Anti-inflammatory and antioxidant properties of Helichrysum italicum

Abstract

The anti-inflammatory and antioxidant activities of the aerial part of Helichrysum italicum extracts have been established in various in-vivo and in-vitro experimental models. The results obtained on the acute oedemas induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) and ethyl phenylpropiolate in the mouse ear, by serotonin and phospholipase A2 (PLA2) in the mouse paw, on chronic inflammation induced by repeated application of TPA in the mouse ear and on the delayed-type hypersensitivity induced by sheep red blood cells suggest that said anti-inflammatory activity is due to the effects of compounds expressed via a corticoid-like mechanism. In addition, the antioxidant activity of the extracts seems to be implicated in this anti-inflammatory activity, as the former inhibits enzymatic and non-enzymatic lipid peroxidation and has free-radical scavenger properties. We conclude that the anti-inflammatory activity of Helichrysum italicum can be explained by multiple effects, including inflammatory enzyme inhibition, free-radical scavenging activity and corticoid-like effects.

Full text

Journal of
JPP 2002, 54: 365–371
# 2002 The Authors
Received June 18, 2001
Accepted November 19, 2001
ISSN 0022-3573
Anti-in ammatory and antioxidant properties of
Helichrysum italicum
Araceli Sala, Mar
õ
´a del Carmen Recio, Rosa Mar
õ
´a Giner,
Salvador Ma´n4 ez, Horacio Tournier, Guillermo Schinella
and Jose
´
-Luis R
õ
´
os
Abstract
The anti-in ammatory and antioxidant activities of the aerial part of
Helichrysum italicum
extracts have been established in various in-vivo and in-vitro experimental models. The results
obtained on the acute oedemas induced by 12-
O
-tetradecanoylphorbol 13-acetate (TPA) and
ethyl phenylpropiolate in the mouse ear, by serotonin and phospholipase A
2
(PLA
2
) in the
mouse paw, on chronic in ammation induced by repeated application of TPA in the mouse ear
and on the delayed-type hypersensitivity induced by sheep red blood cells suggest that said
anti-in ammatory activity is due to the effects of compounds expressed via a corticoid-like
mechanism. In addition, the antioxidant activity of the extracts seems to be implicated
in this anti-in ammatory activity, as the former inhibits enzymatic and non-enzymatic
lipid peroxidation and has free-radical scavenger properties. We conclude that the anti-
in ammatory activity of
Helichrysum italicum
can be explained by multiple effects,
including in ammatory enzyme inhibition, free-radical scavenging activity and corticoid-like
effects.
Introduction
Helichrysum italicum (Roth) G. Don ® l (Asteraceae) is a woody shrub characteristic
of the Mediterranean area. Its ¯ owers and aerial parts are used as a n anti-
in¯ ammatory and anti-allergic phytomedicine (Peris et al 1995 ). Taken in the form
of a ¯ uid extract or as an infusion, the y are eŒective in respiratory disorders with
allergic or infectious components, in skin diseases such as psoriasis or eczema and
in other in¯ ammatory processes. The medicinal properties of this species are
thought to be owed to its ¯ a vonoid, sesquiterpene lactone and essential oil content.
Several studie s on the anti-in¯ ammatory activity of closely related species and
on related topics have been published (Recio et al 1991; de la Puerta et al
1999), but there are few reports on the anti-in¯ ammatory (Ma! n4 ez et al 1990 )
and radical-scavenging (Facino et al 1990; Schinella et al 2002) properties of
H. italicum.
The aim of this study was to investigate the anti-in¯ ammatory and antioxidant
activity of the m ethanolic extract of H. italicum and the fractions obtained from it,
to establish its potential therapeutic value.
Departament de Farmacologia,
Facultat de Farma’ cia, Universitat
de Vale’ ncia, Spain
Araceli Sala, Mar
õ
´
a del Carmen
Recio, Rosa Mar
õ
´
a Giner,
Salvador Ma
´
n4 ez, Jose
´
-Luis R
õ
´
os
Ca
´
tedra de Farmacolog
õ
´
a,
Facultad de Ciencias Me
´
dicas,
Universidad Nacional de la Plata,
La Plata, Argentina
Horacio Tournier, Guillermo
Schinella
Correspondence : J. L. R
õ
´
os
Can
4
avate, Departament de
Farmacologia, Facultat de
Farma’ cia, Universitat de
Vale’ ncia. Avda. Vicent Andre
´
s
Estelle
´
s s/n, 46100 Burjassot,
Spain. E-mail : riosjl!uv.es
Funding : This study was
supported by the Direccio
´
n
General de Ensen4 anza e
Investigacio
´
n Cient
õ
´
 ca of the
Spanish Government (PM98-
0206) and carried out as a part
of the Programa Iberoamericano
de Ciencia y Tecnolog
õ
´
a para el
Desarrollo (CYTED, Project
IV.11).
365

