A new dual inhibitor of arachidonate metabolism isolated from Helichrysum italicum

Article abstract of DOI:10.1016/S0014-2999(02)02954-0

Six acetophenones (1-6) and one γ-pyrone (7), previously isolated from Helichrysum italicum, were tested for their ability to inhibit enzymatic and non-enzymatic lipid peroxidation, the stable 1,1-diphenyl-2-pycryl-hydrazyl free radical, superoxide scavenging and arachidonic acid metabolism. In addition, they were studied in different experimental models such as the chronic inflammation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA), the phospholipase A₂-induced mouse paw oedema test, the carrageenan-induced mouse paw oedema test, and the writhing induced by acetic acid in the mouse. Of the assayed compounds, only 1 inhibited enzymatic lipid peroxidation but had no effect on non-enzymatic lipid peroxidation. None of them scavenged the superoxide radical. Study of the inhibition of arachidonic acid metabolism demonstrated that 1 was an inhibitor of both cyclooxygenase and 5-lipoxygenase, whereas 2 was a selective inhibitor of 5-lipoxygenase. In the assay of phospholipase A₂-induced mouse paw oedema, the γ-pyrone derivative inhibited oedema formation, showing a similar profile to that obtained with cyproheptadine. The acetophenones were effective at 30 and 60 min. In the carrageenan test, acetophenone 1 gave the best results and had analgesic effects in the acetic acid writhing test. In conclusion acetophenone 1 (4-hydroxy-3-(3-methyl-2-butenyl)acetophenone) is a new dual inhibitor of arachidonate metabolism, and could be a useful tool for obtaining anti-inflammatory and analgesic drugs.

Full text of DOI:10.1016/S0014-2999(02)02954-0

European Journal of Pharmacology 460 (2003) 219–226
www.elsevier.com/locate/ejphar
A new dual inhibitor of arachidonate metabolism isolated from
Helichrysum italicum
a
Araceli Sala , M. Carmen Recio , Guillermo R. Schinella , Salvador Manez ,
a
b
a
´
˜
a
Rosa M. Giner , Jose-Luis Rıos
a,
*
´
´
a Departament de Farmacologia, Facultat de Farma`cia, Universitat de Vale`ncia, Av. Vicent Andre´s Estelle´s s/n. 46100 Burjassot, Spain
b Ca´tedra de Farmacolog´ıa, Facultad de Ciencias Me´dicas, Universidad Nacional de La Plata, Argentina
Received 8 August 2002; received in revised form 16 December 2002; accepted 20 December 2002
Abstract
Six acetophenones (1–6) and one g-pyrone (7), previously isolated from Helichrysum italicum, were tested for their ability to inhibit
enzymatic and non-enzymatic lipid peroxidation, the stable 1,1-diphenyl-2-pycryl-hydrazyl free radical, superoxide scavenging and
arachidonic acid metabolism. In addition, they were studied in different experimental models such as the chronic inflammation induced by
12-O-tetradecanoylphorbol 13-acetate (TPA), the phospholipase A2-induced mouse paw oedema test, the carrageenan-induced mouse paw
oedema test, and the writhing induced by acetic acid in the mouse. Of the assayed compounds, only 1 inhibited enzymatic lipid peroxidation
but had no effect on non-enzymatic lipid peroxidation. None of them scavenged the superoxide radical. Study of the inhibition of arachidonic
acid metabolism demonstrated that 1 was an inhibitor of both cyclooxygenase and 5-lipoxygenase, whereas 2 was a selective inhibitor of 5-
lipoxygenase. In the assay of phospholipase A2-induced mouse paw oedema, the g-pyrone derivative inhibited oedema formation, showing a
similar profile to that obtained with cyproheptadine. The acetophenones were effective at 30 and 60 min. In the carrageenan test,
acetophenone 1 gave the best results and had analgesic effects in the acetic acid writhing test. In conclusion acetophenone 1 (4-hydroxy-3-(3-
methyl-2-butenyl)acetophenone) is a new dual inhibitor of arachidonate metabolism, and could be a useful tool for obtaining anti-
inflammatory and analgesic drugs.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Acetophenones; Maltol-glucoside; Arachidonic acid metabolism; Anti-inflammatory compound; Analgesic compound
1. Introduction
and possess different pharmacological activities, such as
antioxidant and anti-inflammatory effects. Several acetophe-
nones were tested in vitro for their effects on superoxide
anion production in human polymorphonuclear neutrophils.
Among them, apocynin (4-hydroxy-3-methoxy-acetophe-
none) and 4-hydroxyethyloxy-3-methoxy-acetophenone
were shown to be the most active inhibitors of the neutrophil
oxidative burst (Stuppner et al., 1995). Apocynin inhibited
superoxide formation by preventing the assembly of the
superoxide-generating enzyme NADPH-oxidase (Stolk et
al., 1994) and also strongly inhibited the formation of
peroxynitrite by murine macrophages (Muijsers et al.,
2000). Lapperre et al. (1999) suggested that the antioxidant
properties of apocynin could be related to the increase in
glutathione synthesis through activation of transcription
factors such as activator protein-1 (AP-1).
