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Phenolic content, anti-inflammatory and antioxidant
activities of Anacyclus clavatus extracts
Hamama Bouriche1*, Abdallah Kherbache1, Seoussen Kada1, Abderrahmane Senator1,
Ibrahim Demirtas2
1Laboratory of Applied Biochemistry, Faculty of SNV, Department of Biochemistry, University Ferhat Abbas Setif 1, Algeria
2Department of Chemistry, Faculty of Science, Çankırı Karatekin University, Çankırı, Turkey
*Corresponding author, E-mail: bouriche_ha@yahoo.fr
Abstract
e anti-inflammatory and the antioxidant activities of methanol (ME) and aqueous (AE) extracts of Anacyclus clavatus were evaluated.
Phenolic constituents in the extracts were screened. Croton oil-induced ear edema in mice, carrageenan-induced paw edema and pleurisy
in rats were evoked. e antioxidant effect was tested by 1,1-diphenyl-2-picrylhydrazyl, ion chelating, lipid peroxidation tests. Both
extracts are rich in phenolic compounds. e application of 2 mg per ear of ME or AE inhibited ear edema by 84 and 83 %, respectively.
e oral treatment of rats with 200 or 400 mg kg–1 of ME reduced paw edema by 64 and 74%, respectively, whereas the inhibition by AE
was by 65 and 80%, respectively. At 400 mg kg–1, the extracts decreased exudation and neutrophil migration into the pleural cavity by
64 and 66%, respectively, while the inhibition by AE was 42 and 55%, respectively. On the other hand, ME exerted scavenging activity
higher than AE, while the AE chelating activity was more than that of ME. However, both extracts had similar inhibitory effect on lipid
peroxidation. In general, A. clavatus may be used as a source of anti-inflammatory and anti-oxidant agents.
Key words: Anacyclus clavatus, anti-inflammatory activity, antioxidant activity, phenolic compounds.
Abbreviations: AE, aqueous extract; BHT, butylated hydroxytoluene; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ME, methanol extract.
Environmental and Experimental Biology (2016) 14: 127–135 Original Paper
DOI: 10.22364/eeb.14.18
Introduction
An imbalance between oxidants and antioxidants leads to
oxidative damage to biological molecules, including lipids,
proteins and nucleic acids. is damage is responsible
for the pathogenesis of several human diseases, such as
neurodegenerative diseases, lung diseases, cardiovascular
diseases, diabetes, and atherosclerosis (Rahman et al.
2012). Moreover, when overproduced, reactive oxygen
species attack tissue and then provoke an inflammatory
response by production of pro-inflammatory mediators
and chemotactic factors, which amplify the inflammation,
resulting in chronic inflammation (Mittal et al. 2014). In
fact, chronic inflammation is another important factor
that may cause or contribute in the pathogenesis of many
diseases. us, scavenging reactive oxygen species and
suppressing their formation, either by inhibiting enzymes
or by chelating trace elements involved in free radical
production, are thought to be an effective means to depress
the level of oxidative stress of organisms.
Usually, phytochemicals possess strong antioxidant
ability as well as anti-inflammatory action, which are
also the basis of other bioactivities and health benefits.
Antioxidants can reduce inflammation via the inhibition of
pro-inflammatory mediators, as well as the increase of the
anti-inflammatory mediator production (Costa et al. 2013;
Moura et al. 2015).
Medicinal plants may offer an alternative source for
the anti-inflammatory and antioxidant drugs, and have
significant effect against several pathologies. Various
bioactive compounds from plants were discovered as a new
medicinal drug (Lahlou 2013).
Anacyclus clavatus Pers. is an aromatic medicinal plant
belonging to the Asteraceae family. It is widely distributed
in the Mediterranean countries and known for its medicinal
properties. In Algerian traditional medicine, the aerial parts
of this plant are used to dispel gas and prevent bloating.
Its leaves and stems are used as digestive herbal tea and as
a traditional remedy against digestive disorders (Benitez,
Gonzalez-Tejero 2010).
