Biomedicine & Pharmacotherapy 84 (2016) 115–122

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Antioxidant, antibacterial, anti-inflammatory and wound healing

effects of Artemisia campestris aqueous extract in rat
Zohra Ghlissia,* , Nadhim Sayarib , Rim Kallelc , Ali Bougatefb , Zouheir Sahnouna
a
Research Unit of Pharmacology and Toxicology of Xenobiotics (UR12 ES13), Faculty of Medicine, University of Sfax, 3029, Tunisie
b
Unit Enzymes & Bioconversion, National School of Engineers of Sfax, Tunisie
c
Anatomopathology Laboratory, Habib Bourguiba University Hospital, 3029, Sfax, Tunisie

A R T I C L E I N F O A B S T R A C T

Article history:
Received 11 July 2016 This study investigated some biological properties of Artemisia campestris aqueous extract (ACAE) as well
Received in revised form 7 September 2016 its global chemical compositions. Twenty four rats were excised on the posterior neck skin area and
Accepted 7 September 2016 divided into 4 groups, treated respectively with: sterile saline, glycerol, CICAFLORA and ACAE. The wound
closure rate, histopathology evolution and the superoxide dismutase (SOD), catalase (CAT) and
Keywords: malondialdehyde (MDA) level in skin tissue were evaluated. Anti-inflammatory activity was studied by
Artemisia campestris L carrageenan-induced rat paw edema. Animals were divided into 3 groups pre-treated respectively with
Wound healing sterile saline, acetylsalicylic acid (AA) and ACAE. The antibacterial activity was tested against six bacteria
Anti-inflammatory
and the antioxidant activity was estimated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power
Antioxidant
and b-carotene activities. Our results demonstrated a significant improvement in wound healing
Antimicrobial
progression and in oxidative stress damage in the wounds tissues of ACAE-treated rats, compared to
control. ACAE-treated rats revealed also a significant inhibition of carrageenan-induced hind paws edema
as confirmed by the histological analysis. In addition to the antioxidant activity, ACAE showed
considerable antibacterial activities. ACAE exhibited important wound healing effect probably due to the
anti-inflammatory, antibacterial and antioxidant activities of its phytochemical contents. Therefore, this
study confirms its popular use and highlights its promise in the development of new drugs.
ã 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction Africa and other similar Mediterranean agro-ecological zones and

Interest in ethnopharmacy as a source of pharmacological
active compounds has increased worldwide, particularly in the
search of new drugs for disease treatment. The use of plants for
medicinal purposes has been practiced for many centuries by a
substantial proportion of the Tunisian population [1]. Due to
economic conditions and availability, plants are the main
medicinal source to many diseases in some developing countries
spatially in the countryside. In Tunisia, traditional plant remedies
were empirically used for treating various diseases and ailments,
however only a small number of them have been scientifically
studied [1].
Artemisia campestris L. (AC) is a perennial aromatic herb
belonging to asteraceae family; it is widespread in Northern
* Corresponding author at: Laboratory of Pharmacology, Faculty of Medicine,
University of Sfax, 3029 Tunisia.
E-mail address: ghlissi_zohra@yahoo.fr (Z. Ghlissi).
is commonly used as an herbal medicine [2,3]. In Tunisia, AC locally
named as “Dgouft”, is an approximately 1 m height shrub
widespread in south-eastern regions. The leaves of this species
are collected in summer and used in Tunisian folk medicine as
decoction for their antispasmodic, anti-inflammatory, anti-rheu-
matic, antimicrobial, anthelmintic and anti-venin properties [1].
Additionnaly, the leaves of AC are also used in the southern of
Tunisia for the treatment of the wounds. The phytochemical
assessment of this species revealed the presence of tannins,
polyphenols, flavonoids, saponosides and essential oils [4,5].
Different compounds have been isolated from the solvent extracts
of this species such as flavonoids, chromones, and acetophenones
[6]. Moreover, interesting in vitro pharmacological activities of AC
have been reported such us antimicrobial, antioxidant, antimuta-
genic [7,8] activities, but it was not too investigated in vivo.
Taking into account its numerous and different uses,
we investigated for the first time the wound healing effect
(much known as traditional use in Tunisia) and the in vivo

http://dx.doi.org/10.1016/j.biopha.2016.09.018
0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
116 Z. Ghlissi et al. / Biomedicine & Pharmacotherapy 84 (2016) 115–122