Helichrysum italicum is a perennial herb used in folk
medicine for its anti-inflammatory properties. In previous
studies (Schinella et al., 2002; Sala et al., 2002), we reported
the antioxidant and anti-inflammatory activities of the aerial
parts of this plant in various in vitro and in vivo exper-
imental models. We have reported the isolation, iden-
tification and topical anti-inflammatory activity of six
acetophenones and one g-pyrone (Fig. 1) against 12-O-
tetradecanoylphorbol 13-acetate (TPA)-induced mouse ear
oedema (Sala et al., 2001).
Acetophenones are a group of phenolic metabolites
present in various taxonomic groups in the plant kingdom
There are some interesting studies in vivo of the anti-
inflammatory and anti-asthmatic effects of diverse natural
* Corresponding author. Tel./fax: +34-965-454-973.
´
E-mail address: riosjl@uv.es (J.-L. Rıos).
0014-2999/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0014-2999(02)02954-0

220
A. Sala et al. / European Journal of Pharmacology 460 (2003) 219–226
glucopyranoside (5), 3-(2-hydroxyethyl) acetophenone-4-O-
h-D-glucopyranoside (6) and maltol h-D-O-glucopyranoside
(7) were isolated from the anti-inflammatory extracts of H.
italicum (Sala et al., 2001). All of them have a purity higher
than 99.9%, according to their mass spectra and nuclear
magnetic resonance data. Acetic acid, allopurinol, ascorbic
acid, butylated hydroxytoluene, calcium ionophore A23187,
carrageenan, cyproheptadine hydrochloride, dexametha-
sone, dimethylsulpoxide, 1,1-diphenyl-2-pycryl-hydrazyl
ethylenediaminetetraacetic acid, glucose-6-phosphate,
glucose-6-phosphate dehydrogenase, glycogen, hexadecyl-
trimethylammonium bromide, hydrogen peroxide, hypoxan-
thine, indomethacin, 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenil-tetrazolium bromide (MTT), nitro blue tetrazolium,
N,N-dimethylformamide, phosphate-buffered saline, phos-
pholipase A2 from Naja mossambica venom, prostaglandin
B2, pyrogallol, TPA, tetramethylbenzidine, 2-thiobarbituric
acid, trichloroacetic acid, Trypan blue, xanthine and xan-
thine oxidase were purchased from Sigma (St. Louis, MO,
USA); Tween 80 was from Fluka Chemika–Biochemika
(Buchs, Switzerland); acetone analytical grade, and meth-
anol of high-performance liquid chromatography gradient
grade were from Baker (Deventer, Holland); ethanol 96j,
methanol, sodium acetate and trifluoroacetic acid, all of
them of analytical grade, were from Panreac (Barcelona,
Spain).
Fig. 1. Chemical structures of compounds isolated from H. italicum: (1),
(1a), (2), (3), (4), (5), (6) and (7).
2.2. Acetylation of acetophenone 1
and synthetic acetophenone derivatives (Muller et al., 1999;
¨
Favier et al., 1998; Dorsch et al., 1994; Ohmi et al., 1994).
Apocynin proved to be very active in rat models of arthritis
or in ulcerative skin lesions in rats (’t Hart et al., 1990,
1992). More recently, Lafeber et al. (1999) described its
effect on inflammation-induced cartilage destruction in
rheumatoid arthritis in a human in vitro experimental model,
accompanied by a decrease in interleukin-1 and tumour
necrosis factor-a production by mononuclear cells.
Acetylation of acetophenone 1 (50 mg) with acetic
anhydride in pyridine afforded, after usual work-up, 48
mg of 4-acetoxy-3-(3-methyl-2-butenyl)acetophenone (1a).
Acetylated compound was purified by gel filtration on a
Sephadex LH-20 column with methanol as eluant.
2.3. Animals
Our study examined the antioxidant and anti-inflamma-
tory activities of the isolated acetophenones on lipid perox-
idation, free radical generation, and arachidonic acid
metabolism and in different in vivo experimental models
of acute and chronic inflammation and peripheral analgesia.
The aim of this paper is to determine the mechanism of
action of the active acetophenones and to establish their
potential as new anti-inflammatory agents.
Groups of six Swiss female mice weighing 25–30 g and
female Wistar rats weighing 180–200 g were used. All
animals were fed on a standard diet ad libitum. Housing
conditions and all in vivo experiments were approved by the
institutional Ethics Committee of the Faculty of Pharmacy
according to the guidelines established by the European
Union on Animal Care (CEE Council 86/609).
2.4. Lipid peroxidation
2. Materials and methods
Rat liver microsomes were prepared using a standard
differential centrifugation technique (Schinella et al., 2000).
