O3Br [M]þ
229.9579, found 229.9581.
s); 13CNMR (75 MHz, DMSO-d6)
d 191.4, 168.1, 165.2, 163.4, 105.3,
93.2, 90.7, 56.1, 55.9; EIMS m/z (rel. int.): 182 (100) [M]þ, 181 (67),
164 (34), 151 (21), 136 (17), 69 (18); HREIMS calcd for C9H10O4 [M]þ
182.0579, found 182.0577.
5.2.6. 3,4-Dihydroxy-5-methoxybenzaldehyde (14)
5-Bromovanilin 13 (20 g, 91 mmol), NaOH (24.5 g, 610 mol)
and copper powder (0.1 g, 1.6 mmol) were slurred into water
(300 mL). The reaction mixture was heated at reflux for
24e27 h. Disodium hydrogen phosphate (0.45 g, 3.2 mmol) was
added at the last half hour of reflux. The reaction was then
cooled at 50 ꢃC, filtered to remove a precipitate of cupric
hydrogen phosphate and acidified with HCl (46 g). The reaction
mixture was extracted with EtOAc and separated by silica gel
column chromatography using n-hexane:EtOAc (6:4) as eluents
to yield 14 (9.5 g, 62%) as a colorless solid, m.p. 132e134 ꢃC; IR
(neat) nmax 3278, 1674, 1593, 1523, 1462, 1334, 1207, 1141, 1091,
5.2.2. 2-(Benzyloxy)-4,6-dimethoxybenzaldehyde (7)
A mixture of 6 (10.0 g, 55.0 mmol), 1-(chloromethyl)benzene
(6.96 g, 55.0 mmol), and anhydrous K2CO3 (3.8 g, 27.5 mmol) in
DMF (50 mL) were stirred at 80 ꢃC for 6 h. The mixture was
extracted with EtOAc and dried, and then concentrated to obtain
suspension liquid. As hexane was added into this liquid, white
solid precipitated and washed with water to obtain 7 (14.2 g,
95%), m.p. 91e93 ꢃC; IR (neat) nmax 2943, 2858, 1678, 1600, 1581,
1458, 1327, 945, 698, 648 cmꢁ1; 1HNMR (300 MHz, CDCl3)
(1H, s, CHO), 7.32 (5H, m), 6.08 (1H, s), 6.02 (1H, s), 5.08 (2H, s),
3.81 (3H, s), 3.77 (3H, s); 13CNMR (75 MHz, CDCl3)
187.1, 165.7,
163.2, 162.9, 135.7, 128.1, 127.5, 126.5, 108.6, 91.2, 90.2, 70.1, 55.5,
55.0; EIMS m/z (rel. int.): 272 [M]þ (23), 243 (40), 181 (35), 92 (14),
91 (100), 61 (40); HREIMS calcd for C16H16O4 [M]þ 272.1049,
found 272.1047.
d 10.38
1002, 840, 717 cmꢁ1
; d 9.68 (1H, s,
1HNMR (300 MHz, DMSO-d6)
d
CHO), 9.50 (1H, s, OH), 9.44 (1H, s, OH), 7.01 (2H, s), 3.81 (3H, s,
OMe); 13CNMR (75 MHz, DMSO-d6)
d 191.3, 148.5, 146.0, 141.1,
127.4, 110.9, 105.0, 56.0; EIMS m/z (rel. int.): 168 [M]þ (100), 167
(79), 125 (18), 97 (16); HREIMS calcd for C8H8O4 [M]þ 168.0423,
found 168.0423.

376
G.-J. Huang et al. / European Journal of Medicinal Chemistry 48 (2012) 371e378
5.2.7. 3-Hydroxy-4,5-dimethoxybenzaldehyde (15)
56.1, 56.0; EIMS m/z (rel. int.): 302 [M]þ (70), 273 (30), 259 (37), 210
(58),197 (29),195 (33), 181 (82), 153 (37), 91 (100), 65 (46); HREIMS
calcd for C17H18O5 [M]þ 302.1154, found 302.1152.
