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Biochemical and Biophysical Research Communications 369 (2008) 761–766

www.elsevier.com/locate/ybbrc

Modulation of inflammatory responses by diterpene acids

from Helianthus annuus L.
Rodrigo Dı́az-Viciedo a, Sonsoles Hortelano b, Natalia Girón a, Jose M. Massó a,
Benjamı́n Rodriguez c, Angel Villar a, Beatriz de las Heras a,*
a
Departamento de Farmacologı́a Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain
b
Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain
c
Instituto de Quı́mica Orgánica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

Received 18 February 2008

Available online 29 February 2008

Abstract

Fractionation of a petroleum ether extract of Helianthus annuus L. led to the isolation of three diterpene acids: grandiflorolic, kaur-
enoic and trachylobanoic acids. These compounds were studied for potential anti-inflammatory activity on the generation of inflamma-
tory mediators in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. At non-toxic concentrations, these compounds reduced,
in a concentration-dependent manner nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor (TNF-a) production, as well
as expression of inducible nitric oxide synthase (NOS-2) and cyclooxygenase-2 (COX-2).
All diterpenoids displayed significant in vivo anti-inflammatory activity and suppressed the 12-O-tetradecanoylphorbol-13-acetate
(TPA)-mouse ear edema. In addition, inhibition of myeloperoxidase (MPO) activity, an index of cellular infiltration, was observed.
In summary, our results suggest that the inhibition of the expression of NOS-2, COX-2 and the release of inflammatory cytokines, is
responsible for the anti-inflammatory effects of the diterpenoids isolated from H. annuus L. which likely contributes to the pharmaco-
logical action of sunflower.
Ó 2008 Elsevier Inc. All rights reserved.

Keywords: Anti-inflammatory activity; Diterpene acids; Helianthus annuus L.; Macrophages; Nitric oxide; Cyclooxygenase-2

Inflammation is a central feature of many pathological
conditions. The pathogenesis of the inflammatory response
involves the sequential activation of signaling molecules,
among which prostaglandins (PGs) and nitric oxide (NO)
are well-known key inflammatory mediators, generated,
respectively, by the inducible isoforms of cyclooxygenase
(COX-2) and NO synthase (NOS-2) [1–3]. Stimulation of
macrophages also produces the release of pro-inflamma-
tory cytokines as TNF-a, which plays a key role in the
induction and perpetuation of inflammation due to auto-
immune reactions by activating T cells and macrophages
[4].
Helianthus annuus L. (Asteraceae), called sunflower, is
an annual herb cultivated primarily for its seeds, which
*
Corresponding author. Fax: +34 91 3941726.
E-mail address: lasheras@farm.ucm.es (B. de las Heras).
are an important source of edible oil. The seed oil, shoots
and herb tincture have been employed for anti-inflamma-
tory, antipyretic, aperitif, diuretic, expectorant and vulner-
ary proposes [5]. Terpenoids are a large and widely
occurring class of natural compounds with a broad spec-
trum of biological activities (antibacterial, antiviral, anti-
inflammatory, cytotoxic, anti-tumor, etc.) [6–12] and in
recent decades the level of research activity into these com-
pounds has increased continuously. In the course of our
search to find compounds possessing potential anti-inflam-
matory activity, we decided to study a petroleum ether
extract of the flower heads of H. annuus in order to isolate
the diterpene metabolites.
Here, we report the isolation of three diterpenes, grandi-
florolic, kaurenoic and trachylobanoic acids, which have
already been reported as constituents of this species [13],
along with their potential anti-inflammatory activities.