Protein contents were quantified by Bradford’s method
using bovine serum albumin as the standard (Bradford,
1976). The antioxidant activity of the products dissolved
in dimethylsulfoxide was tested at a final concentration of
100 AM.
2.1. Test compounds and chemicals
4-Hydroxy-3-(3-methyl-2-butenyl)acetophenone (1), 4-
hydroxy-3-(2-hydroxy-3-isopentenyl) acetophenone (2),
12-hydroxytremetone (bitalin A) (3), 3-(3-methyl-2-buteny-
l)acetophenone-4-O-h-D-glucopyranose (4), 12-hydroxytre-
metone-12-O-h-D-glucopyranoside or bitalin A-12-O-h-D-
Non-enzymatic peroxidation was induced by addition of
FeSO4 (5 AM) and ascorbate (500 AM) (Schinella et al.,

A. Sala et al. / European Journal of Pharmacology 460 (2003) 219–226
221
2000). The products of lipid peroxidation were detected by
measuring the absorbance at 535 nm, using the thiobarbi-
turic acid method. Butylated hydroxytoluene was used as a
positive control. In enzymatic lipid peroxidation, the reac-
tion mixture contained microsomal protein and an NADPH-
generating system. Peroxidation was started by the addition
of 10 Al CCl4 1:4 (v/v) in dimethylsulfoxide. After a 15-min
incubation at 37 jC, thiobarbituric acid-reactive species
were determined as above.
with 0.007% (v/v) trifluoracetic acid; B = water with 0.007%
trifluoracetic acid]; solvent gradient, 50% A to 74% A linear
in 27 min, 50% A isocratic for 12 min; flow 1.0 ml/min. The
results obtained from peak areas were normalised to prosta-
glandin B2 (17 Ag/ml) internal standard and expressed as a
percentage of leukotriene B4 production. Experiments were
performed in triplicate. IC50 value was calculated by means of
linear regression plotted from the inhibition percentages
obtained with four different concentrations.
2.5. Free radical generation
2.8. Assay of cyclooxygenase-1 activity in human platelets
Superoxide radical was generated by enzymatic oxida-
tion of hypoxanthine with xanthine oxidase grade I and was
detected by nitroblue tetrazolium reduction, monitored
spectrophotometrically at 560 nm (Schinella et al., 2000).
Pyrogallol was used as a positive control. The influence on
enzyme activity was evaluated by uric acid formation from
xanthine, and absorbance was measured at 295 nm. Allo-
purinol was used as a positive control. A solution of 1,1-
diphenyl-2-picrylhydrazyl radical in methanol was added to
the compound solutions and activity was determined by
spectrophotometry at 517 nm after 10 min (Schinella et al.,
2000). Butylated hydroxytoluene was used as a reference
compound.
Blood platelets were obtained from healthy human donors
and were separated by sequential centrifugation. The platelets
were incubated in the presence of the test compounds 1 and 2
at 100 AM. Stimulation was performed according to Safayhi
et al. (1995) and Laufer et al. (1995) with 2.5 nM Ca2 + and
1.9 AM calcium ionophore 23187. Separation of 12-hydrox-
yheptadecatrienoic acid (12-HHTrE) was achieved by high-
performance liquid chromatography coupled to diode array
detection. A reverse-phase C18 column was used and eluted
with methanol/water (74:26) containing 0.007% (v/v) tri-
fluoroacetic acid. The results obtained are expressed as
percentages of 12-HHTrE production.
2.9. Mouse ear inflammation induced by multiple topical
applications of TPA
2.6. Cytotoxicity assay
Cytotoxicity of the acetophenones and maltol-glucoside
was measured by the colorimetric assay of Mosmann
(1983). Neutrophils were exposed to the products (100
AM) in a microplate for 30 min, and then 100 Al per well
of a 5 mg/ml solution of MTT was added and incubated at
37 jC until blue deposits were visible. The coloured
metabolite was dissolved in dimethylsulfoxide (100 Al per
well). This reaction was performed in triplicate. Absorbance
was measured at 490 nm using a Labsystems Multiskan
MCC/340 plate reader. Results are expressed in absolute
absorbance readings; a decrease indicated a decrease in cell
viability.
Inflammation was induced by topical application on
alternate days (five applications) of 2 Ag of TPA (20 Al) to
each ear (Stanley et al., 1991). Compounds 1 and 3 (0.5 mg/
ear), and dexamethasone (0.05 mg/ear) were applied topi-
cally twice daily for 4 days. On the last day, the compounds
were applied only in the morning. The mice were killed by
cervical dislocation, and two ear punches from each animal
were taken (n = 5 animals). Details of the method have been
described earlier (Giner-Larza et al., 2001).
2.10. Myeloperoxidase assay
According to the method used by De Young et al. (1989),
each ear sample was placed in an Eppendorf tube with 0.75
ml of 80 mM sodium phosphate buffer (pH = 5.4) containing
0.5% hexadecyltrimethylammonium bromide. Enzyme
activity was determined colorimetrically using a Labsystems
Multiskan MCC/340 plate reader set to measure absorbance
at 620 nm. Details of the method have been described earlier
(Giner-Larza et al., 2001).