A mixture of 14 (25 g, 148.8 mmol), (CH3)2SO4 (18.75 g,
148.8 mmol), and Na2CO3 (17.5 g, 165.1 mmol) was slurred into
acetone and further reflux for 6 h. The resulting crude was further
purified by column chromatography to obtain an oil and this was
readily crystallized to give 15 (19.8 g, 73%), m.p. 65e67 ꢃC; IR (neat)
nmax 3414, 2943, 2843, 1689, 1589, 1504, 1462, 1338, 1203, 1134, 995,
5.2.11. 1-(4-(Benzyloxy)-3-methoxyphenyl)ethanone (19)
Compound 18 (8.3 g, 50 mmol) was dissolved in DMF (50 mL)
and then anhydrous K2CO3 (2.5 g) and benzyl chloride (6.3 g,
50 mmol) were added. The reaction mixture was stirred at 80 ꢃC for
6 h. The resulting crude was dissolved in H2O and extracted with
EtOAc and then was purified by silica gel column chromatography
to obtain 19 (12 g, 94%) as a white powder, m.p. 82e84 ꢃC; IR (neat)
nmax 2873, 1670, 1585, 1512, 1458, 115, 1350, 1276, 1215, 1145, 1076,
; d 9.78 (1H, s,
837, 752, 702 cmꢁ1 1HNMR (300 MHz, DMSO-d6)
CHO), 9.71 (1H, s, OH), 7.03 (2H, s), 3.83 (3H, s, OMe), 3.76 (3H, s,
OMe); 13CNMR (75 MHz, DMSO-d6)
d 191.8, 153.5, 151.0, 141.8, 131.6,
111.0, 104.3, 59.9, 55.8; EIMS m/z (rel. int.): 182 [M]þ (100), 167 (35),
111 (26), 93 (11); HREIMS calcd for C9H10O4 [M]þ 182.0579, found
182.0576.
991, 871, 798, 748 cmꢁ1; 1HNMR (300 MHz, CDCl3)
m), 6.90 (1H, d, J ¼ 9.0 Hz), 5.23 (2H, s), 3.95 (3H, s), 2.55 (3H, s);
13CNMR (75 MHz, CDCl3)
196.6, 152.2, 149.3, 136.1, 130.5, 128.5,
d 7.57e7.33 (7H,
5.2.8. 3-(Benzyloxy)-4,5-dimethoxybenzaldehyde (16)
d
A mixture of 15 (10.0 g, 55 mmol), benzyl chloride (6.96 g,
55 mmol), and anhydrous K2CO3 (3.8 g, 27.5 mmol) in DMF (50 mL)
was stirred at 80 ꢃC for 6 h, and then poured into water and
extracted with EtOAc and dried. It concentrated in vacuo to give 16
(13.8 g, 92%) as a pale yellow oil; IR (neat) nmax 2939, 2831, 1689,
127.9, 127.0, 122.9, 111.9, 110.3, 70.6, 55.8, 26.0; EIMS m/z (rel. int.):
256 [M]þ (39), 92 (26), 91 (100), 65 (24); HREIMS calcd for C16H16O3
[M]þ 256.1099, found 256.1096.
5.2.12. (E)-3-(4-(Benzyloxy)-2,3,6-trimethoxyphenyl)-1-(4-(benzy
loxy)-3- methoxyphenyl)- prop- 2-en-1-one (20)
1585, 1496, 1458, 1384, 1323, 124, 995, 837, 732 cmꢁ1
(300 MHz, CDCl3)
J ¼ 7.2 Hz), 6.98 (2H, m), 4.97 (2H, s), 3.77 (3H, s, OMe), 3.71 (3H, s,
OMe); 13CNMR (75 MHz, CDCl3)
190.4, 153.2, 152.0, 143.5, 135.9,
;
1HNMR
d
9.63 (1H, s, CHO), 7.25 (2H, s), 7.17 (3H, t,
The chalcone 20 was prepared by base-catalyzed condensation
of 1-(4-(benzyloxy)-3- methoxy phenyl)ethanone 19 (1.28 g,
5 mmol) with 4-(benzyloxy)-2,3,6-trimethoxy- benzaldehyde 10
(1.51 g, 5 mmol) in MeOH (30 mL). To a stirred reaction mixture at
d
131.1, 128.0, 127.5, 126.8, 108.3, 106.1, 70.4, 60.3, 55.5; EIMS m/z (rel.
int.): 272 [M]þ (37), 181 (15), 91 (100); HREIMS calcd for C9H10O4
[M]þ 272.1049, found 272.1050.