Phytochemical analysis revealed the presence of
essential oils in the leaves/stems and flower extracts of A.
clavatus (Aliboudhar, Tigrine-Kordjani 2013). Some of
these compounds exhibit antibacterial activity (Hammami
et al. 2013), and antitumour activity (Yi-Qun et al. 2008).
Few studies have been reported that have shown the anti-
inflammatory activity of A. clavatus. e current study
was designed to evaluate anti-inflammatory activity and
antioxidative capacity of different extracts of A. clavatus by
using a series of in vivo and in vitro tests.
Environmental and Experimental Biology ISSN 2255-9582 127
Materials and methods
Chemicals
Indomethacin, ferrosine [3-(2-pyridyl)-5, 6-bis(4-
phenyl-sulfonicacid)-1,2,4-triazine)], FeCl2, FeCl3, EDTA,
trichloroacetic acid, potassium thiocyanate, 1,1-diphenyl-
2-picryl-hydrazyl (DPPH), folin-ciocalteu, gallic acid,
tannic acid, quercetin, K3Fe(CN)6], Na2CO3, Tween 20,
λ-carrageenin, carboxymethyl cellulose, aspirin and croton
oil were purchased from Sigma (Germany). Linoleic
acid and 2,6-di-tert-butyl-4-methylphenol (butylated
hydroxytoluene; BHT) was obtained from Fluka (France).
All other reagents were of analytical grade and supplied
from Panreac (Spain), Riedel-de Haen, Prolabo and Sigma
(Germany).
Plant material
e plant Anacyclus clavatus was collected in May 2012
from Bougaa area in Algeria. e plant was identified and
authenticated taxonomically. A voucher specimen (No.
A.C. 2012-1) was preserved for future reference at the local
Herbarium of Botany, Department of Botany, University of
Sétif 1. e aerial part was air-dried at room temperature,
then reduced to powder and stored away from light until
use.
Animals
Swiss albino mice weighing 25 to 30 g and Albino Wistar
rats weighing 170 to 210 g of either sex were obtained
from the Pasteur Institute of Algiers, Algeria. All animals
were kept to acclimatize under the laboratory conditions
for one week and were provided with standard rodent diet
and water ad libitum. Animals were randomly selected for
different experimental groups (six animals per group) and
fasted overnight prior to the experiments. All procedures
were performed in accordance with European Union
Guidlines for Animal Experimentation (2007/526 /EC).
Preparation of Anacyclus clavatus extracts
Methanol extract (ME) of A. clavatus was obtained by
maceration of the powdered plant (10 g 100 mL–1) with
80% methanol for 24 h under continuous shaking at room
temperature. Aer filtration, the filtrate was concentrated
under reduced pressure at 40 °C. e residue was lyophilized
to give a dark brown powder (yield 19%).
Aqueous extract (AE) of A. clavatus was prepared by
boiling 50 g of powdered plant in 500 mL distilled water
for 20 min, followed by filtration and centrifugation for 10
min. e supernatant obtained was lyophilized to give a
dark brown powder (yield 17%). Both extracts were stored
at –32 °C until use.
Determination of total polyphenol concentration
e concentration of total phenolics in the extracts
was determined according to a modified method of
Li et al. (2007). Briefly, a volume of 100 mL of various
concentrations of extracts solutions was added to 500 mL
of Folin-Ciolcalteu (10%). Aer 4 min, 400 mL of 7.5%
Na2CO3 was added. e mixture was shaken for 2 h at room
temperature and the absorbance was recorded at 765 nm.
Gallic acid was used as a standard. e concentration of
total phenolic compounds in the extracts was determined
as mg of gallic acid equivalent per 1 g of extract (mg GAE
g–1 extract).
Determination of flavonoid concentration
Total flavonoid concentration in the extracts was
determined according to Bahorun et al. (1996). Briefly, 1 mL
of 2% AlCl3 in ethanol was added to 1 mL of the extracts (2
mg mL–1). Aer 10 min of incubation at room temperature,
the absorbance was measured at 430 nm. Quercetin was
used as a standard. Total flavonoid content was expressed
as mg of quercetin equivalent per 1 g of extract (mg QE g–1
extract).
