Kuo-Ping Chao1

Kuo-Feng Hua2
Hsien-Yeh Hsu2
Anti-Inflammatory Activity of Sugiol, A Diterpene Yu-Chang Su3
Isolated from Calocedrus formosana Bark Shang-Tzen Chang4
Original Paper

Abstract ulated macrophages. In addition, sugiol significantly inhibited

LPS-induced ROS production. Our studies suggest that sugiol's
Sugiol is a diterpene which was isolated and purified from alco- efficacy in inhibiting the inflammatory cytokines of IL-1b and
hol extracts of the bark of Calocedrus formosana Florin (Cupres- TNF-a could be attributed to a reduction of the ROS that leads to
saceae). Although sugiol has low inhibitory activity against the a decrease in the phosphorylation of MAPKs.
DPPH radical, it could effectively reduce intracellular reactive
oxygen species (ROS) production in lipopolysaccharide (LPS)- Key words
stimulated macrophages. The present study investigated the po- Calocedrus formosana ´ Cupressaceae ´ sugiol ´ anti-inflammatory
tential anti-inflammatory activity of sugiol, and the relationship activity ´ TNF-a ´ IL-1b ´ MAPKs
between signal transduction and inflammatory cytokines in
vitro. A dose of 30 mM of sugiol was effectively inhibitory for
proIL-1b, IL-1b and TNF-a production, suggesting that sugiol is Abbreviations
bioactive against inflammation. Moreover, sugiol reveals a capa- LPS: lipopolysaccharide
city for suppressing the activation of mitogen-activated protein ROS: reactive oxygen species
kinases (MAPKs), including extracellular signal-regulated kinase MAPK: mitogen-activated protein kinase
300 (ERK), c-Jun NH2-terminal kinase (JNK), and p38 mitogen- ERK: extracellular signal-regulated kinase
activated protein kinase (p38) activated by LPS-stimulation in JNK: C-Jun NH2-terminal protein kinase
J774A.1 murine macrophages. A low dosage of 10 mM of sugiol p38: p38 mitogen-activated protein kinase
completely inhibited ERK1/2 phosphorylation, while 30 mM ef-
fectively inhibited JNK1/2 and p38 phosphorylation in LPS-stim-

Introduction the forestry industry the bark has always been discarded. Many
studies have investigated the bioactivity of sugiol, including a
Calocedrus formosana Florin (Cupressaceae) is an endemic tree strong antitumor activity on EBV-EA induction [1], and weak an-
that grows at elevations of 800 ± 1500 meters in Taiwan's central timicrobial and aldose reductase (AR) inhibitory activities [2],
mountains. Sugiol is abundant in the bark of C. formosana, but in [3]. Many natural products exhibiting a good anti-inflammation

Affiliation
1
Department of Biological Science and Technology, Chung Hwa College of Medical Technology, Tainan, Taiwan
2
Faculty of Medical Technology, Institute of Biotechnology in Medicine, National Yang-Ming University, Taipei,
Taiwan
3
Division of Wood Cellulose, Taiwan Forestry Research Institute, Taipei, Taiwan
4
School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan

Correspondence
Dr. Shang-Tzen Chang ´ School of Forestry and Resource Conservation ´ National Taiwan University ´ No. 1,
Section 4 Roosevelt Road ´ Taipei 106 ´ Taiwan ´ Republic of China ´ Phone: +886-2-236-30231-3196 ´
Fax: +886-2-236-54520 ´ E-mail: [email protected]