0
ꢃC was added a 30% aqueous solution of KOH (30 mL) drop wise
over 30 min. The reaction mixture was kept at room temperature
for 24 h, remove methanol under reduced pressure and extracted
with EtOAc. The resulting crude was purified by column chroma-
tography (benzene:acetone ¼ 9:1) to obtain chalcone 20 as yellow
oil (1.85 g, 69%); IR (neat) nmax 2935, 1674, 1593, 1504, 1458, 1411,
5.2.9. 1-(Benzyloxy)-2,3,5-trimethoxybenzene (9) from 16
To a solution of 16 (10.0 g, 36.8 mmol) in CH2Cl2 at room
temperature, m-CPBA (9.48 g, 55.1 mmol) was added slowly in
portions. The reaction mixture was stirred for 4 h at room
temperature and then the CH2Cl2 was removed under reduced
pressure to obtain ester crude product. It was hydrolyzed by
aqueous 10% NaOH (30 mL) in MeOH (30 mL) at room temperature
for 3 h. The reaction mixture was neutralized with 2 M HCl and
extracted with EtOAc. The resulting crude without purification was
directly used for further methylation with dimethyl sulfate in
Me2CO and anhydrous K2CO3 to obtain 9 in 30%.
1338, 1265, 1022, 802, 744 cmꢁ1 1HNMR (300 MHz, CDCl3)
; d 8.09
(1H, d, J ¼ 15.6 Hz), 7.94 (1H, d, J ¼ 15.6 Hz), 7.67 (1H, d, J ¼ 1.5 Hz),
7.59 (1H dd, J ¼ 1.5, 8.4 Hz), 7.41 (10H, m), 6.93 (1H, d, J ¼ 8.4 Hz),
6.34 (1H, s), 5.26 (2H, s), 5.19 (2H, s), 3.98 (3H, s), 3.94 (3H, s), 3.87
(3H, s), 3.83 (3H, s).
5.2.13. Viscolin (1)
To a solution of chalcone 20 (1.50 g, 2.8 mmol) in a mixture of
EtOAc:MeOH (9:1, 50 mL) was added 10% PdeC (100 mg). This
mixture was stirred at room temperature under H2 gas atmosphere
for 72 h, and then it was filtered. The liquid was concentrated and
further purified by column chromatography over silica gel
(Hexanes:EtOAc ¼ 7:3) to give viscolin 1 (840 mg, 86%) as a color-
less solid, m.p. 122e124 ꢃC; IR (neat) nmax 3425, 2935, 2839, 1600,
5.2.10. 4-(Benzyloxy)-2,3,6-trimethoxybenzaldehyde (10) and 2-
(benzyloxy)-3,4,6- trimethoxybenzaldehyde (11)
In a round-bottomed flask compound 9 (1.37 g, 5.0 mmol) was
suspended in dry DMF (1.83 g, 25 mmol). The reaction flask was
kept at ice-bath (0 ꢃC). To this stirred reaction mixture, phosphorus
oxychloride (3.06 g, 20 mmol) was added drop wise. The reaction
mixture was further kept for 30 min in the cooling bath and then
heated at 80 ꢃC for 3 h. After completion, the reaction mixture was
slowly poured into ice-cold water and then it was basified with 10%
aqueous NaOH to precipitate. The regioisomeric mixture of alde-
hydes 10 (765 mg, 51%) and 11 (245 mg, 16%) were purified with the
aid of silica gel column chromatography. 10: m.p. 76e78 ꢃC; IR
(neat) nmax 2939, 2862, 1678, 1593, 1462, 1396, 1334, 1249, 1199,
1512, 1462, 1423, 1269, 1195, 1149, 1091, 1033, 991 cmꢁ1
(300 MHz, CDCl3)
6.71 (1H, s), 6.32 (1H, s), 5.76 (1H, s, OH), 5.55 (1H, s, OH), 3.87 (3H,
;
1HNMR
d
6.84 (1H, d, J ¼ 8.4 Hz), 6.72 (1H, d, J ¼ 8.4 Hz),
s), 3.85 (3H, s), 3.83 (3H, s), 3.75 (3H, s), 2.62 (4H, t, J ¼ 6.6 Hz), 1.77
(2H, m); 13CNMR (75 MHz, CDCl3)
d 154.2, 151.2, 147.3, 146.2, 143.3,
134.8, 133.5, 120.8, 116.0, 114.0, 111.0, 94.3, 60.8, 60.5, 55.8, 55.6,
35.6, 31.9, 23.1; EIMS m/z (rel. int.): 348 [M]þ (64), 198 (23), 197
(100), 137 (24); HREIMS calcd for C19H24O6 [M]þ 348.1573, found
348.1572.