2.7. Inhibition of leukotriene B4 production by rat
polymorphonuclear leukocytes
Rat peritoneal leukocytes (95% viability) were prepared
according to Safayhi et al. (1995). For 5-lipoxygenase prod-
uct formation from endogenous arachidonic acid, leukocytes
were stimulated at 37 jC for 5 min with the calcium
ionophore A23187 (1.9 AM) and Ca2 + (1.8 mM). The cells
were incubated in the presence of the test compounds at a
final concentration of 100 AM. All incubations, including
controls, were carried out in the presence of 0.5% dimethyl-
sulfoxide. Separation of arachidonic acid products was per-
formed by high-performance liquid chromatography
followed by diode array detection. A reverse-phase (RP-18)
column was used and eluted with mobile phase [A= methanol
2.11. Histology
Ear samples were fixed in 4% neutral-buffered formalin.
Each sample was cut longitudinally into equal halves. Half
of each was embedded in paraffin, cut into 3- to 4-Am
sections and stained with haematoxylin-eosin. Epithelium
Â
thickness was evaluated using an objective
100 and

222
A. Sala et al. / European Journal of Pharmacology 460 (2003) 219–226
expressed as the mean F S.D. of the number of epidermal
layers from the basal to the granulous stratum, inclusive
(Giner-Larza et al., 2001).
treated only with the inflammatory agent. One-way analysis
of variance (ANOVA) followed by Dunnett’s t-test for multi-
ple comparisons of unpaired data was used for statistical
evaluation.
2.12. Phospholipase A2-induced mouse paw oedema
The method was that described by Neves et al. (1993).
Phospholipase A2 from N. mossambica (2 units in 25 Al of
sterile saline) was injected s.c. into the right hind mouse
paw. The left paw received the same volume of vehicle. The
test compounds (80 mg/kg) were injected i.p. 30 min before
phospholipase A2, and the reference drug cyproheptadine
(10 mg/kg) was administered p.o. 60 min prior to inflam-
mation induction. Both the products and the reference drug
were dissolved in Tween 80/ethanol/saline (1:1:10).
Oedema was measured with a plethysmometer (Ugo Basile)
30, 60 and 90 min after challenge and is expressed as the
difference between the right and left paw volumes. The
control group was treated only with phospholipase A2.
Inhibition is expressed as a percentage of control.
3. Results
3.1. Effects on lipid peroxidation and free radical
generation
Acetophenones 1 and 2 (Fig. 1) were the only active
compounds against enzymatic lipid peroxidation in rat liver
microsomes (51% and 25% inhibition, respectively), while
none showed activity in the non-enzymatic lipid peroxidation
test. All the compounds, except acetophenone 4 (29% inhib-
ition), were inactive against the stable 1,1-diphenyl-2-pycryl-
hydrazyl free radical. Finally, no compound exhibited super-
oxide scavenging properties in the hypoxanthine/xanthine
oxidase system or in human leukocytes stimulated with TPA.
2.13. Carrageenan-induced mouse paw oedema
3.2. Effect on leukotriene B4 production by rat polymor-
phonuclear leukocytes
Oedema was induced in the right hind paw by subplantar
injection of carrageenan (3% w/v in saline, 25 Al) (Sugishita
et al., 1981). Compounds 1 and 1a, dissolved in Tween 80/
ethanol/saline (1:1:10) were administered orally at a dose of
150 mg/kg (0.2 ml), 1 h before carrageenan injection. A
group received the reference drug indomethacin (10 mg/kg,
p.o.). Right and left paw volumes were measured with a
plethysmometer (Ugo Basile) 1, 3 and 5 h after inflamma-
tion induction. Oedema is expressed as the difference
between right and left paw volumes, and oedema inhibition
is expressed as the percentage of volume reduction relative
to the control.
None of the tested compounds had cytotoxicity in the
MTT test. Fig. 2 shows the effect on the production of
leukotriene B4 by rat peritoneal leukocytes, which was clearly
reduced by acetophenones 1 and 2 at 100 AM (95% and 44%,
respectively). This effect was concentration dependent, and
IC50 values were therefore determined. The IC50 for aceto-
phenone 1 was nearly four times lower than that for aceto-
phenone 2. The rest of compounds had no activity at 100 AM.
3.3. Effect on cyclooxygenase-1 activity in human platelets
When the compounds were tested for their inhibitory
effect on cyclooxygenase-1 at 100 AM, acetophenone 1
2.14. Writhing test
The experiment was performed according to the
method described by Atta-ur-Rahman et al. (2001) with
minor modifications. Acetophenones 1 and 1a, and
aspirin, a reference peripheral analgesic drug, at 100
mg/kg were administered p.o. and i.p. 1 h or 30 min,
respectively, before the i.p. administration of 3% acetic
acid solution (0.25 ml). Control animals received vehicle
under the same experimental conditions. Immediately after
acetic acid injection, each animal was isolated in an
individual box to be observed for 20 min. The number
of writhings and stretchings was recorded. Analgesic
activity is expressed as the percentage of writhing reduc-
tion relative to the control.