Received June 22, 2004 ´ Accepted November 15, 2004

Bibliography
Planta Med 2005; 71: 300±305 ´  Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2005-864094
ISSN 0032-0943
capacity have been isolated from plants, but there have been chemicals and solvents used in this study were of the highest
few discussions about their molecular mechanism. Results quality available.
from our preliminary study demonstrated that the n-hexane
fractions of alcoholic extracts from C. formosana, from which Extraction and purification
large amounts of sugiol were isolated, exhibited a significant The bark particles of C. formosana (10 kg d. w.) were treated with
anti-inflammatory capacity (unpublished results). In this study, ethanol (95 % v/v, 10 days repeated 3 times) at room temperature,
we have isolated sugiol (Fig. 1) and investigated its biological and then the extracts were concentrated to give the alcoholic ex-
functions, including antioxidant and anti-inflammatory activ- tracts (AE; ca. 410 g). 205 g of AE were successively extracted
ities in macrophages. with n-C6H14, CH2Cl2, EtOAc, BuOH and H2O, which then yielded
about 64.8 g of n-C6H14-soluble fraction. The n-hexane fraction
Innate immunity is triggered by pathogen-associated molecular was applied on the top of a 160 g silica gel column, then eluted
patterns shared by groups of microbial pathogens, which can be with n-hexane/EtOAc (95/5, 90/10, 85/15, 80/20, 75/25, 70/30,
recognized by pattern recognition receptors in host cells [4]. The 60/40, 40/60, 20/80, 0/100) followed by elution with EtOAc/etha-
lipopolysaccharide (LPS), or endotoxin, represents a well-known nol (100/0, 90/10, 80/20, 70/30, 60/40, 50/50, 40/60, 20/80, 0/

Original Paper
pathogen-associated molecular pattern, localized on Gram-neg- 100) to give 22 subfractions. Each eluted volume of subfraction
ative bacteria cell walls [5]. LPS activates macrophages by bind- was 1000 mL, except for the H1 subfraction, which was 4000
ing to toll-like receptor 4 and stimulates the production of in- mL. Each collected subfraction was rotary evaporated at 50 8C to
flammatory cytokines, including TNF-a and IL-1b proteins. While dryness, resulting in a white precipitate (678 mg) from subfrac-
mediation of inflammation against pathogen infection by TNF-a tion H5. Recrystallization of this precipitate with ethanol gave
and IL-1b proteins could be beneficial to the host, overexpression sugiol which was then purified by semi-preparative HPLC (Hita-
of these cytokines may cause serious disease, including septic chi L-7100 pump, a UV detector model Jasco UV 975, l = 254 nm)
shock. Hence, suppression of TNF-a and IL-1b protein production on a Zorbax SIL column (25 cm in length, 4.6 mm i. d., 5.0 mm).
could aid in the treatment of septic shock. The pure sugiol (> 98 %) was obtained at tR = 5.14 min. The se-
paration conditions were as follows: flow rate, 2 mL/min; mobile
It is well known that the mitogen-activated protein kinases phase, EtOAc/EtOH = 1/1. The structure of sugiol was confirmed
(MAPKs) play an important role in regulating cell growth and by comparison of its physical and spectral data (optical rotation,
survival, including mitogenic and stress responses. Our recent EI-MS, 1H-NMR) with previously reported values [7]. The optical
study revealed that LPS stimulated reactive oxygen species rotation was determined using a Horiva STA-300 polarimeter.
(ROS) production, MAPKs activation, and proIL-1b/IL-1b protein [a]D25: + 28.38 (c 1.0, CHCl3). The NMR spectra were recorded on a
expression in murine macrophages [6]. This prompted us to in- Bruker Avance-300 MHz FT-NMR. 1H-NMR (CDCl3): d = 0.99 (3H,
vestigate whether sugiol affects LPS-induced signal transduction s, H-18), 1.00 (3H, s, H-19), 1.19 (3H, s, H-20), 1.20 (3H, d, J = 7.0
in the regulation of cytokine production. Herein we present evi- Hz, H-16), 1.24 (3H, d, J = 7.0 Hz, H-17), 1.83 (dd, J = 4.5, 13.0 Hz,
dence that sugiol inhibited LPS-induced TNF-a and IL-1b expres- H-5), 2.18 (dd, J = 13.0, 18.0 Hz, H-6b), 2.62 (1H, dd, J = 4.5, 1.8 301
sion as well as ROS and MAPKs activation in macrophages. This is Hz, H-6a), 3.13 (1H, sept, J = 7.0 Hz, H-15), 6.68 (1H, s, H-11),
the first report to demonstrate that sugiol has an anti-inflamma- 7.89 (1H, s, H-14). The mass spectra (MS) were obtained on a Fin-
tory activity in macrophages. nigan MAT-95S mass spectrometer. EI-MS: m/z calcd. for
C20H28O2 : 300; found: 300.