1041, 979, 910, 802, 748 cmꢁ1
(1H, s, CHO), 7.41 (5H, m), 6.30 (1H, s), 5.21 (2H, s), 3.96 (3H, s), 3.84
(3H, s), 3.81 (3H, s); 13CNMR (75 MHz, CDCl3)
188.0, 158.5, 158.2,
; d 10.30
1HNMR (300 MHz, CDCl3)
d
5.3. Bioassay
156.8, 136.2, 135.8, 128.7, 128.3, 127.2, 112.7, 93.3, 70.9, 62.1, 61.1,
56.0; EIMS m/z (rel. int.): 302 [M]þ (20), 229 (18), 187 (22), 167 (21),
149 (45), 91 (100), 77 (15); HREIMS calcd for C17H18O5 [M]þ
302.1154, found 302.1155. 11: m.p. 81e83 ꢃC; IR (neat) nmax 2939,
1681, 1593, 1462, 1369, 1334, 1249, 1207, 1138, 1041, 975, 902, 806,
5.3.1. Chemicals and antibodies
LPS (endotoxin from Escherichia coli, serotype 0127:B8), Carr (l-
carrageenin), Indo (indomethacin), MTT (3-[4,5-dimethylthiazol-2-
yl]- 2,5-diphenyltetrazolium bromide) and other chemicals were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). TNF-
purchased from Biosource International Inc. (Camarillo, CA, USA).
Anti-iNOS, anti-COX-2, and anti- -actin antibody (Santa Cruz, USA)
and a protein assay kit (Bio-Rad Laboratories Ltd., Watford, Herts,
748, 648 cmꢁ1; 1HNMR (300 MHz, CDCl3)
d
10.27 (1H, s, CHO), 7.47
(2H, d, J ¼ 9.0 Hz), 7.35 (3H, m), 6.28 (1H, s), 5.14 (2H, s), 3.95 (3H, s),
3.89 (3H, s), 3.82 (3H, s); 13CNMR (75 MHz, CDCl3)
188.0, 159.1,
158.6, 155.4, 136.6, 136.0, 128.6, 128.4, 128.2, 112.9, 91.8, 76.5, 61.1,
a was
d
b

G.-J. Huang et al. / European Journal of Medicinal Chemistry 48 (2012) 371e378
377
U.K.) were obtained as indicated. MAPK/extracellular signal-
eregulated kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK)/
stress-activated protein kinase, and p38 MAPK proteins and phos-
phorylated proteins were purchased from Cell Signaling Technology
(Beverly, MA). Poly-(vinylidene fluoride) membrane (Immobilon-P)
was obtained from Millipore Corp. (Bedford, MA, USA).
5.3.9. Antioxidant enzyme activity Measurements
Total SOD activity was determined by the inhibition of cyto-
chrome c reduction [24]. Total CAT activity was based on that of
Aebi [25]. Total GPx activity in cytosol was determined according to
Paglia and Valentine’s method [26].
5.3.10. Protein Lysate preparation and Western blot analysis
Total protein was extracted with a RIPA solution (radioimmuno-
precipitation assay buffer) at ꢁ20 ꢃC overnight. We used BSA
(bovine serum albumin) as a protein standard to calculate equal
5.3.2. Animals
Male imprinting control region (ICR) mice (6e8 weeks) were
obtained from the BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). The
animals were kept in plexiglass cages at a constant temperature of
22 ꢂ 1 ꢃC, and relative humidity of 55 ꢂ 5% with 12 h darkelight
cycle for at least 2 week before the experiment. They were given
food and water ad libitum. All experimental procedures were per-
formed according to the National Institutes of Health (NIH) Guide
for the Care and Use of Laboratory Animals. After a 2 week adap-
tation period, male ICR mice (18e25 g) were randomly assigned to
four groups (n ¼ 6) of the animals in the study. The control group
receives normal saline (i.p.). The other three groups include a Carr-
treated, a positive control (Carr þ Indo) and viscolin administered
groups (Carr þ viscolin).
total cellular protein amounts. Protein samples (30
mg) were
resolved by denaturing sodium dodecyl sulfateepolyacrylamide gel
electrophoresis (SDSePAGE) using standard methods, and then
were transferred to PVDF membranes by electroblotting and
blocking with 1% BSA. The membranes were probed with the
primary antibodies at 4 ꢃC overnight, washed three times with
PBST, and incubated for 1 h at 37 ꢃC with horseradish peroxidase
conjugated secondary antibodies. The membranes were washed
three times and the immunoreactive proteins were detected by
enhanced chemiluminescence (ECL) using hyperfilm and ECL
reagent (Amersham International plc., Buckinghamshire, U.K.). The
results of Western blot analysis were quantified by measuring the
relative intensity compared to the control using Kodak Molecular
Imaging Software and represented in the relative intensities.