Fig. 2. Inhibition concentration-50 (IC50) of 1 and 2 on leukotriene B4
production from endogenous arachidonic acid in Ca2 + ionophore-
stimulated rat peritoneal polymorphonuclear leukocytes. Concentrations
assayed ranging from 12.5 to 200 AM. Data are expressed as means
F S.E.M. percentages of metabolite formation with respect to controls:
n = 4–6.
2.15. Statistical analysis
Inhibition percentages were calculated from the differ-
ences between drug-treated animals and control animals

A. Sala et al. / European Journal of Pharmacology 460 (2003) 219–226
223
Â
Fig. 3. Effect of acetophenones 1 and 3 (0.5 mg/ear 7) on myeloperox-
idase activity following ear inflammation induced by repeated application
Â
of TPA. ** P < 0.01 (Dunnett’s t-test). Dexamethasone 0.05 mg/ear 7.
was the only one to reduce the production of 12-HHTrE
by 59%.
Fig. 5. Effect of 1 and its acetylated derivative 1a (150 mg/kg, p.o.) on
carrageenan-induced mouse paw oedema. Footpad oedema was induced 1 h
later by injection of carrageenan (3% w/v in saline). Footpad volume was
measured 1, 3 and 5 h after irritant injection. Each point represents the mean
from five to six increases in footpaw volume and the vertical lines indicate
the S.E.M. Statistically significant difference with respect to the control is
expressed as ** P < 0.01 (Dunnett’s t-test).
3.4. Effect on mouse ear inflammation induced by multiple
topical applications of TPA
Acetophenones 1 and 3 were studied in a model of
chronic inflammation induced by TPA. Neither inhibited
oedema formation (data not shown), but they did signifi-
cantly inhibit myeloperoxidase activity by 57% and 71%,
respectively (Fig. 3). The histological study demonstrated
that none of the compounds affected the inflammatory
process (data not shown).
3.6. Effect on carrageenan-induced mouse paw oedema
Acetophenone 1 was the only compound active against
the cyclooxygenase pathway in isolated human platelets.
This compound and its acetylated derivative (1a) were
assayed in the carrageenan (Fig. 5) and the peripheral
analgesia (Fig. 6) tests. Both compounds exhibited signifi-
cant anti-inflammatory activity, inhibiting paw oedema by
51% and 75%, respectively, after the first hour. Later
measurements gave similar results for both compounds,
with 71% and 79% inhibition at 3 h, and 66% and 70%
inhibition at 5 h, respectively.
3.5. Effect on phospholipase A2-induced mouse paw
oedema
All the substances, except acetophenone 2, which was
isolated in a small quantity, were studied in vivo against the
phospholipase A2-induced mouse paw oedema (Fig. 4). One
hour after phospholipase A2 injection, the most active
compound was acetophenone 5 (65% oedema reduction)
followed by compounds 6 and 7 (57% and 52% of oedema
inhibition, respectively). These compounds were still active
90 min after phospholipase A2 subplantar injection.
3.7. Analgesic effect
Finally, acetophenones 1 and 1a were tested in the acetic
acid writhing test and both compounds showed analgesic
Fig. 4. Anti-inflammatory effect of acetophenones (1–6) and compound 7 (80 mg/kg, i.p.) and cyproheptadine (10 mg/kg, p.o.) on phospholipase A2-induced
paw oedema 30, 60 and 90 min after irritant injection. PLA2: Phospholipase A2. Each point represents the mean for five to six animals and the vertical lines
indicate the S.E.M. **P < 0.01 (Dunnett’s t-test).

224
A. Sala et al. / European Journal of Pharmacology 460 (2003) 219–226
suggest that the antioxidant properties of our acetophenones
are not involved in their mechanism of anti-inflammatory
activity.
Hydroxyacetophenone derivatives have been described
as inhibitors of chemotaxis of polymorphonuclear granulo-
cytes (Muller et al., 1999; Ohmi et al., 1994). Methyl or
¨
methoxy group substitution in C-3 resulted in pronounced
inhibitory effects (Muller et al., 1999). In our studies,
¨
histology of the ear sample obtained following the chronic
inflammation induced by repeated application of TPA dem-
onstrated that acetophenones 1 and 3 had no effect on the
inflammation or on the neutrophil infiltration (data not
shown). These results suggest that the myeloperoxidase
activity inhibition observed in the chronic model of inflam-
mation is due to direct inhibition of the enzyme. In a
previous paper, Van den Worn et al. (2001) studied the
inhibition of this enzyme by different structurally related
compounds, and only two inhibited the release of myelo-
peroxidase and/or its activity, but in both cases, the com-
pounds were acid derivatives. There is no direct chemical
relationship between these active compounds and our ace-
tophenones.
Fig. 6. Analgesic effects of 1 and its derivative 1a in the writhing test.
Compounds were administered p.o. and i.p. at 100 mg/kg. Aspirin was
administered at the same dose only p.o. ** P < 0.01; * P < 0.05 (Dunnett’s
t-test). n = 5 animals.
properties after oral and intraperitoneal administration.