366
Araceli Sal a et al
Materials and Methods
Plant material, extraction and identi cation
Aerial parts of Helichrysum ita licum (Roth) G. Don ® l
(Asteraceae) were collected in Chiva (Valencia, Spain)
in June 1996.A specimen was deposited in the herbarium
of the Department of Pharmacology, University of
Valencia (Burjassot, Spain).
Air-dried and powdered aerial parts of H. italicum
(400 g) were percolated with methanol (3.5 L) at room
temperature, and the solution was evaporated under
reduced pressure, leaving a residue of 55 g. This was re-
dissolved in water and fractionated with solvents of
increasing polarity to obtain four subextracts : n-hexane
(10 g), dichloromethane (22 g), ethyl acetate (7 g) and n-
butanol (4 g). The fractions were subjected to a chro-
matographic analysis by thin-layer chromatography
(TLC) and high-performance liquid chromatography
(HPLC), and the major compounds were identi® ed by
comparison with standards previously isolated a nd
identi® ed by Recio et al (1991) from H. stoechas. TLC
analysis was performed on SiO
2
using dichloro-
methane± methanol (90 : 10) or dichloromethane± ethyl
acetate (70:30; 97:3) as mobile phases. HPLC± diode
array detector (HPLC-DAD) analysis was performed
using a Merck Hitachi HPL C system (L-6200 pump)
equipped with an L-3000 Photodiode array detector and
a pre-packed analytical column (12.5¬ 0.7 mm) of
Lichrospher RP-18 (5 l m). The following conditions
were used: as eluents, H
2
O tri¯ uoroacetic acid 0.05%
(A), methanol tri¯ uoroacetic acid 0.05% (B). Elution
pro® le : 0± 5 min 70% A, 5± 15 min 50 % A, 15± 29 min
30 % A, 29± 30 min 70% A ; ¯ ow rate 1 mL min

1
,
column pressure 60± 80 bar and the UV detector was set
at 280 nm.
Chemicals and animals
Chemicals
Ascorbic acid, arachidonic acid 99 % , butylated hy-
droxytoluene, cyproheptadine hydrochloride, dexa-
methasone, dimethyl sulfoxide (DMSO), 2,2-dinitro-
¯ uorobenzene (DNFB), 2,2-diphenyl-1-picrylhydrazyl
(DPPH), hexadecyltrimethylammonium bromide
(HTAB), hypoxanthine, indometacin, nitro blue tetra-
zolium, nordihydroguaiaretic acid (NDGA ), oxazolone,
phospholipase A
2
(PLA
2
) from Naja mossambica
venom, pyrogallol, sheep red blood cells (SRBC), 12-O-
tetradecanoylphorbol 13-acetate (TPA), 2-thiobarbit-
uric acid, trichloroacetic acid, xanthine and xanthine
oxidase were purchased from Sigma Chemical Co. (St
Louis, MO); ethyl phenylpropiolate (EPP) and Tween
80 from Fluka Chemika-Biochemika (Buchs, Swit-
zerland) ; methanol (HPLC grade) from Merck (Darm-
stadt, Germany); acetone, butanol, dichloromethane,
ethanol 96° , ethyl acetate, hexane, methanol, and tri-
¯ uoroacetic acid from Panreac (Barcelona, Spain).
Animals
Groups of six Swiss female mice, 25± 30 g, were used. All
mice had free access to a standard diet. Housing con-
ditions and all in-vivo experiments were approved by
the institutional Ethical Committee of the Faculty of
Pharmacy according to the guidelines established by the
European Union on Animal Care (CEE Council
86
}
609).
Extracts and reference drug dissolution
For topical application, the hexane, dichloromethane
and ethyl acetate extracts (1 mg
}
ear), NDGA
(2 mg
}
ear), indometacin (0.5 mg
}
ear) and dexa-
methasone (0.05 mg
}
ear) were dissolved in 20 l L
acetone, and methanol and butanol extracts (1 mg
}
ear)
in 20 l L 80% aqueous ethanol. For subcutaneous ap-
plication, the hexane, dichloromethane and ethyl acetate
extracts (100 mg kg