0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.02.104
762 R. Dı́az-Viciedo et al. / Biochemical and Biophysical Research Communications 369 (2008) 761–766
Our results show that these diterpenoids clearly contribute
to the pharmacological action of the sunflower by inhibit-
ing NOS-2, COX-2 expression and TNF-a release.
Materials and methods
Materials. Aspirin, arachidonic acid, indomethacin, LPS (lipopoly-
saccharide) from Escherichia coli and TPA (12-O-tetradecanoylphorbol
acetate) were from Sigma-Aldrich (St. Louis, MI). Western blot reagents
(polyvinylidene difluoride membranes and ECL kit) were from GE
Healthcare (Buckinghamshire, UK). Antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA) and Cayman Chemical Co. (Ann Arbor,
MI). Kits for TNF-a and PGE2 were from GE Healthcare (Bucking-
hamshire, UK). For pharmacological studies, diterpenoids were dissolved
in DMSO and subsequently diluted in PBS before use.
Plant material. Helianthus annuus L. was cultivated in a farm at Picazo
de Júcar, Cuenca province, Spain, and flower heads were collected in the
summer of 2005. A voucher specimen (No. HA-0125) has been deposited
in the Herbarium of the Pharmacology Department, School of Pharmacy,
Universidad Complutense of Madrid, Spain.
Isolation and identification of diterpenoids from H. annuus L. Dried and
powdered H. annuus L. flower heads without seeds (1 kg) were extracted in
a Soxhlet with petroleum ether (bp 50–70 °C) for 5 days. Evaporation of
the solvent yielded an extract (44 g) which was saponified (KOH 50 g,
MeOH 350 mL, benzene 50 mL and water 50 mL, 4 h under reflux). The
reaction mixture was diluted with water (400 mL), acidified with HOAc
until, pH ffi2, and then extracted with Et2O (4  200 mL). The ether
extract was dried (Na2SO4), filtered and evaporated in vacuo giving a
residue (39.1 g). Aliphatics were clathrated by dissolving the residue in
boiling MeOH (500 mL) and urea (118 g). The filtrate, after removal of the
solvent, yielded a residue (21.4 g) which was subjected to column chro-
matography [Si gel Merck 60 (0.040–0.063) 500 g, elution gradient from
100% petroleum ether to 1:1 petroleum ether–EtOAc] collecting 44 frac-
tions (100 mL each). Fractions 7–14, eluted with 9:1 petroleum ether–
EtOAc, furnished white crystals (2.8 g) of a mixture of kaurenoic and
trachylobanoic acids [13] and further elution with 1:1 petroleum ether–
EtOAc (fractions 28–44) yielded pure grandiflorolic acid (760 mg). A part
(1 g) of the mixture of kaurenoic and trachylobanoic acids was rechro-
matographed [Si gel column impregnated with 15% (w/w) of AgNO3,
150 g, 7:1 petroleum ether–EtOAc as eluent, fractions of 50 mL each]
yielding trachylobanoic acid (fractions 28–51, 206 mg), mixtures of both
diterpenoids (fractions 52–60, 422 mg) and kaurenoic acid (336 mg, frac-
tions 61–90). The isolated compounds were identified by their physical
(mp, [a]D) and spectroscopic (IR, 1H and 13C NMR and mass spectra)
data, which were identical to those reported in the literature: grandiflorolic
acid (ent-15b-hydroxy-16-kauren-19-oic acid) [13,14] kaurenoic acid (ent-
16-kauren-19-oic acid) [13,15] and trachylobanoic acid (ent-tracyloban-19-
oic acid) [13,16,17]. To the best of our knowledge, this is the first report on
the chromatographic resolution of a mixture of trachylobanoic and
kaurenoic acids without derivatization.
Cell culture. The murine macrophage cell line RAW 264.7 was main-
tained in RPMI 1640 medium supplemented with 10% fetal bovine serum
(FBS), L-glutamine (1 mM), penicillin (100 U/ml) and streptomycin
(100 lg/ml) in a humidified 5% CO2 atmosphere, as previously described
[9].
Cell viability assay. The mitochondrial-dependent reduction of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to forma-
zan [18] was used to exclude a cytotoxic effect of the diterpenoids at the
tested concentrations. To this end, cells were seeded in 96-well flat bot-
tomed microplates and were grown to confluence. Subsequently, cells were
incubated with vehicle or test compound for 48 h.
Nitric oxide (NO) and TNF-a production in RAW 264.7 macrophages.
Macrophage cells were seeded in 24-well plates and were incubated with
250 ng/ml of LPS at 37 °C for 24 h in the presence of test compounds or
vehicle. The release of NO into phenol red-free medium was determined
from the amount of accumulated nitrite, measured spectrophotometrically
with the Griess reagent. The absorbance at 548 nm was compared with a
NaNO2 standard. The accumulation of TNF-a in culture medium was
measured in duplicate with a commercial kit. Readings were compared to