However, although similar behaviour was observed p.o.,
different responses were noted after intraperitoneal admin-
istration, with inhibition values of 75% and 92%, respec-
tively.
Six of the compounds isolated from H. italicum were
assayed in the phospholipase A2 test, and the results
demonstrated a net effect against this irritant. Of these,
compound 7, a g-pyrone glucoside, was the only one to
be effective throughout the inflammatory process. The
mechanism responsible for this effect may be a direct
inhibition of the enzyme, a blockage of mast cell degranu-
lation, or an antagonism of the liberated vasoamines (Cirino
et al., 1989). The behaviour of compound 7 was similar to
that of cyproheptadine, the reference drug used, which is a
serotonin and histamine antagonist. A structural relationship
can be established between chemical composition and
pharmacological response. In general, glycosylation had
no effect at 30 min but the response at 90 min was greater
than that of the corresponding aglycones. However, the free
forms responded quickly against the oedema induced by the
irritant agent. Only acetophenone 4 was ineffective at all the
different times investigated, but its corresponding aglycone
was active at 30 and 60 min after challenge.
4. Discussion
In a previous paper, Sala et al. (2001) reported the anti-
inflammatory activity of some of the acetophenones isolated
from H. italicum against TPA-induced ear oedema. In this
study, we demonstrate the effects of these compounds in
different experimental models in order to establish their
potential as anti-inflammatory agents.
Some acetophenones have previously been reported to be
inhibitors of reactive oxygen species production, and a
relationship between their chemical structure and pharma-
cological activity has been established (Dorsch et al., 1994;
Stuppner et al., 1995; Van den Worn et al., 2001). Apocynin
and its derivatives are the best known of the agents with this
type of structure. It is a potent inhibitor of the NADPH
oxidase involved in superoxide anion generation in stimu-
lated human neutrophils, has low toxicity and does not
interfere with other neutrophil functions. Our data demon-
strate that the acetophenones isolated from H. italicum have
no antioxidant properties and only compound 1 inhibited
enzymatic lipid peroxidation, while it had no effect on non-
enzymatic peroxidation. The most relevant difference
between apocynin and acetophenone 1 is the presence of a
methoxy group at C-3 in the former and a butenyl group in
the latter. Various studies of structure–activity relationships
of acetophenones have highlighted the relevance of methox-
ylation in C-5 and its interference with the signal trans-
duction that mediates neutrophil activation. Our findings,
together with the results obtained with apocynin derivatives,
Arachidonic acid metabolism was inhibited only by
acetophenones 1 and 2. At 100 AM, acetophenone 1 totally
inhibited leukotriene B4 production by 5-lipoxygenase in rat
neutrophils stimulated with calcium ionophore, while its
effect on the production of 12-HHTrE by cyclooxygenase-1
in human platelets, also stimulated with the same agent, was
more moderate. Acetophenone 2 was a more specific
inhibitor of 5-lipoxygenase than of cyclooxygenase-1,
although its potency was about five times lower than that
of acetophenone 1.
The latter compound was tested in the carrageenan
mouse paw oedema model in order to explore its anti-
inflammatory effect. This test is a good tool for investigat-
ing the in vivo effects of cyclooxygenase inhibitors, princi-
pally its constitutive form or cyclooxygenase-1, which is the

A. Sala et al. / European Journal of Pharmacology 460 (2003) 219–226
225
biological source of prostaglandin E2 production during the
inflammatory process (Smith et al., 1998). Some acetophe-
nones, such as tremetone, exhibited anti-inflammatory
activity in the acute inflammatory process induced by
carrageenan in the paw mouse (Favier et al., 1998). The
effects of acetophenone 1 3 h after the subcutaneous
injection of carrageenan were similar to those observed
for indomethacin, although we noted that the effects of
acetophenone 1 were longer lasting, reducing the inflam-
mation in the later phase (5 h). These results confirm those
previously reported by Malaviya et al. (2000) for related
acetophenones in a study of their effects on the production
of prostaglandin E2 in vitro as well as in vivo in the
carrageenan air pouch test.
IV.11. GS is member of CIC Provincia de Buenos Aires,
Argentina.
References
Atta-ur-Rahman, Choudhary, M.I., Thomsen, W.J., 2001. Bioassay Tech-
niques for Drug Development. Harwood Academic Publishers,
Amsterdam, p. 100.
Ballou, L.R., Botting, R.M., Goorha, S., Zhang, J., Vane, J.R., 2000. No-
ciception in cyclooxygenase isozyme-deficient mice. Proc. Natl. Acad.
Sci. U. S. A. 97, 10272–10276.
Berkenkopf, J.W., Weichman, B.M., 1988. Production of prostacyclin in
mice following intraperitoneal injection of acetic-acid, phenylbenzoqui-
none and zymosan—its role in the writhing response. Prostaglandins
36, 693–709.