1
) were dissolved in olive oil, and
methanol and butanol extracts (100 mg kg

1
) and dexa-
methasone (0.5 mg kg

1
) in ethanol± saline (1 : 1 9). For
oral application, the extracts (200 mg kg

1
) a nd cypro-
heptadine (10 mg kg

1
) were dissolved in Tween 80±
ethanol± H
2
O (1 :1:10). The antioxidant activity of the
extracts dissolved in DMSO was tested at a concen-
tration of 100 l g mL

1
. Extracts in the hydroxyl radical
test were dissolved in 2% Tween 80.
12-
O
-Tetradecanoylphorbol-13-acetate (TPA)-,
arachidonic acid-, and ethyl phenylpropiolate
(EPP)-induced mouse ear oedema
Experimental methods hav e been previously described
by Recio et al (2000). An oedema was induced in the
right ear by topical applicati on with a micropipette of
an irritant agent dissolved in acetone (20 l l). The
oedema was expressed as the diŒerence between the
ear’s thickness before and after induction of in¯ am-
mation.
Phospholipase A
2
(PLA
2
)- and serotonin-induced
hind-paw mouse oedema
The PLA
2
from Naja mossambica venom (1.18 U) in
saline (25 l L) was injected subcutaneously into the
mouse right hind paw. The left paw received the same
volume of the saline solution. Samples were admin-

367
Anti-inammatory and antioxidant properties of
Helichrysum italicum
istered orally 60 min before challenge, and the oedema
was measured 30, 60 and 90 min after challenge (Neves
et al 1993).
Serotonin was injected into the right hind paw 3 h
after administration of the test compounds. The left
paw received the same volume of the saline solution.
The oedema was measured 12 min after its induction
(Recio et al 2000).
Mouse ear in ammation induced by multiple
topical applic ations of TPA
In¯ ammation was induced in each ear by topical ap-
plication of 2 l g of TPA (20 l L) on alternate days (5
applications). Extracts and dexamethasone w ere applied
topically twice daily for four days. On the last day the
compounds were applied only in the morning. The mice
were killed by cervical dislocation, and two ear punches
were taken from each mouse (n ¯ 5 mice). Eight samples
placed in HTAB were frozen for the myeloperoxidase
assay. Details of the methods have be en described in an
earlier study (Recio et al 2000).
Oxazolone- and DNFB-induced contact-
delayed-type hypersensitivity (DTH)
The oxazolone-induced DTH test was performed ac-
cording to Recio et al (2000). The DNFB-induced DTH
test was performed according to Go! ngora et al (2000).
Ear swelling w as assessed 24 and 96 h after e ach chal-
lenge. Samples and dexamethasone were applied top-
ically 2, 24, 48 and 72 h after challenge.
Sheep red blood cell (SRBC)-induced DTH
The experiment was performed according to the pro-
tocol referred to previously (Go
!
ngora et al 2000). The
oedema w as measured 18, 24 and 48 h after challenge.
The extracts and dexamethasone were administered
intraperitoneally immediately before and 16 h a fter
challenge.
Oedema measurements
Ear swelling was measured using a micrometer
(Mitutoyo Series 293). The oedema was expressed as the
increase in ear thickness due to the oedematous agent.
The percentage of oedema inhibition was expressed as
the reduction in thickness with respect to that of the
control group treated only with the oedema inductor.
The paw oedema was measured by means of a plethys-
mometer (Ugo Basile) and was expressed a s the diŒer-
ence between the rig ht and left paw.
Lipid peroxidation
Liver microsomes were prepared using a standard diŒer-
ential centrifugation technique (Schinella et al 2000).
Protein content was qua nti® ed by Bradford’ s method
using bovine serum albumin a s the sta ndard (Bradford
1976).
Non-enzymatic peroxidation was induced by FeSO
4
(5 l m ) and ascorbate (500 l m ) (Schinella et al 2000).
The products of lipid peroxidation were detected mea-
suring the absorbance at 535 nm usi ng the 2-thio-
barbituric acid method. Butylated hydroxytoluene was
used as a positive control. In enzymatic lipid peroxi-
dation, the reaction mixture contained microsomal pro-
tein and an NADPH-generating system (Schinella et al
2000). Peroxidation was started by 10 l L CCl
4
1: 4 (v
}
v)
in DMSO. After 15 min incubation at 37° C, thio-
barbituric acid reactive substances were determined as
above.
To determine cytochrome P-450 2E1 activity, micro-
somal protein was pre-incubated for 10 min in
100 l g mL