Determination of tannin concentration
Tannin concentration was determined using the hemoglobin
precipitation assay according to Hagerman, Butler (1989).
Tannic acid was used as standard. An aliquot of 0.5 mL
of each extract was mixed with 0.5 mL of hemolyzed
bovine blood. e mixture was reacted for 20 min at room
temperature, and then subjected to centrifugation at 4000
rpm for 10 min at 4 °C. e absorbance was measured at
576 nm and tannic content was expressed as mg tannic acid
equivalent per g of extract (TAE g–1 extract).
HPLC-TOF/MS analysis
HPLC-TOF/MS analysis of A. clavatus extracts was carried
out as described elsewhere (Abay et al. 2015). is HPLC
method was developed and validated to analyze phenolic
acids and flavonoids in the plant extracts. An Agilent
Technology of 1260 Infinity HPLC System was coupled
with a 6210 Time of Flight (TOF) LC/MS detector and
ZORBAX SB-C18 (4.6 × 100 mm, 3.5 μm) column. Mobile
phases A and B were ultra-pure water with 0.1% formic acid
and acetonitrile, respectively. Flow rate was 0.6 mL min–1
and column temperature was 35 °C. Injection volume was
10 μL. e solvent program was as follow: 0 to 1 min 10% B;
1 to 20 min 50% B; 20 to 23 min 80% B; 23 to 25 min 10%
B; 25 to 30 min 10% B. Ionization mode of the HPLC-TOF/
MS instrument was negative and operated with a nitrogen
gas temperature of 325 °C, nitrogen gas flow of 10.0 L min–1,
nebulizer of 40 psi, capillary voltage of 4000 V and finally,
fragmentor voltage of 175 V. For sample analysis, dried
crude extracts (200 ppm) were dissolved in methanol at
room temperature. Samples were filtered passing through a
PTFE (0.45 μm) filter by an injector to remove particulates.
Croton oil induced ear edema in mice
Croton-oil induced ear edema was evoked according to
128
H. Bouriche, A. Kherbache, S. Kada, A. Senator, I. Demirtas
Manga et al. (2004). Cutaneous inflammation was induced in
the inner surface of the right ear of mice (6 mice per group)
by application of 15 µL acetone containing 80 µg croton oil
as an irritant. Treated animals received topically 2 mg per
ear of methanol, aqueous extract of A. clavatus (disolved in
acetone/water) or 0.5 mg per ear of indomethacin, used as
reference drug. e thickness of ears was measured before
and 6 h aer the induction of inflammation using a dial
calliper. e edema was expressed as an increase in the ear
thickness due to croton oil application.
Carrageenan induced paw edema in rats
Paw edema was induced by injecting 0.1 mL of 1%
λ-carrageenan into the subplantar region of the right
hind paw of rats (Winter et al., 1962). One hour before
carrageenan injection, rats received orally 200 and 400 mg
kg–1 of A. clavatus methanol, aqueous extract (prepared
in saline solution) or 200 mg kg–1 aspirin (suspended in
CMC 1%). Rats of control group were injected with 0.1 mL
λ-carrageenan and received orally only the vehicle before
the injection. To assess the edema the injected paw was
measured using a plethysmometer (UGO Basile, Varese,
Italy) initially (V0) and 1, 2, 3, 4, 5, and 6 h aer carrageenan
injection (Vt). Inflammation was calculated as the increase
in volume of the paw aer treatment subtracted from the
basal volume. Results were expressed as percentage of
inhibition of edema, calculated according to the following
equation:
% inhibition = [(Vt – V0) control – (Vt – V0) treated] /
(Vt – V0) control] × 100.
Carrageenan-induced pleurisy in rat
e carrageenan-induced pleurisy in rats was assessed
according to Cuzzocrea et al. (1998). Treated rats (six rats
per group) were administered orally 2 mL (400 mg kg–1)
of methanol or aqueous extract of A. clavatus (prepared
in saline solution), one hour before the intra-pleural
injection of 0.2 mL of the λ-carrageenan 1%. Rats of the
untreated control group were treated orally with 2 mL of
saline solution. Animals were lightly anaesthetized with
chloroform and submitted to a skin incision at the level
of the le sixth inter-costal space. e underlying muscle
was dissected and saline solution (0.2 mL) containing 1% λ
-carrageenan (0.2 mL) was injected into the pleural cavity.