It had been demonstrated that the writhing response to
intraperitoneal injection of acetic acid in mice is produced
by the release of prostacyclin synthesised by cyclooxyge-
nase-1 in the abdominal cavity (Berkenkopf and Weichman,
1988). The analgesic potency of some non-steroid anti-
inflammatory drugs correlates closely with increasing selec-
tivity for cyclooxygenase-1 rather than for cyclooxygenase-
2 (Warner et al., 1999; Ballou et al., 2000). In a parallel
assay, we tested the potential effect of acetophenone 1 as an
analgesic agent and obtained encouraging results when it
was assayed in the writhing test. Acetophenone 1 was
effective after oral and intraperitoneal administration, with
an effect similar to that obtained with the reference drug
aspirin, at the same dose.
Bradford, M.M., 1976. A rapid and sensitive method for quantification of
micrograms quantities of protein utilizing the principle of protein-dye
binding. Anal. Biochem. 72, 248–254.
Cirino, G., Peers, S.H., Wallace, J.L., Flowers, R.J., 1989. A study of
phospholipase A2-induced oedema in rat paw. Eur. J. Pharmacol. 166,
505–510.
De Young, L.M., Kheifets, J.B., Ballaron, S.J., Young, J.M., 1989. Edema
and cell infiltration in the phorbol ester-treated mouse ear are tempo-
rally separate and can be differentially modulated by pharmacological
agents. Agents Actions 26, 335–341.
Dorsch, W., Mu¨ller, A., Christoffel, V., Stuppner, H., Antus, S., Gottsegen,
A., Wagner, H., 1994. Antiasthmatic acetophenones—an in vivo study
on structure activity relationship. Phytomedicine 1, 47–54.
Favier, L., Tonn, C., Guerreiro, E., Rotelli, A., Pelzer, L., 1998. Anti-
inflammatory activity of acetophenones from Ophryosporus axilliflo-
rus. Planta Med. 64, 657–659.
´
Giner-Larza, E.V., Man˜ez, S., Recio, M.C., Giner, R.M., Prieto, J.M., Cer-
To complete this study, we synthesised an acetylated
derivative of acetophenone 1 (1a) and assayed it in the same
experimental protocols used for acetophenone 1. In this
case, the effects were similar to those obtained for aceto-
phenone 1 in the paw oedema test, but the activity of
acetophenone 1a was higher at 1 h. Both compounds
showed similar properties in the writhing test after oral or
parenteral administration, showing a more marked effect in
the latter case, in particular the acetylated derivative (1a).
The behaviour of both compounds in this assay was similar
to that of aspirin, when administered orally at the same dose
as the reference drug.
´
´
´
da-Nicolas, M., Rıos, J.L., 2001. Oleanonic acid, a 3-oxotriterpene from
Pistacia, inhibits leukotriene synthesis and has anti-inflammatory activ-
ity. Eur. J. Pharmacol. 428, 137–143.
Lafeber, F.P., Beukelman, C.J., van der Worm, E., van Roy, J.L., Vianen,
M.E., van Roon, J.A., van Dijk, H., Bijlsma, J.W., 1999. Apocynin, a
plant-derived, cartilage-saving drug, might be useful in the treatment of
rheumatoid arthritis. Rheumatology (Oxford) 38, 1088–1093.
´
Lapperre, T.S., Jimenez, L.A., Antonicelli, F., Drost, E.M., Hiemstra, P.S.,
Stolk, J., MacNee, W., Rahman, I., 1999. Apocynin increases gluta-
thione synthesis and activates AP-1 in alveolar epithelial cells. FEBS
Lett. 443, 235–239.
Laufer, S., Neher, K., Bayer, B., Homman, J., Reutter, E., Tries, S., 1995.
In vitro test system for the evaluation of dual cyclooxygenase and 5-
lipoxygenase inhibitors. Pharm. Pharmacol. Lett. 4, 166–169.
Malaviya, R., Malaviya, R., Uckun, F.M., 2000. Anti-inflammatory activ-
ity of 2,4,6-trihydroxy-a-p-methoxyphenyl-acetophenone (compound
D-58). Dermatology 201, 337–342.
These results confirm the potential of H. italicum as a
medicinal plant to be used in different inflammatory and
allergic diseases, and confirm the potential of its isolated
compound, 1, as a new dual anti-inflammatory and analge-
sic drug. This compound could be a useful tool for obtaining
new analgesic and anti-inflammatory agents, following
minor structural modifications.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and sur-
vival: applications to proliferation and cytotoxicity assays. J. Immunol.
Methods 65, 55–63.
Muijsers, R.B.R., van der Worm, E., Folkerts, G., Beukelman, C.J., Kos-
ter, A.S., Postma, D.S., Nijkamp, F.P., 2000. Apocynin inhibits perox-
ynitrite formation by murine macrophages. Br. J. Pharmacol. 130,
932–936.
Muller, A.A., Reiter, S., Heider, K.G., Wagner, H., 1999. Plant-derived
¨
Acknowledgements
acetophenones with antiasthmatic and anti-inflammatory properties:
inhibitory effects on chemotaxis, right angle light scatter and actin
polymerization of polymorphonuclear granulocytes. Planta Med. 65,
590–594.