1
of each extract, and the catalytic activity
was then assessed using p-nitrophenol hydrolase as the
selective enzyme assay (Lee & Forkert 1994).
Free radical generation
Superoxide radical was generated by enzymatic oxi-
dation of hypoxanthine with xanthine oxidase grade I
and was detected by nitro blue tetrazolium reduction
viewed spectrophotometrically at 560 nm (Schinella et
al 2000). The in¯ uence on enzyme activity w as evaluated
by uric acid formation from xanthine and absorbance
was measured at 295 nm. Pyrogallol was used as a
positive control.
The hydroxyl radical was produced in a Fenton
reaction (Schinella et a l 2000) with diŒerent concen-
trations of test material at a volume of 1 mL. Deoxy-
ribose degradation by hydroxyl radicals was measured
by using the thiobarbituric acid method. DMSO was
used as a positive control.
A 2,2-diphenyl-1-picrylhydrazyl radical (DPPH ) sol-
ution in methanol was added to the extract solutions
and activity was determined by spectrophotometry at
517 nm after 10 min (Schinella et al 2000). Butylated
hydroxytoluene was used as reference compound.
Statistics
Statistical analysis was performed using one-way analy-
sis of variance followed by Dunnett’ s t-test for multiple
comparisons, and Student’ s t-test for single com-
parisons.

368
Araceli Sal a et al
Results
Ursolic acid, 4 « -hydroxy-3« -(3-methyl-2-butenyl)aceto-
phenone and the ¯ avonoid gnaphaliin were identi® ed by
TLC and HPL C-DAD analysis as the main compounds
in the dichloromethane fraction by comparison with
previously isolated standards (Recio et al 1991). Other
phenolics were detected by HPLC-DAD analysis, and
had a UV spectrum pro® le characteristic of ¯ av onoids.
The ethy l acetate and butanol fractions are formed
mainly by high-polarity ¯ avonoids and acetophenone
derivatives. In the hexane fraction, only lipids, sitosterol
and other analogous compounds were detected by chro-
matographic analysis.
The results obtained with the total methanol extract
and i ts fractions ± hexane, dichloromethane, ethyl acet-
ate and butanol ± on diŒerent mouse models of topical
acute in¯ ammation are presented in Figure 1. A ll the
samples inhibited the TPA-induced ear oedema, whereas
only the ethyl acetate and butanol extracts were active
against the EPP-induced ear oedema. None of these
extracts, however, reduced the in¯ ammation provoked
by topical application of arachidonic acid (data not
shown).
Figure 2 shows the eŒect of the extracts and dexa-
methasone on vascular leakage induced by serotonin,
3 h after administration to mice. Al l the extracts reduced
the oedema forma tion, but the ethyl acetate extract was
the most active and gave a percentage reduction in
oedema vol ume similar to that of dexamethasone.
300
200
100
0
Thickness (m
±
s.e.m.)
TPA EPP
Control
Methanol
Hexane
Dichloromethane
Ethyl acetate
Butanol Reference
Figure 1 Anti-in¯ ammatory eŒect of
Helichrysum italicum
extracts
(1 mg
}
ear) on acute 12-
O
-tetradecanoylphorbol 13-acetate (TPA)-
and ethyl phenylpropiolate (EPP)-induced ear oedemas in mice.
Values are mean³ s.e.m. (n ¯ 6); **
P
!
0
.
01 (Dunnett’s
t
-test) com-
pared with control. Reference drugs: indometacin (0
.
5 mg
}
ear) in
TPA test, dexamethasone (0
.
05 mg
}
ear) in EPP test.
0
Paw volume (L
±
s.e.m.)
Serotonin
Methanol
Hexane
Dichloromethane
Ethyl acetate
Butanol Dexamethasone
25
50
75
Figure 2 Anti-in¯ ammatory eŒect of
Helichrysum italicum
extracts
(100 mg kg