TNF-a standards in culture medium containing diterpenoids to control for
potential effects of these compounds on the enzyme immunoassay.
Determination of cyclooxygenase-2 activity. RAW macrophages were
resuspended in RPMI 1640 culture medium containing aspirin (300 lM
and incubated at 37 °C for 2 h) to inactivate endogenous COX activity.
Thereafter, cells were washed twice, resuspended in RPMI 1640 containing
10% FBS and incubated with LPS 250 (ng/ml) at 37 °C for 24 h to induce
COX-2. After centrifugation the cells were sonicated at 4 °C in an ultr-
asonicator at maximum potency, and microsomes were prepared by cen-
trifugation at 2000g for 5 min at 4 °C followed by centrifugation of the
supernatant at 100,000 for 100 min at 4 °C. Microsomes (40 lg protein/
tube) were used as a source of COX-2, and were incubated for 30 min at
37 °C in 50 mM Tris–HCl, pH 7.4, supplemented with 5 lM arachidonic
acid and test compound or vehicle in the presence of 2 lM hematin and
1 mM L-trytophan. The reaction was terminated by boiling (5 min), and
PGE2 was determined with an enzyme immunoassay kit (GE Healthcare)
[19].
Preparation of cytosolic extracts. RAW 264.7 cells were washed twice
with ice-cold buffer A (10 mM Hepes, pH 7.9; 1 mM EDTA, 1 mM
EGTA, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, 2 lg/ml aprotinin,
10 lg/ml leupeptin, 2 lg/ml TLCK (Na-Tosyl-L-lysine chloromethyl
ketone hydrochloride), 5 mM NaF, 1 mM NaVO4, 10 mM Na2MoO4)
containing 120 mM NaCl and scraped off the plate. Cells were lysed at
4 °C with buffer A supplemented with 0.5% Nonidet P-40 with continuous
shaking. After centrifugation of the cell lysate the supernatant was stored
at 80 °C (cytosolic extract). Protein content was assayed with the Bio-
Rad protein reagent. All cell fractionation steps were carried out at 4 °C.
Western blot analysis. Cytosolic protein extracts were size-separated by
10% SDS–PAGE. Gels were blotted onto polyvinylidene difluoride
membrane (GE Healthcare) and probed with the following antibodies:
anti-NOS-2 and anti-b-actin (Santa Cruz Biotechnology); and anti-COX-2
(Cayman Chemical Co). Blots were submitted to sequential re-probing
with antibodies after treatment with 100 mM b-mercaptoethanol and 2%
SDS in TBS, and heating at 60 °C for 30 min. Blots were developed by
ECL according to the manufacturer’s instructions (GE Healthcare). b-
Actin was used as a loading control for cytosolic extracts.
RNA analysis and real-time quantitative PCR. Total RNA was
extracted with Trizol reagent (Life Technologies, Inc.). Real-time quan-
titative PCR (SYBRgreen) analysis was performed with an ABI 7900
sequence detector as described [20]. Briefly, 1 lg of total RNA was reverse
transcribed with random hexamers using the Taqman reverse transcription
reagent kit (Applied Biosystems) according to the manufacturer’s proto-
col. Each Taqman reaction (20 ll) contained 25 ng of cDNA, 500 nM
forward primer, 500 nM reverse primer, and 10 ll of Power SYBR Green
PCR Master mix (Applied Biosystems). PCR thermocycling parameters
were 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Each sample was run in duplicate, and all samples were analyzed in par-
allel for the expression of the housekeeping gene 36B4 (acidic ribosomal
phosphoprotein P0), used as an endogenous control to normalize the
expression level of target genes. Fold induction was determined from
average replicate values. Statistical analysis of mRNA expression was by
two-tailed, homoscedastic t-test. Primer sequences are available on
request.
Mouse ear edema. All experiments were approved by the institutional
Animal Care and Use Committee and were carried out in accordance with
the declaration of Helsinki and EC guidelines for the handling and use of
laboratory animals. Male Swiss mice (20–25 g) were housed in tempera-
ture-controlled rooms (22–23 °C) until use. Food and water were supplied
ad libitum and each experimental group contained six animals. 12-O-
Tetradecanoylphorbol acetate (TPA) (2.5 lg) dissolved in 20 ll of acetone
was applied in 10 ll volumes to the inner and outer surfaces of the right
ear. Test compounds were applied topically in acetone 30 min before TPA
administration. Animals were killed by cervical dislocation after 4 h, and
equal sections of both ears were punched out and weighed. The degree of
edema was indicated by the increase in the weight of the right ear punch
over the left. Ear sections were homogeneized in 750 ll saline and, after
 R. Dı́az-Viciedo et al. / Biochemical and Biophysical Research Communications 369 (2008) 761–766 763