For rats of the negative control group, 0.2 mL of sterile
0.9% NaCl instead of the λ-carrageenan solution was
injected in their pleural cavity and they were not treated
with any other substance. e skin incision was closed with
a suture and the animals were allowed to recover. Four
hours aer the injection of λ-carrageenan, rats were killed
and their chests were carefully opened, and the pleural
cavity is subsequently washed with 2 mL of heparinized
saline solution. e exudate and washing solution were
removed by aspiration and the total volume was measured.
Any exudate, contaminated with blood was discarded.
e amount of exudates was calculated by subtracting the
volume injected (2 mL) from the total volume recovered.
e leukocytes in the exudate were suspended in PBS and
counted with an optical microscope aer vital Trypan blue
staining.
DPPH radical scavenging assay
e free radical scavenging activity of A. clavatus methanol
and aqueous extracts was measured using 1,1-diphenyl-2-
picryl-hydrazil (DPPH), according to the method described
by Que et al. (2006) with slight modifications. A volume
of 500 mL of the DPPH solution (0.1 mM) was added to
500 mL of extract solutions or standard (BHT) at different
concentrations, the mixture was incubated in the dark for
30 min at room temperature, and then the absorbance was
recorded at 517 nm. e percentage of DPPH scavenging
activity was calculated by the following equation:
Scavenging activity (%) = [(Ac – At) / Ac] × 100,
where Ac is the absorbance of the control (in the absence of
the abstracts) and At is the absorbance of the test sample (in
the presence of the extracts).
Metal chelating activity
e chelating of ferrous ions by methanol and aqueous
extracts of A. clavatus was estimated by the method of Li et
al. (2007). Briefly, extract samples at different concentrations
were added to a solution of 0.6 mmol L–1 FeCl2 (50 µL). e
reaction was initiated by the addition of 50 µl of ferrozine
(5 mM) and the mixture was shaken vigorously and le
standing at room temperature for 10 min. Absorbance of the
solution was then measured spectrophotometrically at 562
nm. e percentage of inhibition of ferrozine–Fe2+ complex
formation was calculated. EDTA was used as a reference.
e percentage of chelating activity was calculated using
the following equation:
Chelating activity (%) = [(Ac – At) / Ac] × 100,
where Ac is the absorbance of control (in the absence of the
abstracts) and At is the absorbance of the test sample.
Total antioxidant activity
e total antioxidant activity of methanolic and aqueous
extracts of A. clavatus was determined according to the
thiocyanate method using a linoleic acid system (Gulcin
et al. 2005) with slight modifications. e linoleic acid
emulsion was prepared by mixing 0.028 g linoleic acid,
0.028 g Tween 20 and 10 mL phosphate buffer (0.04 M,
pH 7.0). e mixture was then homogenized, and 0.6
mL phosphate buffer containing 0.6 µL plant extract and
BHT (50 µg mL–1) were mixed with 0.6 ml of linoleic acid
emulsion. e mixed solutions were incubated at 25 °C in
the dark for 96 h. e peroxide level was determined aer
reaction of 20 µL of sample solution with 20 µL of FeCl2
(20 mM in 3.5% HCl) and 20 µL thiocyanate (30%). e
absorbance of each mixture was determined at 500 nm
aer 3 min of reaction and every 24 h for 96 h. During
linoleic acid oxidation, the peroxides formed oxidize Fe2+ to
Fe3+. e Fe3+ form a complex with SCN– and this complex
129
Anti-inflammatory and antioxidant activities of Anacyclus clavatus extracts
has a maximum absorbance at 500 nm. erefore, high
absorbance indicated high linoleic acid oxidation. e
percent inhibition of lipid peroxidation was calculated
according to the following equation:
Peroxidation inhibition (%) = [(Ac – At) / Ac] × 100,
where Ac is the absorbance of control (in the absence of the
abstracts) and At is the absorbance of the test sample.