1
) a nd dexamethasone(0
.
5 mg kg

1
) on serotonin-induced
paw oedema in mice. Values are me an³ s.e.m. (n ¯ 6); **
P
!
0
.
01,
*
P
!
0
.
05 (Dunnett’s
t
-test) compared with control.
0
Paw volume (L
±
s.e.m.)
Phospholipase A
2
Methanol
Hexane
Dichloromethane
Ethyl acetate
Butanol Cyproheptadine
25
50
75
100
Figure 3 Anti-in¯ ammatory eŒect of
Helichrysum italicum
extracts
(200 mg kg

1
) and cyproheptadine (10 mg kg

1
) on PLA
2
-induced
paw oedema in mice 60 min after subcutaneous injection of PLA
2
.
Values are mean³ s.e.m. (n ¯ 6 ; **
P
!
0
.
01 (Dunnett’s
t
-test) com-
pared with PLA
2
.
Subplantar injection of PLA
2
produced a time-de-
pendent hind paw oedema that reached a maximum
after 60 min. All the samples (200 mg kg

1
, p.o.), except
the hexane extract, inhibited the paw oedema (Figure 3),
the methanol and butanol extracts being the most active.
In chronic in¯ ammation induced by TPA, the meth-
anol extract inhibited the oedema (65% inhibition) and
leucocyte in® ltration (58 % inhibition) to a lesser extent
than dexamethasone (85 % and 86% inhibition, re-
spectively). After fractioning the methanol extract, only
those fractions obtained with hexane (44% inhibition)

369
Anti-inammatory and antioxidant properties of
Helichrysum italicum
Table 1 Anti-in¯ ammatoryeŒect of
Helichrysum italicum
extracts (100
l
g mL

1
) on lipid peroxidation in rat liver microsomes stimulated with
Fe
2
+
}
ascorbate or CCl
4
}
NADPH.
Fe
2
+
/ascorbate CCl
4
/ NADPH
Malondialdehyde (nm ol) % Inhibition Malondialdehyde (nmol) % Inhibition
Control 40
.
7³ 0
.
7 ± 7
.
4³ 0
.
4 ±
Hexane 34
.
4³ 1
.
0
ns
16 2
.
8³ 0
.
1** 62
Dichloromethane 1
.
5³ 0
.
6** 96 1
.
0³ 0
.
0** 87
Ethyl acetate 1
.
5³ 0
.
1** 96 1
.
5³ 0
.
1** 80
Butanol 2
.
1³ 0
.
3** 95 4
.
0³ 0
.
2** 46
Butylated hydroxytoluene 1
.
2³ 0
.
1** 97 0
.
9³ 0
.
1** 88
Values are mean³ s.e.m. (n ¯ 3); **
P
!
0
.
01 (Dunnett’s
t
-test) compared with control.
0
Paw volume (L
±
s.e.m.)
Sheep red
blood cells
Methanol
Hexane
Dichloromethane
Ethyl acetate
Butanol
Dexamethasone
25
50
75
100
18 h 24 h
Figure 4 Anti-in¯ ammatory eŒect of
Helichrysum italicum
extracts
(50 mg kg

1
) and dexamethasone (10 mg kg

1
) on sheep red blood cell
(SRBC)-induced paw oedema in mice at 18 and 24 h, respectively.
Values are mean³ s.e.m. (n ¯ 5); **
P
!
0
.
01 (Dunnett’s
t
-test) com-
pared with PLA
2
.
and dichloromethane (48 % inhibition) signi® cantly re-
duced the ear oedema, w hile all inhibited leucocyte
recruitment (40± 66% , depending on the type of extract).
The SRBC-induced DTH in¯ ammation reached its
maximum at 18 h and disappeared 48 h after challenge.
Treatment with the drugs immediately before, and 16 h,
after challenge was eŒective in reducing the paw oedema
(Figure 4). However, none of the drugs displayed activity
against the contact-DTH induced by oxazolone or
DNFB.
The ac tivity of the methanol extract has been reported
by Schinella et a l (2002) in a previous screening. In this
study we demonstrate the activity of the diŒerent frac-
Table 2 Anti-in¯ ammatory eŒect of
Helichrysum italicum
extracts
(100
l
g mL