centrifugation at 10,000g for 15 min at 4 °C, myeloperoxidase activity

(MPO) was measured in aliquots of supernatants of TPA-treated ear A B
Trachylobanic acid Trachylobanic acid
homogenates by a modification of the method of Suzuki et al. [21,22]. The
reaction mixture contained 50 ll supernatant, 150 ll phosphate buffered 120 120

TNF-α release(%)
saline, 20 ll 0.22 M NaH2PO4 (pH 5.4), 20 ll 0.026 (v/v)% H2O2 and 20 ll 100 100

Viability %
18 mM tetramethylbenzidine in 8% (v/v) aqueous dimethylformamide. 80 80
After 10 min reaction at 37 °C, 30 ll 1.46 M sodium acetate, pH 3.0 was 60 60 * * *
added and absorbance was read at 620 nm in a microtiter plate reader. 40 40 *
Statistical analysis. Data are presented as means ± SD (n = 3–4). 20 20
Statistical significance was estimated with Student’s t-test for unpaired 0 0
observations. A P < 0.05 was considered significant. In studies of Western 0 1 5 10 20 0 1 5 10 20
blot analysis, a linear correlation between increasing amounts of input Concentration μM Concentration μM
protein and signal intensity was observed (correlation coefficients higher
than 0.84).
Kaurenoic acid Kaurenoic acid
120 120

TNF-α release (%)

Results 100 100

Viability %
80 80
The diterpenoids selected for evaluation of potential 60 60 * * *
anti-inflammatory activity are shown in Fig. 1. At the con- 40 40 *
20 20
centrations assayed (1–20 lM), these compounds did not
0 0
exert any cytotoxic effects during the 48 h incubation per- 0 1 5 10 20 0 1 5 10 20
iod as indicated by MTT reduction assay (Fig. 2A). Concentration μM Concentration μM
To analyze the anti-inflammatory effects of the three
diterpenoids we next examined whether the release of Grandiflorolic acid Grandiflorolic acid
inflammatory mediators induced by LPS was affected by 120 120

TNF-α release (%)

these compounds in macrophages. To determine the effect 100 100
Viability %

of these compounds on cytokine release RAW macrophage 80 80

cultures were pretreated with the diterpenoids for 30 min 60 60 * * *
and subsequently incubated with LPS for 24 h. At concen- 40 40 *
20 20
trations not affecting cell viability, all diterpenoids signifi- 0 0
cantly inhibited the release of TNF-a in a concentration- 0 1 5 10 20 0 1 5 10 20
dependent manner (Fig. 2B). Concentration μM Concentration μM
We also investigated the effects of the three diterpenoids
Fig. 2. Dose-dependent effects of diterpenoids on cytotoxicity and
on LPS-induced release of the pro-inflammatory mediators
cytokine release. (A) Macrophages were pretreated with diterpenoids (1–
NO and PGE2. As shown in Fig. 3, we found that pretreat- 20 lM) and then left untreated for 48 h. At the end of treatment, the
ing cells with diterpenoids (1–20 lM) also reduced dose- extent of reduction of MTT (0.5 mg/ml) to formazan was spectrometri-
dependently NO release (Fig. 3A) and PGE2 (Fig. 3B). cally measured in a microplate reader (OD550 nm). (B) Macrophages were
To further analyze the signaling pathways modulated by pre-incubated for 30 min with diterpenoids (1–20 lM) and then stimulated
with 250 ng/ml LPS for 24 h. The accumulation of TNF-a in the culture
these diterpenoids, we studied whether their inhibitory
medium was determined in duplicate by ELISA. Results are the
effects on the pro-inflammatory mediators (PGE2 and means ± SD of three independent experiments carried out in triplicate.
NO) are related to NOS-2 and COX-2 modulation, as *
P < 0.05, vs. LPS.
markers of the activation process, using Western blot. In

unstimulated RAW 264.7 cells, levels of NOS-2 and

Fig. 1. Structures of diterpenoids isolated from Helianthus annuus L.
COX-2 proteins were undetectable. As Fig. 3C shows,
diterpenoids assayed at 20 lM potently inhibited the
expression of NOS-2 and were less efficient regarding the
effects on COX-2 (Fig. 3D). Moreover, analysis of NOS-2
and COX-2 mRNA by quantitative PCR revealed that
these compounds inhibited LPS-induced expression, once
again showing greater activity on NOS-2 induction
(Fig. 3E and F).
Evaluation of the in vivo anti-inflammatory activity of
these molecules was performed in the TPA-induced mouse
ear edema model. Topical application of diterpenoids sig-
nificantly reduced the extent of swelling induced by TPA
in a similar manner (Fig. 4A), and to a lesser extent that
the reference drug indomethacin. This topical anti-inflam-
matory activity was also confirmed by measuring MPO
764 R. Dı́az-Viciedo et al. / Biochemical and Biophysical Research Communications 369 (2008) 761–766