Statistical analysis
Results were expressed as mean ± SD in vitro and as mean
± SE in vivo. e significance of differences between control
and the various tests was determined by an ANOVA test
followed by a Dunnett/Tukey test for multiple comparisons.
e differences were considered statistically significant at p
≤ 0.05.
Results
Polyphenol, flavonoid and tannin content
Table 1 shows that methanol extract of A. clavatus was
richer in polyphenols and tannins than the aqueous extract,
while the aqueous extract was richer in flavonoids than
methanol extract.
HPLC-TOF/MS analysis
HPLC-TOF/MS analysis revealed the presence of phenolic
acids and flavonoids in the A. clavatus extracts (Table 2
and Fig. 1). However, methanol extract was richer than
the aqueous one. Chlorogenic acid was detected as major
constituent (1839.77 mg kg–1 of plant) of ME, while
apigetrin was detected as a major flavonoid (505.74 mg kg–1
of plant) in this extract. Moreover this extract contained
high amounts of apegenin, diosmin and quercetin-3-
β-D-glucoside. Several compounds (caffeic acid, rutin,
quercetin-3-β-D-glucoside, sinapic acid, polydatine and
apigenin) were detected as traces in AE.
Effect of Anacyclus clavatus extracts on croton oil-induced
ear edema in mice
e mice in the control group that received only the croton
oil solution developed aer 6 hours an edema characterized
by an increased thickness (94 ± 9.7 μm). e treatment
with 2 mg per ear of the methanolic extract induced a very
significant reduction (p < 0.001) of inflammation compared
to the control mice group. e increase in thickness aer 6
hours was 20 ± 8.3 µM, which corresponded to an inhibition
of 84%. e aqueous extract exerted almost the same effect
as the methanol extract (83%) (Fig. 2). is inhibition was
better than that exerted by 0.5 mg per ear of indomethacin
(70%), used as a reference anti-inflammatory agent.
Effect of Anacyclus clavatus extracts on carrageenan-
induced paw edema
e subplantar injection of carrageenan in the control group
caused edema with a maximum volume (78%) aer 4 to 6
h of injection. e oral pretreatment of rats by extracts of
Table 1. Polyhenols, tannins and flavonoids content of A. clavatus methanol extract (ME) and aqueous extract (AE). Values are mean ±
SD (n = 3). GAE, gallic acid equivalents; QE, quercetin equivalents; TAE, tannic acid equivalents
Extract Polyphenols (mg GAE g–1 extract) Flavonoids (mg QE g–1 extract) Tannins (mg TAE g–1 extract)
ME 131.30 ± 6.88 9.96 ± 0.43 39.21 ± 6.55
AE 79.06 ± 3.24 16.39 ± 1.38 31.14 ± 2.27
Table 2. Phenolic compounds in methanol and aqueous extracts of A. clavatus determined by HPLC-TOF/MS. RT, retention time; –,
trace
Coumpound RT ME mg phenolic kg–1 plant AE mg phenolic kg–1 plant
Gentisic acid 4.39 24.60 23.69
Chlorogenic acid 6.03 1839.77 180.17
4-hydroxybenzoic acid 6.59 41.75 17.65
Protocatechuic acid 6.96 26.64 22.34
Caffeic acid 7.68 21.41 –
Syringic acid 8.04 28.06 24.45
Vanillic acid 8.69 12.99 10.30
Rutin 9.51 0.92 –
Quercetin-3-β-D-glucoside 10.03 51.98 –
Naringin 10.60 4.59 0.07
Sinapic acid 10.61 18.46 –
Polydatine 10.76 4,29 –
Diosmin 11.04 128.99 43.05
Apigetrin 11.31 505.74 26.50
Cinnamic acid 15.40 – 12.21
Apigenin 16.48 173.75 –
130
H. Bouriche, A. Kherbache, S. Kada, A. Senator, I. Demirtas
A. clavatus or aspirin significantly prevented the formation
of edema. Indeed, the treatment of rats with 200 or 400 mg
kg–1 of methanol extract inhibited the edema aer 6 h by
64 and 74%, respectively. At 400 mg kg–1 the inhibition was
close to that of aspirin, which was used as a standard anti-
inflammatory agent. At the same doses, the aqueous extract
exerted an anti-edematous effect with inhibition rates of 65
and 80%, respectively (Fig. 3).