1
in DMSO) on 2,2-diphenyl-1-picrylhydrazyl (DPPH)
reduction.
DPPH % Inhibition
A
Control 0
.
340³ 0
.
010 ±
Hexane 0
.
229³ 0
.
001** 33
Dichloromethane 0
.
037³ 0
.
005** 89
Ethyl acetate 0
.
019³ 0
.
001** 94
Butanol 0
.
023³ 0
.
001** 93
Butylated hydroxytoluene 0
.
031³ 0
.
006** 91
A ¯ absorbance units. Values are mean³ s.e.m. (n ¯ 3); **
P
!
0
.
01
(Dunnett’s
t
-test) compared with control.
tions obtained by liquid± liquid extraction from the
methanol extract. Table 1 shows the activities of the
extracts against enzymatic and non-enzymatic lipid per-
oxidation i n rat liver microsomes. When tested in the
system dependent on Fe
2
+
}
ascorbate at a concentration
of 100 l g mL

1
, all the fractions except the hexanic
showed anti-peroxidative activity, and all the fractions,
without exception, inhibited lipid peroxidation in the
CCl
4
}
NADPH system.
The eŒects of the extracts on p-nitrophenol hydroxyl-
ase (cytochrome P-450 2E1) activity were evaluated, but
the fractions did not inhibit thi s enzymatic activity (data
not shown). The potential antioxidant activity of the
plant extracts was assessed on the basis of the scavenging
activity of the stable DPPH free radical, and all the
fractions, except the hexanic, reduced this ra dical in the
same way as did the reference compound (Table 2).
Superoxide scavenging was only observed with the
dichloromethane and butanol fractions. The eŒect ob-
served in this test was not related to the inhibition of

370
Araceli Sal a et al
xanthine oxidase activity, except in the case of the ethyl
acetate fraction, which was able to inhibit the activity of
the enzyme.
The extracts were unable to scavenge the hydroxyl
radical produced in the Fe
3
+
-EDTA H
2
O
2
system i n
the presence of ascorbate. The inclusion of ethyl acetate
and butanol extracts in the reaction mixture increased
deoxyribose degradation in this system, both in the
presence and absence of ascorbate (data not shown).
Discussion
In this study, the anti-in¯ ammatory activity of the
methanol extract from Helichrysum italicum and its four
sub-extracts was demonstrated in diŒerent experimental
models of in¯ ammation. The most active extract was
dichloromethane in the chronic TPA test, ethyl acetate
in the EPP and serotonin tests and butanol in the acute
TPA and PLA
2
tests.
Interesting data were obtained from the EP P test. The
total methanol extract showed no inhibitory activity
and the two extracts with less polarity (hexane and
dichloromethane) increased the oedema, whereas the
two polar extracts signi® cantly reduced the oedema
formation. These data suggest the possible presence of
compounds in the ethyl acetate and butanol extracts
which have a mechanism of action similar to that of
corticoids. This would explain why the total extract
(methanol) was not eŒective in this experimental model
of in¯ ammation.
In the acute TPA assay all the extracts inhibited
formation of the oedema, the eŒect noted being similar
in part to that obtained in the EPP test. In fact, the ethyl
acetate and butanol extracts were once again the most
eŒective, but in this case the extracts with less polarity
were also active, though to a lesser extent than the
methanol extract. In two previous papers, the anti-
oedematous eŒect of ursolic acid and 4« -hydroxy-3« -
(3-methyl-2-butenyl)acetophenone isolated from H.