A B
Trachylobanic acid Trachylobanic acid
120 120 Kaurenoic acid
Kaurenoic acid
Grandiflorolic acid

PGE2 release (%)

100 Grandiflorolic acid 100

NO release(%)
80 * 80
* * * * *
* *
60 * 60 * * *
*
40 * * 40 ** ** **
20 ** ** ** 20
0 0
0 1 5 10 20 0 1 5 10 20
Concentration μM Concentration μM

C D
- + + + + LPS - + + + +
LPS
Trachylobanic acid - - + - - Trachylobanic acid - - + - -
Kaurenoic acid - - - + - Kaurenoic acid - - - + -
Grandiflorolic acid - - - - + Grandiflorolic acid - - - - +
COX-2
NOS-2

β-actin β-actin

E F
Fold induction, NOS-2 mRNA

Foldinduction, COX-2 mRNA

100 100
** **
80 80

60
b
60
*a *a *a
40
* * b
* b 40

20 20

0 0
control LPS TA KA GA control LPS TA KA GA

Fig. 3. Diterpenoids inhibit PGE2 and NO production through inhibition of COX-2 and NOS-2 expression. RAW 264.7 mouse macrophages were pre-
incubated for 30 min with diterpenoids (1–20 lM) and then stimulated with 250 ng/ml LPS for 24 h. The accumulation of nitrite (A) and PGE2 (B) in the
culture medium was determined in duplicate with the Griess reagent or by ELISA, respectively. Results are the means ± SD of three independent
experiments carried out in triplicate. *P < 0.05, **P < 0.01 vs. LPS. (C and D) Expression of NOS-2 and COX-2 proteins was detected by Western blot in
cells pre-incubated with diterpenoids (20 lM) for 30 min and then stimulated with 250 ng/ml LPS for 24 h. b-Actin content was used as a loading control.
Results show a representative experiment of three. (E and F) Macrophages were pre-incubated as in A and stimulated with 250 ng/ml LPS for 4 h. TA
(Trachylobanoic acid), KA (Kaurenoic acid), GA (Grandiflorolic acid). Relative expression of NOS-2 and COX-2 mRNA was determined by real-time
quantitative PCR. Results show the means ± SD of three independent experiments carried out in triplicate. *P < 0.05, **P < 0.01 vs. Control; a, P < 0.05,
b, P < 0.01 vs. LPS.

activity, a biochemical marker of neutrophil infiltration these compounds. H. annuus L., (Asteraceae) is an annual
[22,23], in ear biopsies. TPA-induced high levels of MPO herb whose seed oil, shoots and herb tincture have been
activity detected at 4 h after application, and this activity employed for anti-inflammatory purposes, although the
was also significantly attenuated by pretreatment with active components responsible for the anti-inflammatory
diterpenoids (Fig. 4B). Fig 4 demonstrates a high correla- effects of this plant have not been identified. Recently,
tion between diterpenoid inhibition of edema and the neu- the isolation of several triterpene glycosides from a metha-
trophil influx as assessed biochemically by accumulation of nol extract of ligulate flower petals of H. annuus has been
MPO. described. These triterpenes exhibited anti-inflammatory
activity against TPA-induced inflammation in mice,
Discussion although the mechanisms involved in this process have
not been evaluated [24]. In the present study, we report
The search for natural products with anti-inflammatory the isolation of three natural diterpenoids and the evalua-
activity has increased enormously in recent years. Diterpe- tion of their potential anti-inflammatory activities testing
noids have been identified as having a broad spectrum of their ability to inhibit two main inflammatory signaling
biological activities, including antibacterial, antiviral, pathways, the NO production and PGE2 release.
anti-inflammatory, cytotoxic, anti-tumor, etc. [6,7,9,23]. In murine macrophage RAW 264.7 cells, LPS induces
Indeed, many of them have been used for therapeutic pur- NOS-2, and then NO production. Therefore, this macro-
poses for centuries, but few reports have examined the phage cell line provides an excellent model for drug
mechanisms involved in the anti-inflammatory actions of screening and for evaluation of potential inhibitors on
 R. Dı́az-Viciedo et al. / Biochemical and Biophysical Research Communications 369 (2008) 761–766 765