The authors wish to thank the Spanish government for
its financial support (DGESIC, PM98-0206). We are
indebted to the Centre de Transfusions de la Comunitat
Valenciana (Valencia, Spain) for its generous supply of
human blood. This study is a part of the CYTED project
Neves, P.C.A., Neves, M.C.A., Bella Cruz, A., Sant’ana, A.E.G., Yuner,
R.A., Calixto, J.B., 1993. Differential effects of Mandevilla velutina
compounds on paw oedema induced by phospholipase A2 and phos-
pholipase C. Eur. J. Pharmacol. 243, 213–219.

226
A. Sala et al. / European Journal of Pharmacology 460 (2003) 219–226
Ohmi, N., Tani, C., Yamada, K., Fukui, M., 1994. Pharmacological profile
of a novel orally active leukotriene B4 antagonist, SM-15178. Inflam-
mation 18, 129–140.
teristics of the inhibition of NADPH oxidase activation in neutrophils
by apocynin, a methoxy-substituted catechol. Am. J. Respir. Cell Mol.
Biol. 11, 95–102.
Safayhi, H., Sailer, S., Havens, M., 1995. Mechanism of 5-lipoxygenase
inhibition by acetyl-11-keto-h-boswellic acid. Mol. Pharmacol. 47,
1212–1216.
Stuppner, H., Reinisch, O., Wiedermann, C.J., Wagner, H., 1995. Aceto-
phenones-compounds from plant origin with inhibitory effects on neu-
trophil in vitro respiration burst activity. Phytomedicine 4, 283–286.
Sugishita, E., Amagaya, S., Ogihara, Y., 1981. Anti-inflammatory testing
methods: comparative evaluation of mice and rats. J. Pharm. Dyn. 4,
565–575.
´
´
Sala, A., Recio, M.C., Giner, R.M., Man˜ez, S., Rıos, J.L., 2001. New
acetophenone glucosides isolated from extracts of Helichrysum italicum
with anti-inflammatory activity. J. Nat. Prod. 64, 1360–1362.
´
Sala, A., Recio, M.C., Giner, R.M., Man˜ez, S., Tournier, H., Schinella,
´
G., Rıos, J.L., 2002. Anti-inflammatory and antioxidant properties of
’t Hart, B.A., Simons, J.M., Knaan-Shanzer, S., Bakker, N.P., Labadie,
R.P., 1990. Antiarthritic activity of the newly developed neutrophil
oxidative burst antagonist apocynin. Free Radic. Biol. Med. 9,
127–131.
Helichrysum italicum. J. Pharm. Pharmacol. 54, 365–371.
´
Schinella, G.R., Troiani, G., Davila, V., de Buschiazzo, P.M., Tournier, H.,
2000. Antioxidant effects of an aqueous extract of Ilex paraguariensis.
Biochem. Biophys. Res. Comm. 269, 357–360.
’t Hart, B.A., Elferink, J.G., Nibbering, P.H., 1992. Effect of apocynin on
the induction of ulcerative lesions in rat skin injected with tubercle
bacteria. Int. J. Immunopharmacol. 14, 953–961.
Schinella, G.R., Tournier, H.A., Prieto, J.M., Mordujovich de Buschiazzo,
´
P., Rıos, J.L., 2002. Antioxidant activity of anti-inflammatory plant
Van den Worn, E., Beukelman, C.J., Van den Berg, A.J.J., Kroes, B.H.,
Labadie, R.L., Van Dijk, H., 2001. Effects of methoxylation of apocynin
and analogs on the inhibition of oxygen species production by stimulated
human neutrophils. Eur. J. Pharmacol. 433, 225–230.
extracts. Life Sci. 70, 1023–1033.
Smith, C., Zhang, Y., Kobolat, C.M., Muhammad, J., Zweifel, B.S.,
Shaffer, A., Talley, J.J., Masferrer, J.L., Seibert, K., Isakson, P.C.,
1998. Pharmacological analysis of cyclooxygenase-1 in inflammation.
Proc. Natl. Acad. Sci. U. S. A. 95, 13313–13318.
Warner, T.D., Giuliano, F., Vojnovic, I., Bukasa, A., Mitchell, J.A., Vane,
J.R., 1999. Nonsteroid drug selectivities for cyclooxygenase-1 rather
than cyclooxygenase-2 are associated with human gastrointestinal
toxicity. A full in vitro analysis. Proc. Natl. Acad. Sci. U. S. A.
96, 7563–7568.
Stanley, P.L., Steiner, S., Havens, M., Tramposch, K.M., 1991. Mouse skin
inflammation induced by multiple topical applications of 12-O-tetrade-
canoylphorbol 13-acetate. Skin Pharmacol. 4, 262–271.
Stolk, J., Hiltermann, T.J., Dijkman, J.H., Verhoeven, A.J., 1994. Charac-