Materials and Methods Cell culture

The J774A.1 cells were propagated in RPMI 1640 medium supple-
Materials mented with 10 % heat-inactivated fetal bovine serum and 2 mM
The samples of C. formosana were collected in September 2003 L-glutamine (Life Technologies, Inc., MD) and cultured in a 37 8C,

from the Lien Hua-Chin Research Center located in Nantou Coun- 5 % CO2 incubator. Sugiol was dissolved in dimethyl sulfoxide
ty in central Taiwan. The species was identified by Mr. Yen-Ray (DMSO) and the final DMSO concentration was 0.1 % in all cul-
Hsui of the Taiwan Forestry Research Institute, and voucher spe- tures containing this agent; the same amount of vehicle was add-
cimens (reg. #WCCL-04-B006) were deposited at the laboratory ed to the control cultures.
of wood chemistry (School of Forestry and Resource Conserva-
tion, National Taiwan University). Murine macrophage J774A.1 MTT assay for cell viability
cells were obtained from ATCC (Rockville, MD); LPS (from The cytotoxicity of sugiol was assessed using the microculture
Escherichia coli 0111:B4), SB203580, curcumin (> 95 %), monoclo- tetrazolium (MTT) assay [8]. After culturing on 96 well plates
nal anti-MAP kinase, activated (diphosphorylated ERK1/2) anti- for 24 h, the cells were washed twice and incubated with 100 mL
body, monoclonal anti-JNK kinase, activated (diphosphorylated of 1 mg/mL of MTT for 2 h at 37 8C. The medium was discarded
JNK) antibody, and monoclonal anti-p38 MAP kinase, activated and 100 mL lysis buffer were then added. After 30 min incubation,
(diphosphorylated p38) antibody were obtained from Sigma Co. the absorbance was measured at 570 nm using a microplate
Mouse TNF-a and IL-1b enzyme-linked immunosorbent assay reader.
(ELISA) kits were purchased from R & D Systems, Inc. (Minneapo-
lis, MN). Anti-IL-1b polyclonal antibody, anti-ERK1 polyclonal Measurement of cytokine production
antibody, anti-JNK1 polyclonal antibody, anti-p38 polyclonal an- To investigate the inhibitory effect of sugiol on TNF-a and proIL-
tibody, anti-rabbit IgG-HRP, and anti-mouse IgG-HRP were ob- 1b/IL-1b protein production from LPS-stimulated J774A.1 cells,
tained from Santa Cruz Biotechnology (Santa Cruz, CA). The other the cells were pretreated with sugiol (5 to 30 mM) for 30 min at

Chao K-P et al. Anti-Inflammatory Activity of ¼ Planta Med 2005; 71: 300 ± 305
 37 8C, followed by LPS (1 mg/mL) treatment for the indicated Fig. 1 Chemical structure of sugiol from
times. The levels of TNF-a and IL-1b protein in supernatants and C. formosana.
proIL-1b protein in cell lysates were quantified by ELISA and by
Western blot, respectively [6]. Histograms represent quantifica-
tion by PhosphorImager of proIL-1b in J774A.1 cell samples with
ImageQuaNT software from Molecular Dynamics.