stoechas in the TPA test (Recio et al 1991) was reported,
as was the inhibition of leucocyte eicosanoid generation
and the radical scavenging activity of gnaphaliin (de la
Puerta et al 1999). These compounds are present in the
dichloromethane extract, and appear to be among the
constituents implicated in these pharmacological pro-
perties. With respect to the ethyl acetate and butanol
extracts, their activity may be due to the glycosidic
¯ avonoids and acetophenones detected as major com-
pounds.
All the extracts inhibited the paw oedema induced by
serotonin and PLA
2
. The paw oedema induced by PLA
2
is mediated by mast cell degranulation and histamine
}
serotonin release. For this reason, anti-PLA
2
drugs
which block the eŒect of the enzyme against the me m-
brane phospholipids and H
1
-antihistamine and anti-
serotonin drugs are eŒective in this test. In our study all
the extracts were active, while the methanol ex tract was
again the most eŒective and only the butanol fraction
had a similar eŒect. However, in the serotonin-induced
paw oedema, the ethyl acetate fraction was the most
active.
The methanol extract clearly inhibited the chronic
in¯ ammation induced after application of TPA on the
mouse ear, but had no eŒec t on acute a rachidonic-acid-
induced ear oedema and the DTH reaction induced by
oxazolone or DNFB. However, SRBC-DTH was clearly
decreased by the methanol extract and some of its
fractions which we assayed. All the fractions improved
the results obtained with the total extract at 18 and 24 h,
but only the butanol fraction was clearly more eŒective
at 48 h. In addition, this extract showed similar eŒects at
the diŒerent times points assayed.
Taken together, these results sug gest that the anti-
in¯ ammatory a ctivity of H. italicum is due to diŒerent
compounds, some of which are polar principles and are
present in the butanol fraction. In addition, they seem to
have some corticoid-related mechanisms. A direct in-
teraction of some compounds with PLA
2
or serotonin
receptor could be considered for further study.
The role of oxygen-derived free radicals in the in¯ am-
matory process is well known. Free radicals are im-
plicated in the activation of nuclear factor j B (NFj B)
and p38-mitogen-activated protein kinase, which in-
duces the transcription of pro-in¯ ammatory enzymes
such as cyclooxygenase 2 and nitric oxide synthase, and
in¯ ammatory cytokines such as interleukin-1b (IL-1b ),
IL-2 and tumour necrosis factor a (TNFa ). On the
other hand NFj B is itself activated by some of these
cytokines (Winrow et al 1993; Sahnoun et al 1998 ;
Bowie & O ’Neill 2000; Schoonbroodt & Piette 2000).
Previously, we showed that the methanol extract
inhibits the enzymatic and non-enzymatic l ipid per-
oxidation in model membranes and also acts as a
superoxide radical scavenger (Schinella et al 2002). In
this study, we demonstrated that all the fractions sig-
ni® cantly inhibited the microsomal lipid peroxidation.
The hexane extract was a weak scavenger of the stable
DPPH radical, but had no scavenging eŒect on anion
superoxide and hydroxyl radicals. The dichloromethane
and butanol extracts showed signi® cant scavenging ac-
tivity on superoxide anion and DPPH radicals. The
ethyl acetate extract also showed antioxidant activity
against the DPPH radical. When examined in the Fe
3
+
-