Effect of Anacyclus clavatus extracts on carrageenan-
induced pleurisy in rats
e rats of the control group, which received orally
saline solution, have developed aer 4 h an acute pleurisy
characterized by an exudate volume of 0.55 ± 0.07 mL
(Fig. 4A). is exudate contained 26.70 ± 1.13 × 106 PMNs
(Fig. 4B). e pretreatment with 400 mg kg–1 of ME and
AE decreased significantly the development of the pleurisy.
Indeed, the methanol extract inhibited the exudation and
the polymorphonuclear leukocyte migration by 64 and
66%, respectively, compared with rats of the control group.
At the same dose, aqueous extract inhibited the exudation
by 42% and the number of migrating polymorphonuclear
leukocytes into the exudates by 55%. is inhibitory activity
was less than that observed with aspirin at 200 mg kg–1,
Fig. 1. HPLC chromatograms of phenolic compounds in A.
clavatus methanol (A) and aqueous (B) extracts..
Fig. 3. Effect of A. clavatus extracts on carrageenan-induced paw
edema in rat. e edema was induced by sub-plantar injection of
0.1 mL of carrageenan 1% in the rat pre-treated orally with 200 mg
kg–1 and 400 mg kg–1 of methanolic extract (ME), aqueous extract
(AE) and 100 mg kg–1 body weight of aspirin. e control received
only the saline solution. Each value represents the increase in
volume of the injected paw at different times aer injection of
carrageenan. Values are means ± SE (n = 6).
Fig. 2. Effect of A. clavatus extracts on croton oil-induced
ear edema in mice. Mice were treated with 2 mg per ear of
methanolic extract (ME), aqueous extract (AE) or 0.5 mg per ear
of indomethacin (Ind). Control group received only sterile saline
solution. Edema is expressed as mean thickness of ears before and
6 h aer croton oil application. Values are expressed as means ± SE
(n = 6). ***, p < 0.001 vs control, NS, not significant.
131
Anti-inflammatory and antioxidant activities of Anacyclus clavatus extracts
which decreased the exudation by 98% and the number of
polymorphonuclear leukocytes by 78% (Fig. 4 A,B).
DPPH free radical scavenging activity
A. clavatus aqueous and methanol extracts showed a
concentration-dependent anti-radical activity (Fig 5). At
100 µg mL–1, methanol extract exerted maximum activity
(90%). is effect was better than that of the standard BHT,
which in turn was better than that of aqueous extract. e
IC50 of ME, AE and BHT was 28.30 ± 3.45, 68.98 ± 1.64 and
44.36 ± 3.10, respectively.
Metal chelating activity
Results showed that both extracts of A. clavatus exerted
chelating activity. However, the chelating activity of
aqueous extract was better than that of the methanol
extract. Indeed, at 300 µg mL–1, AE exerted 88% inhibition,
whereas ME reached this percentage of inhibition only at
600 µg mL–1 (Fig. 6). EDTA used as a standard chelator
exerted high chelation activity (99%) at only 14 µg mL–1.
e IC50 obtained with AE, ME and EDTA was 74.64 ±
11.68, 152.93 ± 1.67, and 5.97 ± 0.20, respectively.
Total antioxidant activity
Fig. 7 shows the kinetics of linoleic acid peroxidation in the
presence and absence of ME, AE and BHT. e absorbance
of the samples was stable throughout the 96 h of incubation,
indicating strong antioxidant activity compared to
the control. At 50 µg mL–1, ME and AE inhibited lipid
peroxidation by 75%. is value is statistically similar
to that of BHT. At the same concentration, BHT exerted
inhibition of 77% (Fig. 7).
Fig. 4. Effect of A. clavatus extraxts on λ-carrageenan-induced
pleurisy in rats. Rats were pretreated orally with 400 mg kg–1
methanol (ME), aqueous (AE) extracts or 200 mg kg–1 aspirin.
e control was pretreated with normal saline solution and
then injected by λ-carrageenan. A, volume of exudate aspirated
from the pleural cavity 4 h aer the injection of λ-carrageenan.