Measurement of MAPK activation

To investigate the inhibitory effect of sugiol on MAPK activation in
LPS-stimulated J774A.1, the cells were pretreated with sugiol (5 to shows the structure of sugiol, and it was characterized as de-
30 mM) for 30 min at 37 8C, followed by LPS (1 mg/mL) treatment scribed previously [7].
for 15 min. The phosphorylation levels of ERK1/2, JNK1/2, and
p38 were quantified by Western blot with antibody against To investigate whether sugiol exhibits immunomodulation ac-
diphosphorylated ERK1/2, antibody against diphosphorylated tivity in macrophages, J774A.1 cells were pretreated with the in-
Original Paper

JNK, and antibody against diphosphorylated p38, respectively [6]. dicated concentrations of sugiol for 30 min and challenged with
Histograms represent quantification by PhosphorImager of phos- LPS for 6 h. As shown in Fig. 2A, bioactive TNF-a protein was se-
pho-ERK1/2, phospho-JNK1/2, and phospho-p38 in J774A.1 cell creted from J774A.1 cells in response to LPS challenge as meas-
samples with ImageQuaNT software from Molecular Dynamics. ured by ELISA. Interestingly, LPS-induced TNF-a secretion was
dramatically reduced in sugiol pretreated macrophages. Specifi-
Measurement of intracellular ROS production cally, 60 ng/mL TNF-a was secreted from LPS-stimulated cells,
Intracellular ROS stimulated by LPS was measured by detecting and LPS-induced TNF-a secretion was reduced to 30 and 20 ng/
the fluorescent intensity of either 2¢,7¢-dichlorofluorescein di- mL by 20 and 30 mM sugiol, respectively. SB203580 (positive con-
acetate (DCFH) or the improved analogue carboxyl-DCFH (CM- trol), a pharmacological antagonist that inhibits the activation of
DCFH) (Molecular Probes, Inc., Eugene, OR) oxidized product, p38, had a significant effect on TNF-a secretion [12]. Further-
DCF (or CM-DCF), as described [9]. Briefly, 0.5 ± 1.0 ” 106 J774A.1 more, no cytotoxic effect was observed after cells were treated
cells/mL were grown in serum- and phenol red-free RPMI medi- with sugiol plus LPS for 24 h as measured by MTT assay (data
um (starvation medium) for 24 h, and then were preincubated not shown). These results indicate that the inhibition of TNF-a
with 2 mM CM-DCFH, and sugiol or 10 mM NAC, at 37 8C for 30 secretion by sugiol is not due to cell death. In addition, cells
min in the dark. To these were added fresh starvation medium were pretreated with sugiol for 30 min, followed by LPS chal-
containing LPS for additional incubation for the indicated times. lenge for 24 h. As shown in Fig. 2B, sugiol showed a dose-depen-
The relative fluorescent intensity of the fluorophore CM-DCF, dent inhibitory effect on LPS-induced IL-1b secretion. We found
which was formed by peroxide oxidation of the non-fluorescent that 135 pg/mL IL-1b protein was secreted by LPS-stimulated
302 precursor, was detected at an excitation wavelength of 485 nm cells, and by using 5, 10, 20, and 30 mM sugiol, this was reduced
and an emission wavelength of 530 nm with a fluorometer, Cyto- to 90, 85, 60, and 45 pg/mL respectively. 1 mM SB20380 reduced
fluor 2300 (Millipore Inc., Bedford, MA). CM-DCFH with starva- LPS-induced IL-1b protein secretion to 40 pg/mL.
tion medium was used as a blank control [6].
The molecular mechanism of IL-1b protein secretion was then in-
DPPH (1,1-diphenyl-2-picrylhydrazyl) assay vestigated by detection of proIL-1b protein (IL-1b precursor, 34
Sugiol's reductive activities on the DPPH assay were measured as kD) expression via Western blotting analysis. As shown in Fig. 3,
follows. The reaction mixture contained 1000 mL of 0.1 mM incubation of J774A.1 with LPS for 6 h significantly increased the
DPPH-ethanol solution, 450 mL of 0.05 M Tris-HCl buffer (pH expression of proIL-1b protein 5-fold compared with untreated
7.4), and 50 mL of test samples or control (ethanol). After incuba- control cells. Sugiol, at concentrations of 20 and 30 mM, reduced
tion for 30 min, the reduction in DPPH was measured by reading LPS-induced proIL-1b protein expression 4- and 2.5-fold, respec-
the optical absorption at 517 nm [10]. Curcumin was used as a tively. In addition, SB203580 reduced LPS-induced proIL-1b ex-
positive reference in this experiment [11]. The inhibition ratio pression 1.8-fold at 1 mM.
(%) was calculated using the following equation: % Inhibitio-
n = [(absorbance of control ± absorbance of test sample)/absor- Our previous study demonstrated that MAPKs play important
bance of control] ” 100. roles in the regulation of proIL-1b/IL-1b expression in LPS-stim-
ulated J774A.1 cells [6]. Therefore, we investigated whether LPS-
Statistical analysis mediated activation of one or more of these MAPKs is altered in
Statistical differences between the experimental groups were ex- the J774A.1 macrophages pre-exposed to sugiol. As demonstrat-
amined by analysis of variance, and statistical significance was ed in Fig. 4, LPS strongly induced activation of ERK1/2, JNK1/2,
determined at p < 0.05. The experiments were conducted three and p38 in J774A.1 cells. Interestingly, LPS-mediated activation
times, or as indicated, and all data are expressed as mean  S.D. of ERK1/2, JNK1/2, and p38 was greatly inhibited in the J774A.1
cells previously exposed to sugiol. Specifically, LPS-induced
ERK1/2 phosphorylation, and sugiol showed a significant inhibi-
Results tory effect on ERK1/2 phosphorylation at concentrations above
10 mM, yet SB203580 had no observed effect. In addition, the
Sugiol (> 98 % pure), colorless crystals, was purified from the pre- LPS-induced phosphorylated JNK1/2 was significantly inhibited
cipitates of subfraction H5 and the yield was about 50 mg. Fig. 1 by sugiol concentrations ranging from 10 to 30 mM, and comple-