anti-inflammatory activity of the aqueous extract of AC at a dose of 2.4.3. Determination of total tannins content
10%. Its in vitro antibacterial and antioxidant activities, as well as Condensed tannins were determined according to the method
some global chemical compositions (polyphenols, tannins and of Julkunen-Titto [11]. An aliquot (50 ml) of each extract or standard
flavonoids) were equally studied. solution was mixed with 1.5 ml of 4% vanillin (prepared with
MeOH), then 750 ml of HCl (12 M) were added. The well-mixed
solution was incubated in the dark at ambient temperature for
2. Materiel and methods
20 min. The absorbance against blank was read at 500 nm. Catechin
was used to make the standard curve. The results were expressed
2.1. Reactifs
as mg catechin equivalents (CE)/g extract).
1,1-Diphenyl-2-picrylhydrazyl (DPPH), ethylene diamine tetra-
2.5. In vitro antioxidant activity
acetic acid (EDTA), trichloroacetic acid (TCA), thiobarbituric acid
(TBA) and nitro blue tetrazolium (NBT) were purchased from Sigma
2.5.1. DPPH activity
Chemical Co. (St. Louis, MO, USA). Butylated hydroxyanisole (BHA)
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scaveng-
and all other chemicals were of analytical grade. Acetylsalicylic
ing potential of ACAE was determined by the method of Kirby and
acid (AA) and “CICAFLORA” cream were purchased from local
Schmidt [12] with some modifications. A volume of 500 ml of each
pharmaceutical industry.
extract at different concentrations (10–50 mg/ml) was added to
375 ml of 99% ethanol and 125 ml of DPPH solution (0.2 mM in
2.2. Animals
ethanol) as free radical source. The mixtures were incubated for
60 min in the dark at room temperature. Scavenging capacity was
Male Wistar rats (250–270 g) were used in the experiment. The
measured spectrophotometrically by monitoring the decrease in
rats were caged under controlled conditions of light (12 h light/
absorbance at 517 nm. In its radical form, DPPH has an absorption
dark cycles), room temperature (23
1  C) and relative humidity
band at 517 nm which disappears upon reduction by an antiradical
(50%
10%) with free access to food and water ad libitum. All
compound. Lower absorbance of the reaction mixture indicated
animal procedures were conducted in strict conformity with the
higher free radical-scavenging activity. Butylated hydroxyanisole
local Institute Ethical Committee Guidelines for the Care and Use of
(BHA) was used as positive control. DPPH radical-scavenging
laboratory animals of our institution.
activity was calculated as:
2.3. Plant material DPPH radical  scavenging activity ð%Þ
A control þ Ablank  A sample
¼  100
Fresh leaves of Artemisia campestris were collected at Septem- A control
ber 2015 in the region of Bir Ali Ben Khalifa in Sfax city, Tunisia.
where A control, A blank and A sample are, respectively, the
Leaves were washed, air-dried and powdered using an electric
absorbance of the control reaction (containing all reagents except
blender. Powdered plant material was extracted by maceration in
the sample), the ACAE without DPPH solution and the AC aqueous
water (10 g/100 ml) at ambient temperature for 24 h with
extract with DPPH solution. The test was carried out in triplicate.
continuous stirring. The extracts were filtrated with Whatmann
Millipore filter paper and concentrated to dryness with a rotary
2.5.2. Reducing power determination
evaporator at 40  C.
The reducing power of ACAE was determined by the method of
Yildirim et al. [13]. Sample solutions (0.5 ml) containing different
2.4. Global chemicals assessment
concentrations of dried extract (10–50 mg/ml) were mixed with
1.25 ml of 0.2 M phosphate buffer (pH 6.6) and 1.25 ml of 10 g/l
2.4.1. Determination of total phenolic content
potassium ferricyanide solution. The mixtures were incubated for
The total phenolic content in ACAE extract was determined with
30 min at 50  C. After incubation, 1.25 ml of 100 g/l TCA was added
Folin–Ciocalteau reagent using the method of Chen et al. [9]. A
and the reaction mixtures were centrifuged for 10 min at 3000 g. A
standard curve must be first plotted using gallic acid as a standard.
1.25 ml aliquot of the supernatant from each sample mixture was
Different concentrations of gallic acid were prepared in methanol,
mixed with 1.25 ml of distilled water and 0.25 ml of 1.0 g/l ferric
and their absorbances were recorded at 750 nm. 100 ml of diluted
chloride solution in a test tube. After a 10 min reaction time, the
sample was added to 2 ml of 2% Na2CO3 aqueous solution. After
absorbance was measured at 700 nm. Higher absorbance of the
2 min, 100 ml of 50% Folin–Ciocalteau reagent was added. The final
reaction mixture indicated higher reducing power. The control was
mixture was shaken and then incubated at room for 30 min in the
conducted in the same manner, except that distilled water was
dark at room temperature. The absorbance of all samples was
used instead of sample. Values presented are the mean of triplicate
measured at 750 nm and the results are expressed in mg gallic acid
analyses.
equivalents per gram extract (mg GAE/g extract).
2.5.3. b-carotene bleaching assay
2.4.2. Determination of total flavonoids content
The ability of ACAE to prevent b-carotene bleaching was
The total flavonoids content in ACAE was determined by the
determined respectively by the methods Koleva et al. [14]. A stock
method of Djeridane et al. [10], using a method based on the
solution of b-carotene/linoleic acid mixture was prepared by