B, number of polymorphonuclear leukocytes (PMNs) migrated
into exudates 4 h aer the injection of λ-carrageenan. Results are
expressed as mean ± SE (n = 6). ***p < 0.001; **p < 0.01 vs the
control. .
Fig. 5. Free radical scavenging activity of methanolic extract (ME),
aqueous extract (AE) of A. clavatus and butylated hydroxytoluene
(BHT). Values are means ± SD (n = 3)..
Fig. 6. Ferrous ion chelating activity of methanolic extract (ME),
aqueous extract (AE) of A. clavatus and EDTA. Values are means
± SD (n = 3)..
132
H. Bouriche, A. Kherbache, S. Kada, A. Senator, I. Demirtas
Discussion
Anti-inflammatory drugs and synthetic antioxidants are
oen associated with several adverse effects on human
health, such as gastrointestinal ulcers and cardiovascular
risk (Al-Saeed 2011), and liver damage and carcinogenesis
(Gulcin et al. 2005). erefore, the development of
alternative anti-inflammatory agents and antioxidants
mainly from natural sources has attracted considerable
attention. Medicinal plants may offer a safer and an effective
alternative treatment for inflammatory and oxidative-stress
related diseases. In this context, the anti-inflammatory
and the antioxidant properties of Anacyclus clavatus were
investigated.
To study the inflammatory processes, a croton oil-
induced ear edema experimental model is wide used. In
this model, edema events are triggered by protein kinase
C, which leads to phospholipase A2 activation and then
the release of a variety of bioactive eicosanoids, which are
implicated in the development of inflammatory events
(Cuzzocrea 1998). Also, protein kinase C promotes various
immune mediators such as cytokines and chemokines,
which increase and maintain the inflammatory response
(Kim et al. 2013). Croton oil is able to activate protein kinase
C, which in turn activates other enzymatic cascades, such
as cyclooxygenase 2 and inducible nitric oxide synthase
(Aquila et al. 2009). is cascade of events stimulates
vascular permeability, vasodilation, polymorphonuclear
leukocyte migration, histamine and serotonin release
and activates synthesis of eicosanoids by cyclooxygenase
and 5-lipoxygenase enzymes (Cuzzocrea 1998). Local
pre-treatment of ear mice with A. clavatus methanol and
aqueous extracts reduced significantly the size of the ear
edema (Fig. 2). is effect was better than that exerted by
indomethacin, used as standard anti-inflammatory drug to
inhibit cyclooxygenase 1 and 2, the formation of exudate
and the production of the pro-inflammatory mediators
such as TNF α, IL-6 and PGE2 (Bidaut-Russell 2008). e
activity observed with the studied extracts is probably due
to the presence of active substances that can cross the skin
barrier and exert anti-inflammatory effect (Manga et al.
2004). Flavonoids and polyphenols are likely candidates for
this effect (Gonzalez et al. 2011; Zhong et al., 2012).
In the carrageenan-induced paw edema model, the
subcutaneous injection of carrageenan into the rat paw
produced plasma exudation associated with the migration
of neutrophils into the inflated site and increased
arachidonic acid product release (Cuzocrea 1998). Oral
administration of A. clavatus extracts elicited a significant
reduction of paw edema formation at all assessment
times (Fig. 3), which indicated that these extracts contain
compounds that may act as anti-inflammatory agents by
inhibiting the release the pro-inflammatory mediators. In
fact, Romier-Crouzet et al. (2009) reported the inhibition
of inflammatory mediators by polyphenolic plant extracts.
Moreover, oral administration of A. clavatus extracts
significantly attenuated the development of pleurisy (Fig.