Chao K-P et al. Anti-Inflammatory Activity of ¼ Planta Med 2005; 71: 300 ± 305
 mM), a potent antioxidant, quickly reduced the release of LPS-in-
duced ROS. Interestingly, we found that cells pretreated with su-
giol significantly reduced LPS-stimulated ROS production in the
initial 30 min, falling at almost constant values (Fig. 5B).

Discussion

Pathogen infection causes significant mortality mediated by the

up-regulation of inflammatory cytokines (i. e., TNF-a and IL-1b)
or by other factors. The inflammatory cytokine TNF-a, in particul-
ar, plays a complex and central role in responses to infection [13].
Sugiol is a diterpene, which abounds in woody plants [7], [14]. Our
results demonstrate that sugiol inhibited the production of TNF-a

Original Paper
and IL-1b protein in LPS-stimulated macrophages. A number of
studies show that natural products in herbal medicines can have
a remarkable effect on anti-inflammation. For instance, the me-
thanolic extracts of Cordyceps pruinosa have been proven to have
a strong inhibitory capacity on LPS-induced TNF and IL-1b expres-
sion in murine macrophage cell line RAW 264.7 [15]. A similar in-
hibitory capacity has been observed in the diterpene tanshinone
IIA that was isolated from Salvia miltiorrhiza [16]. In this study,
we found that sugiol could effectively inhibit IL-1b and TNF-a pro-
teins expression in LPS-stimulated J774A.1 cells in a dose-depen-
dent manner (Figs. 2A and B). Our results suggest that the inhibi-
tory capacity of sugiol in LPS-induced TNF-a and IL-1b expression
may offer an advantage in protecting the host from endotoxic
shock. However, the effect of sugiol on LPS-induced cytokines pro-
duction in vivo needs further investigation.