A 120
100
80 * *

% edema
* * *
60 *
40
** ** **
20
***
0
0.25 0.5 1 0.25 0.5 1 0.25 0.5 1

TPA Trachylobanic acid Kaurenoic Acid Grandiflorolic Acid Indo

(mg/ear) (mg/ear) (mg/ear)

B 120

100
% MPO activity

80
* *
60 * *
** * **
40 *
** ***
20

0
0.25 0.5 1 0.25 0.5 1 0.25 0.5 1

TPA Trachylobanic acid Kaurenoic Acid Grandiflorolic Acid Indo

(mg/ear) (mg/ear) (mg/ear)

Fig. 4. Inhibitory effects of diterpenoids and indomethacin on TPA-mouse ear oedema and myeloperoxidase (MPO) activity. (A) The indicated amounts
of diterpenoid or indomethacin (1 mg/ear) were applied topically to one ear of mice, followed by application of TPA (2.5 lg/ear). Animals were killed after
4 h, and the percentage edema was determined from the ratio of the weights of identical ear tissue sections from the test and control (vehicle-treated) ears.
(B) Mice were treated as in A and MPO activity was determined in tissue homogenates. Results in A and B show the means ± SD of three independent
experiments carried out in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs. treatment with TPA alone.

the pathway leading to the induction of NOS-2. The
reactive free radical NO synthesized by NOS-2 is a major
macrophage-derived inflammatory mediator and also has
been reported to be involved in the development of
inflammatory diseases [25]. The production of TNF-a is
crucial for the synergistic induction of NO synthesis in
IFN-c and/or LPS-stimulated macrophages. TNF-a elic-
its a number of physiological effects including septic
shock, inflammation and cytotoxicity [26]. Moreover, a
large body of evidence suggests that PGs are involved
in various pathophysiological processes, including inflam-
mation and carcinogenesis, and inducible COX-2 is
mainly responsible for the production of large amounts
of these mediators [27]. Hence, the inhibition of pro-
inflammatory cytokines or iNOS and COX-2 expression
in inflammatory cells, offers us a new therapeutic strategy
for the treatment of inflammation. Based on this infor-
mation, we have evaluated the effects of three natural
diterpenoids isolated from H. annuus L. on LPS-induced
NO, PGE2 and TNF-a production in murine macro-
phage RAW 264.7 cells in an attempt to define their
potential as anti-inflammatory agents. We have shown
that low concentrations (10 lM) of these compounds
inhibit the production of NO and PGE2 in LPS-stimu-
lated macrophages. These inhibitory effects were accom-
panied by concentration-dependent decreases in the
expression of NOS-2 and COX-2 protein and mRNA,
indicating that the impaired release of NO and PGE2
can be attributed to inhibition of NOS-2 and COX-2
expression at the transcriptional level. Supporting these
data, diterpenoids efficiently inhibited cytokine release
(TNF-a) by LPS-stimulated macrophages. Furthermore,
the inhibition of the LPS-stimulated expressions of these
molecules was not due to diterpenoids cytotoxicity, as
assessed by MTT assay.
In addition to these data, these compounds also showed
in vivo anti-inflammatory activity, when tested in mice.
They prevented the extent of swelling in the TPA-induced
ear edema and inhibited MPO activity, indicating that a
control on leukocyte migration participated in the
observed topical anti-inflammatory activity.
In summary, the data presented in this study demon-
strate that these diterpenoids have anti-inflammatory activ-
ity in macrophages, as indicated by inhibition of NOS-2
and COX-2 expression and activity and impaired cytokine
release. The low cytotoxicity of these compounds in cell
culture and their effectiveness in an in vivo animal model
of inflammation indicate that these substances may con-
tribute to the anti-inflammatory activity attributed to the
sunflower.
766 R. Dı́az-Viciedo et al. / Biochemical and Biophysical Research Communications 369 (2008) 761–766
Acknowledgments
S.H. is a FIS program investigator and is supported by Plan
Nacional de Investigación Cientı́fica, Desarrollo e Innovación
tecnológica (I+D+I) and Instituto de Salud Carlos III. CNIC
is supported by the Spanish Ministry of Health and Consumer
Affairs and the Pro-CNIC Foundation.
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