5.3.3. Cell culture
A murine macrophage cell line RAW 264.7 (BCRC No. 60001)
was purchased from the Bioresources Collection and Research
Center (BCRC) of the Food Industry Research and Development
Institute (Hsinchu, Taiwan). Cells were cultured in plastic dishes
containing Dulbecco’s Modified Eagle Medium (DMEM, Sigma, St.
Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) in
a CO2 incubator (5% CO2 in air) at 37 ꢃC and subcultured every 3
days at a dilution of 1:5 using 0.05% trypsine0.02% EDTA in Ca2þ-,
Mg2þ- free phosphate-buffered saline (DPBS).
5.3.11. Histological examination
For histological examination, biopsies of paws were taken 5 h
following the intraplantar injection of Carr. The tissue slices were
fixed in Dietric solution (14.25% ethanol, 1.85% formaldehyde, 1%
acetic acid) for 1 week at room temperature, dehydrated by graded
ethanol and embedded in Paraplast (Sherwood Medical). Sections
(7
mm thick) were deparaffinized with xylene and stained with
trichromic Van Gieson, and antigen retrieval was performed with
citrate buffer, then blocked with 5% normal goat serum in PBS and
incubated with rabbit anti-COX-2 and anti-iNOS in PBS with 5%
normal goat serum. The sections were incubated with biotinylated
goat anti-rabbit IgG. After washing in PBS, sections were processed
with the Dako kit (Dako REALTM envision TM detection system).
Thus some sections were stained with hematoxylin and eosin,
while others were processed for iNOS and COX-2 immunohis-
tochmistry staining. All samples were observed and photographed
with BH2 Olympus microscopy. The numbers of neutrophils were
counted in each scope (400 ꢄ ) and thereafter obtain their average
count from five scopes of every tissue slice in hematoxylin and
eosin stain.
5.3.4. Cell viability
Cells (2 ꢄ 105) were cultured in 96-well plate containing DMEM
supplemented with 10% FBS for 1 day to become nearly confluent.
Then cells were cultured with viscolin in the presence of 100 ng/mL
LPS for 24 h. After that, the cells were washed twice with DPBS and
incubated with 100
cell viability. The medium was then discarded and 100
sulfoxide (DMSO) was added. After 30-min incubation, absorbance
at 570 nm was read using a microplate reader.
m
L of 0.5 mg/mL MTT for 2 h at 37 ꢃC testing for
L dimethyl
m
5.3.5. Measurement of nitric oxide/nitrite
NO production was indirectly assessed by measuring the nitrite
levels in the cultured media and serum determined by a colori-
metric method based on the Griess reaction [23].
5.3.12. Statistical analysis
Data are expressed as mean ꢂ standard error of the mean (SEM).
Statistical evaluation was carried out by one-way analysis of vari-
ance (ANOVA followed by Scheffe’s multiple range tests). Statistical
significance is expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.
5.3.6. Carr-induced edema
The Carr-induced hind paw edema model was used for deter-
mination of anti-inflammatory activity according to the previous
report [23].
Acknowledgments
5.3.7. Lipid peroxidation assay: malondialdehyde (MDA) formation
Determination of MDA from Carr-induced edema foot by the
thiobarbituric acid reactive substances (TBARS) was used an index
of the extent of lipid peroxidation [23].
This work was supported by a grant of National Science Council,
Taiwan, Republic of China, awarded to T. S. Wu. This study is sup-
ported in part by National Cheng Kung University (HUA100-3-2-
232).
5.3.8. Measurement of serum TNF-
a
and PGE2 by an enzyme-linked
and PGE2 were
Immunosorbent assay (ELISA)
Cell culture medium or serum levels of TNF-
a
Appendix. Supplementary Data
determined using a commercially available ELISA kit (Biosource
International Inc., Camarillo, CA) according to the manufacturer’s
Figs. S1eS6 were provided in the supporting information.
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.12.008.
instruction. The concentrations of TNF-a and PGE2 were were
expressed as pg/mL.

378
G.-J. Huang et al. / European Journal of Medicinal Chemistry 48 (2012) 371e378
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