371
Anti-inammatory and antioxidant properties of
Helichrysum italicum
EDTA H
2
O
2
system, the butanol and ethyl acetate
extracts increased deoxyribose degradation in the pres-
ence and absence of ascorbate. The ability to stimulate
deoxyribose degradation as a result of Fe
3
+
reduction
would indicate the pro-oxidant properties of some c om-
pounds present in the extracts, probably due to redox
cycling of the iron (Aruoma et al 1992; Puppo 1992). In
a previous paper, F acino et al (1990) described the
radical scavenger activity of three ¯ avonoids isolated
from H. italicum, tetra hydroxychalcone-2« -glucoside,
kaempferol-3-glucoside and naringenin-glucoside, all of
which inhibited lipid peroxidation induced by ADP
}
Fe
2
+
or CCl
4
in the presence of NADPH, while the total
extract was clearly more a ctive than the isolated ¯ avo-
noids. We obtained the most notable eŒect in the
CCl
4
}
NADPH system when the dichloromethane and
ethyl acetate extracts were assayed. Cytochrome P450-
linked activities are involved in NADPH-induced mi-
crosomal lipid peroxidation. The extracts did not inhibit
the hydroxylation of p -nitrophenol, which suggests tha t
the preventive action of the extracts are due to their
inhibition of the initiation of lipid peroxidation by acting
as scavengers of free radical species.
Conclusions
The anti-in¯ ammatory activity of our plant extracts
could be due to the synergistic eŒect of pro-in¯ am-
matory enzyme inhibition, free radical scavenging ac-
tivities or corticoid-like eŒects. The results of this study
suggest that the anti-in¯ ammatory activity of the same
extracts can be explained, at least in part, by their
antioxidant properties. Although the active principles
responsible for the antioxidant activity of the tested
extracts have not yet been identi® ed, we propose these
extracts as a useful source of compounds that help to
increase the overall antioxidant capacity of an organism,
and thereby protect it against lipid peroxidation induced
by oxidative stress.
References
Aruoma, O. I., Halliwell, B., Aeschbach, R., Lo
$
ligers, J. (1992)
Antioxidant and pro-oxidant properties of active rosemary con-
stituents: carnosol and carnosic acid.
Xenobiotica
22 : 257± 268
Bowie, A., O’ Neill, L. A. J. (2000) Oxidative stress and Nuclear
Factor-
j
B activation.
Biochem
.
Pharmacol
. 59 : 13± 23
Bradford, M. M. (1976) A rapid and sensitiv e method for quan-
ti® cation of micrograms quantities of protein utilizing the principle
of protein-dye binding.
Anal
.
Biochem
. 7 2 : 248± 254
de la Puerta, R., Forde r, R. A., Hoult, J. R. S. (1999) Inhibition of
leukocyte eicosanoid generation and radical scavenging activity by
gnaphaliin, a lipophilic ¯ avonol isolated from
Helichrysum picardii
.
Planta Med
. 65 : 507± 511
Facino, R. M., Carini, M., Franzoi, L., Pirola, O., Bosisio , E. (1990)
Phytochemical characterization and radical scavenger activity of
¯ av onoids from
Helichrysum italicum
G. Don (Compositae).
Pharm
.
Res
. 2 2 : 709± 721
Go! ngora, L., Ma! n
4
ez, S., Giner, R. M., Recio, M. C., Rõ ! os, J. L.
(2000) On the activity of tri¯ uoperazine and palmitoylcarnitine in
delayed hypersensitivity models.
Life Sci
. 66 : 183± 188
Lee, R. P., Forkert, P. (1994)
In vitro
biotransformation of 1,1-
dichloroethylene by hepatic cytochrome P-450 2E1 in m ice.
J
.
Pharmacol
.
Exp
.
Ther
. 270: 371± 376
Ma
!
n4 ez, S., Alcaraz, M. J., Paya
!
, M., Rõ
!
os, J. L., Hancke, J. L. (1 990)
Selectedextracts from medicinalplants as anti-in¯ ammatoryagents.
Planta Med
. 56 : S656
Neves, P. C. A., Neves, M. C. A., Bella Cruz, A., Sant’ana, A. E. G.,
Yunes, R. A., Calixto, J. B. (1993) DiŒerential eŒects of
Mandevilla
velutina
compounds on paw oedema induced by phospholipase A
2
and phospholipase C.
Eur
.
J
.
Pharmacol
. 243 : 213± 219
Peris, J. B., Stu
$
bing, G., Vanaclocha, B. (1995)
Fitoterapia aplicada
.
MICOF, Valencia, pp 470± 471
Puppo, A. (1992) EŒect of ¯ avonoids on hydroxyl radical formation
by Fenton type reactions; in ¯ uence of the iron chelator.
Phyto
-
chemistry
31 : 85± 88
Recio, M. C., Giner, R. M., Terencio, M. C., Sanz, M. J., Rõ ! os, J. L.
(1991) Anti-in¯ ammatory activity of
Helichrysum stoechas
.
Planta
Med
. 57 : A56± 57
Recio, M. C., Giner, R. M., Uriburu, L., Ma! n4 ez, S., Cerda! , M., De la
Fuente,J. R., R õ ! os, J. L. (2000)
In vivo
activity of pseudoguaianolide
sesquiterpene lactones in acute and chronic in¯ ammation.
Life Sci
.
66 : 2509± 2518
Sahnoun, Z., Jamoussi, K., Zeghal, K. M. (1998) Free radicals and
antioxidants: physiology, human pathology and therapeutic as-
pects.
Therapie
53 : 315± 339
Schinella, G. R., Troiani, G., Davila, V., de Buschiazzo, P. M.,
Tournier, H. A. (2000) Antioxidant eŒects of aqueous extract of
Ilex paraguariensis
.
Biochem
.
Biophys
.
Res
.
Com
. 269: 357± 360
Schinella, G. R., Tournier, H. A., Prieto, J. M., Mordujovich de
Buschiazzo, P., Rõ
!
os, J. L. (2002) Antioxidant activity of anti-
in¯ ammatory plant extracts.
Life Sci
. 70 : 1023± 1033
Schoonbroodt, S., Piette, J. (2000) Oxidative stress interference with
the nuclear factor-
j
B activation pathways.
Biochem
.
Pharmacol
.
60 : 1075± 1083
Winrow, V. R., Winyard, P. G., Morris, C. J., Blake, D. R. (1993)
Free radicals in in¯ ammation: second messengers and mediators
of tissue destruction.
Br
.
Med
.
Bull
. 49: 506± 522