303

Fig. 2 Sugiol inhibits TNF-a and IL-1b protein expression in LPS-stim-

ulated macrophages. J774A.1 cells were pretreated with the indicated
concentration of sugiol for 30 min prior to incubation with 1 mg/mL LPS
for indicated time. Culture media were assayed by using a specific ELI-
SA. A Dose-dependent incubation of sugiol on LPS-induced TNF-a. B
Dose-dependent incubation of sugiol on LPS-induced IL-1b. SB203580
(1 mM) was used as a positive control. The error bar for all experiments
was mean  SD (n = 3).

tely inhibited at 30 mM. Finally, sugiol, at concentrations above

20 mM, reduced LPS-induced p38 phosphorylation to the basal
level, and SB203580 also inhibited p38 phosphorylation. These
results indicate that sugiol inhibits LPS-induced MAPKs activa-
tion in a concentration-dependent manner.

To investigate the antioxidant activity of sugiol, DPPH and intra- Fig. 3 Sugiol inhibits proIL-1b protein expression in LPS-stimulated
cellular ROS production assays were conducted. We found that at macrophages. J774A.1 cells were pretreated with the indicated con-
dosages over 100 mg/mL sugiol had the lowest inhibitory activity centration of sugiol for 30 min prior to incubation with 1 mg/mL LPS
against the DPPH radical, where inhibition was always below for 6 h. ProIL-1b protein expression level was analyzed by Western
10 %, (Fig. 5A). The inhibitory activity of curcumin was better blot. The histogram represents quantification by PhosphorImager of
LPS-stimulated proIL-1b in J774A.1 cell sample using ImageQuaNT
than that of sugiol. Furthermore, LPS stimulation of cells rapidly software from Molecular Dynamics. SB203580 (1 mM) was used as a
induced significant ROS when compared with that of LPS-un- positive control. One of four experiments is presented. * p < 0.05; **
treated control samples. In contrast, pretreatment with NAC (10 p < 0.01 versus LPS alone.

Chao K-P et al. Anti-Inflammatory Activity of ¼ Planta Med 2005; 71: 300 ± 305
 itor of ROS [6]. This illustrates that the bioactivity of sugiol an-
tioxidant analysis ex vivo is more distinct than in the cell-free
model.

To gain further insights into mechanisms of sugiol-mediated in-

hibition of LPS-induced TNF-a and proIL-1b/IL-1b protein ex-
pression, we then examined the intracellular signaling mol-
ecules involved in the LPS signaling pathways in sugiol-treated
macrophages. By utilizing certain specific pharmacological an-
tagonists such as PD98059, SP600125, and SB203580 that inhibit
the activation of MEK1, JNK, and p38, respectively, we have dem-
onstrated that MAPKs, including ERK1/2, JNK1/2, and p38 play
important roles in LPS-induced proIL-1b/IL-1b protein expres-
sion [6]. This prompted us to investigate whether sugiol affects
Original Paper

LPS-induced MAPKs activation. The results showed that sugiol

inhibited ERK1/2, JNK1/2, and p38 phosphorylation in LPS-stim-
ulated macrophages. Hence, we suggest that the inhibitory effect

Fig. 4 Sugiol inhibits MAPKs activation in LPS-stimulated macropha-

ges. J774A.1 cells were pretreated with the indicated concentration of
sugiol for 30 min prior to incubation with 1 mg/mL LPS for 15 min. The
phosphorylation levels of ERK1/2, JNK1/2, and p38 were analyzed by
Western blot with anti-diphosphorylated ERK1/2, anti-diphosphorylat-
ed JNK1/2 or anti-diphosphorylated p38 monoclonal antibody, respec-
304 tively. The histogram represents quantification by PhosphorImager of
LPS-stimulated ERK1/2, JNK1/2 and p38 phosphorylation in J774A.1
cell sample using ImageQuaNT software from Molecular Dynamics.
SB203580 (1 mM) was used as a positive control. One of three experi-
ments is presented. * p < 0.05; ** p < 0.01 versus LPS alone.