4 A,B), by inhibiting plasma exudation as well as leukocyte
recruitment to the inflated site aer 4 h following the
induction of pleurisy by λ-carrageenan. is result may
attribute to the phyto-constituents of the extracts that are
able to reduce the production of mediators involved in the
development of the acute inflammatory reaction. Indeed,
phytochemical screening showed that both studied extracts
are rich in flavonoids, polyphenols and tannins. ese
compounds are good inhibitors of serotonin, histamine and
leukocyte migration (Middleton et al. 2000). Flavonoids
have been found to have anti-inflammatory activity in both
proliferative and exudative inflammation phases, and they
inhibit histamine, cytokine, prostaglandin and leukotriene
release (Park et al. 2008; Rathee et al. 2009). Furthermore,
phenolic compounds are a very effective treatment against
inflammatory disorders (Gonzalez 2011).
e observed anti-inflammatory effects of A. clavatus
extracts may be due also to the presence of antioxidant
compounds. In fact, reactive oxygen species generated
during inflammation by phagocytic cells and during
the metabolism of arachidonic acid can activate the
phospholipase A2, which releases more arachidonic acid
from the phospholipid membrane, which is subsequently
transformed into pro-inflammatory prostaglandins and
leukotrienes (Geronikaki, Gavalas, 2006). is suggestion is
supported by the obtained antioxidant results. Indeed, both
extracts of extracts of A. clavatus exhibited a significant
anti-radical, iron chelating, and anti-lipid peroxidation
activity.
e DPPH radical scavenging activity of the two extracts
Fig. 7. Kinetics of the inhibition of lipid peroxidation by
methanolic extract (ME), aqueous extract (AE) of A. clavatus and
butylated hydroxytoluene (BHT). Values are means ± SD (n = 3).
133
Anti-inflammatory and antioxidant activities of Anacyclus clavatus extracts
was concentration-dependent (Fig. 5). Methanolic extract,
which showed the highest content of phenolic compounds
exhibited the highest scavenging activity (90%). ere is
a close positive correlation between phenolic content and
antioxidant activity (Zhao et al. 2014). e antioxidant
activity of phenolic compounds is mainly due to their redox
properties, which can play an important role in absorbing
and neutralising free radicals by their hydrogen donating
ability (Prasad et al. 2010).
e metal chelating activity is based on chelating of Fe2+
ions by the reagent ferrozine, which leads to the formation
of ferrozine-Fe2+ ions complex (Dinis et al. 1994). A. clavatus
aqueous extract exhibited a chelating activity higher than
the methanol extract (Fig. 6). is result indicates that the
aqueous constituents are more able to inhibit the formation
of ferrous complex with the reagent ferrozine, suggesting
the chelating activity of these extract and capture of the
ferrous ions before ferrozine. It has been reported that
chelating agents are effective as secondary antioxidants,
as they reduce the redox potential, thereby stabilizing the
oxidized form of the metal ions (Gulcin et al. 2007).
Lipid peroxidation is proceeded by radical-mediated
abstraction of the hydrogen atom from a methylene
carbon in a polyunsaturated fatty acid side chain (Yin et
al. 2011), and the inhibitory effects on lipid peroxidation
and autoxidation of linoleic acid have been attributed
to the radical scavenging activity (Bajpa et al. 2014).
Anacyclus clavatus extracts were similarly able to reduce
linoleic acid peroxidation (Fig. 7). is ability to modify
lipid peroxidation is linked not only to the structural
characteristics of the antioxidants agents but also to their
ability to interact with and penetrate the lipid bilayer
(Salcedo et al. 2014). It has been shown that the structure
and the lipophilicity of polyphenols are determinant factors
of antioxidant properties of these compounds in the lipid
layer of the membrane (Djeridane et al. 20010). Phenolic
compounds are the main class of natural antioxidants and
there is a close positive correlation between the phenolic
content and antioxidant activity of plant extracts (Zhao et
al. 2014).
In conclusion, Anacyclus clavatus extracts exhibit anti-
inflammatory and antioxidant activities, and phenolic
constituents could be responsible for these activities. is
plant may be considered as new promising source for
functional foods and pharmaceuticals.
Acknowledgements
e authors are thankful to the Algerian Ministry of High
Education for providing facilities to carry out the research work.
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Anti-inflammatory and antioxidant activities of Anacyclus clavatus extracts
Received 22 June 2016; received in revised form 2 September 2016; accepted 15 September 2016