Next, to assess whether the inhibitory effect on IL-1b protein

production was regulated by transcriptional regulation, we used
Western blotting to detect proIL-1b (IL-1b precursor) protein ex-
pression. The inhibitory effect of sugiol on LPS-induced proIL-1b
protein expression was dose-dependent and required more than
20 mM for a significant inhibitory effect (Fig. 3). However, sugiol
at a concentration of 5 mM showed a significant inhibitory effect
on IL-1b protein secretion (Fig. 2B). These results suggest that su-
giol inhibits LPS-induced IL-1b secretion at the transcriptional
level, and perhaps at the post-transcriptional, interleukin-1 con-
verting enzyme-catalyzed level [6].

Nevertheless, our results show that sugiol has a low antioxidant

ability that is not consistent in different bioassays (Fig. 5A and Fig. 5 Antioxidant activity of sugiol. A Free-radical scavenging activity
Fig. 5B). In a tissue culture system, sugiol displayed a strong of sugiol measured using the DPPH assay: (*) curcumin; (l) sugiol.
anti-oxidation activity as analyzed by CM-DCF. However, in a Results are mean  SD (n = 3). B Reduced intracellular ROS capacity
cell-free system we observed no significant anti-oxidation ac- of sugiol in LPS-stimulated macrophages. J774A.1 cells were pre-incu-
bated with CM-DCFH (2 mM) and sugiol or 10 mM NAC for 30 min, fol-
tivity of sugiol as analyzed by DPPH. This result indicates that
lowed by substitution with medium. Cells were treated with 1 mg/mL
if a DPPH assay system is used, we may overlook some biologi- LPS for the indicated time points. The relative fluorescence intensity
cally significant anti-oxidation components. Sugiol's bioactiv- of fluorophore CM-DCF was detected as described in Materials and
ity is comparable with that of NAC at 10 mM, which is an inhib- Methods. The data are representative experiments (n = 6).

Chao K-P et al. Anti-Inflammatory Activity of ¼ Planta Med 2005; 71: 300 ± 305
of sugiol on LPS-induced cytokines production may result from 5
Christian RH, Chris Whitfield. Lipolysaccharide endotoxins. Annu Rev
the inhibition of MAPKs activation. Biochem 2002; 71: 635 ± 700
6
Hsu HY, Wen MH. Lipopolysaccharide-mediated reactive oxygen spe-
cies and signal transduction in the regulation of interleukin-1 gene ex-
According to our previous studies, LPS activated all three MAPKs pression. J Biol Chem 2002; 277: 22131 ± 9
including ERK1/2, JNK1/2 and p38 in J774A.1 cells within 15 ± 30 7
Wang SY, Wu JH, Shyur LF, Kuo YH, Chang ST. Antioxidant activity of
min [6]. Sugiol, unlike other specific MAPK inhibitors, has a abietane-type diterpenes from Taiwania crytomerioides Hayata. Holz-
forschung 2002; 56: 487 ± 92
broad inhibitory activity on ERK1/2, JNK1/2, and p38. This led us 8
Wang YY, Khoo KH, Chen ST, Lin CC, Wong CH, Lin CH. Studies on the
to investigate whether sugiol affected the signal molecules up- immuno-modulating and antitumor activities of Ganoderma lucidum
stream of MAPKs. In a previous study [6], NAC, an ROS scavenger, (Reishi) polysaccharides: functional and proteomic analyses of a fu-
inhibited LPS-induced MAPKs activation as well as proIL-1b/IL- cose-containing glycoprotein fraction responsible for the activities.
Bioorg Med Chem 2002; 10: 1057 ± 62
1b expression, which indicated that ROS was upstream of MAPKs 9
Wan CP, Myung E, Lau BH. An automated micro-fluorometric assay for
and regulated proIL-1b/IL-1b expression in LPS-stimulated mac- monitoring oxidative burst activity of phagocytes. J Immunol Methods
rophages [6]. In the present study, we found that sugiol signifi- 1993; 159: 131 ± 8
10
cantly inhibited by ROS production in LPS-stimulated macropha- Wang SY, Wu JH, Cheng SS, Lo CP, Chang HN, Shyur , Chang ST. Antiox-

Original Paper
idant activity of extracts from Calocedrus formosana leaf, bark and
ges. Our experiments showed that the pretreatment of macro- heartwood. J. Wood Sci 2004; 50: 422 ± 6
phages with sugiol effectively decreased ROS production and 11
Priyadarsini KI, Maity DK, Naik GH, Kumar MS, Unnikrishnan MK, Sa-
prevented LPS-induced production of the three MAPKs as well tav JG, Mohan H. Role of phenolic O-H and methylene hydrogen on the
as proIL-1/IL-1 expression. Our results indicate that sugiol-medi- free radical reactions and antioxidant activity of curcumin. Free Radic
Biol Med. 2003; 35: 475 ± 84
ated inhibition of TNF-a and proIL-1b/IL-1b protein expression 12
Ropert C, Almeida IC, Closel M, Travassos LR, Ferguson MA, Cohen P,
and MAPKs phosphorylation may be, at least in part, due to the Gazzinelli RT. Requirement of mitogen-activated protein kinases and
scavenger activity of ROS [17]. I kappa B phosphorylation for induction of proinflammatory cytokines
synthesis by macrophages indicates functional similarity of receptors
triggered by glycosylphosphatidylinositol anchors from parasitic pro-
tozoa and bacterial lipopolysaccharide. J Immunol 2001; 166: 3423 ±
Acknowledgements 31
13
Ware CF. The TNF superfamily. Cytokine Growth Factor Rev 2003; 14:
This study was supported by a grant (NSC-92-2313-B-002-042) 81 ± 4
14
Ulubelen A, TopcËu G, Sönmez U, Choudhary MI, Atta-Ur-Rahman .
from the National Science Council of Taiwan. Abietane diterpenes from Salvia napifolia. Phytochemistry 1995; 40:
861 ± 4
15
Kim KM, Kwon YG, Chung HT, Yun YG, Pae HO, Han JA, Ha KS, Kim TW,
References Kim YM. Methanol extracts of Cordyceps pruinosa inhibits in vitro and
in vivo inflammatory mediators by suppressing NF-kB activation. Tox-
1
icology and Applied Pharmacology 2003; 190: 1 ± 8
Iwamoto M, Minami T, Tokuda H, Ohtsu H, Tanaka R. Potential antitu- 16
Jang SI, Jeong SI, Kim KJ, Kim HJ, Yu HH, Park R, Kim HM, You YO. Tan-
mor promoting diterpenoids from the stem bark of Thuja standishii. shinone IIA from Salvia miltiorrhiza inhibits inducible nitric oxide syn-
Planta Medica 2003; 69: 69 ± 72 305
2
thase expression and production of TNF-a, IL-1b and IL-6 protein in
Tezuka Y, Kasimu R, Basnet P, Namba T, Kadota S. Aldose reductase in- activated raw 264.7 cells. Planta Medica 2003; 69: 1057 ± 9
hibitory constituents of the root of Salvia miltiorrhiza Bunge. Chem 17
Haddad JJ, Land SC. Redox/ROS regulation of lipopolysaccharide-in-
Pharm Bull 1997; 45: 1306 ± 11 duced mitogen-activated protein kinase (MAPK) activation and
3
Politi M, Braca A, Tommasi ND, Morelli I, Manunta A, Battinelli L, Max- MAPK-mediated TNF-alpha biosynthesis. Br J Pharmacol 2002; 135:
xanti G. Antimicrobial diterpenes from the seeds of Cephalotaxus 520 ± 36
harringtonia var. drupacea. Planta Medica 2003; 69: 468 ± 70
4
Medzhitov R, Janeway CA. Innate immunity: the virtues of a nonclonal
system of recognition. Cell 1997; 91: 295 ± 8

Chao K-P et al. Anti-Inflammatory Activity of ¼ Planta Med 2005; 71: 300 ± 305