formation of a complex flavonoid–aluminium, having the maxi-
dissolving 0.5 mg of b-carotene, 25 ml of linoleic acid and 200 ml of
mum absorbance at 430 nm. Rutin was used to make the
Tween 40 in 1 ml of chloroform. The chloroform was completely
calibration curve. About 1 ml of diluted sample was mixed with
evaporated under vacuum in a rotatory evaporator at 40  C, then
1 ml of 2% aluminium trichloride (AlCl3) methanolic solution. After
100 ml of bi-distilled water were added, and the resulting mixture
incubation at room temperature for 15 min, the absorbance of the
was vigorously stirred. The emulsion obtained was freshly
reaction mixture was measured at 430 nm with a Shimadzu
prepared before each experiment. Aliquots (2.5 ml) of the
UVmin1240 UV–vis spectrophotometer and the total flavonoid
content was expressed in mg rutin equivalent (RE) per g of extract.
b-carotene/linoleic acid emulsion were transferred to test tubes
containing different ACAE concentrations. After two hours
 Z. Ghlissi et al. / Biomedicine & Pharmacotherapy 84 (2016) 115–122
incubation at 50  C, the absorbance of each sample was measured
at 470 nm. BHA was used as positive standard. The control tube
contained no sample. Antioxidant activity in b-carotene bleaching
model in percentage was calculated with the following equation:
1  ðA0  At Þ
Antioxidant activity% ¼  0   100
A0At
0 2.8.1. Experiment protocol
where A0 and A’0 are the absorbance of the sample and the blank,
respectively, measured at time zero, and At and A’t are the
absorbance of the sample and the blank, respectively, measured
after 2 h. Tests were carried out in triplicate. The same procedure
was repeated with BHA as positive control.
2.6. In vitro antibacterial activity
The antibacterial activity of ACAE was tested against six
bacterial strains: Staphylococcus aureus (ATCC 25923), Micrococcus
luteus (ATCC 4698), Escherichia coli (ATCC 25922), Klebsiella
pneumoniae (ATCC 13883), Salmonella enterica (ATCC 43972) and
Listeria monocytogenes (ATCC 43251). Antibacterial activity was
determined by means of agar well diffusion assay according to
Vanden Berghe and Vlietinck method [15]. ACAE (50 mg) were
dissolved in 1 ml dimethylsulfoxide (DMSO) (100%). Culture
suspension (200 ml) of the tested microorganisms 106 colony-
forming units (cfu)/ml of bacteria were spread on Luria Bertani
agar. Then a 50 mg of AC aqueous extract was dissolved in 100%
dimethylsulfoxide (DMSO) at 100 mg/ml and added (60 ml) to wells
of 6 mm diameter, which were punched in the agarose layer.
Ampicillin (50 mg/ml) was used as a positive control and DMSO as a
negative control. The Petri dishes were first kept for 1 h at 4  C to
allow the diffusion of the extract and then incubated for 24 h at
37  C. Antibacterial activity was measured as the diameter of the
clear zone of growth inhibition. All tests were carried out for three
sample replications and the mean diameter of the inhibition zone
was recorded.
2.7. In vivo anti-inflammatory test
2.7.1. Experimental design
The rats were divided into 3 groups (n = 6) and were treated
respectively with 1 ml/kg of sterile saline, 150 mg/kg of acetylsa-
licylic acid (AA) and 200 mg/kg of ACAE [16]. After 1 h of
intraperitoneal injection of drug and plant extract, 0.1 ml of
carrageenan (1% in normal saline) was subcutaneously injected
into the foot pad of the rat right hind paw [17].
2.7.2. Edema volume measurement
Edema was measured using a plethysmometer before the
administration of carrageenan and at several time-points after
injection of inflammatory mediators or the irritants. Edema was
expressed in millimeters as the differences between the tests in the
control paws using the following formula [18]:
Ft  F0
Edema rate ð%Þ ¼  100
F0
where F0 and Ft are the foot volume measured respectively at time
0 and at time t (3 and 7 h) after injection of carrageenan.
2.7.3. Histological analysis
Animals were sacrificed 7 h after the carrageenan injection and
muscles samples were then removed The sub-plantar muscles
were fixed in 10% neutral buffered formalin solution, embedded in
paraffin wax, cut into 5 mm-thick sections, and stained with
hematoxylin–eosin for evaluation under light microscopy. For
polymorphonuclear (PMN) quantification, square with 200 mm
117
were selected within the microscope image area from the
inflammatory site.
2.8. Wound healing activity
The back of each rat was shaved under anesthesia (50 mg/kg b.w
ketamine) and disinfected with alcohol. Then a uniform wound
area with 2 cm diameter was excised from the napes of the rats
using a round seal, as described by Morton et al. [19]. The rats were
divided into 4 groups (n = 6) and treated respectively with sterile
saline, glycerol (dilution matrix of ACAE), “CICAFLORA” (reference
drug) and ACAE. Each rat was housed separately and was treated
every day.
2.8.2. Wound area measurement
During the wound healing period, the wound areas were traced
manually using transparent paper. Wound areas were then
measured using a computer software application for design and
drafting (Auto CAD RL 14). Wound contraction, expressed as a
percentage of reduction of original wound size, was calculated
using the following equation [20]:
A0  An
Wound closure ð%Þ ¼  100
A0
where A0 refers to the initial wound size and An to the wound size
at a specific day.
2.8.3. Oxidant stress parameters in wound tissue homogenate
The skin tissues were weighed (500 mg), minced, and
homogenized in 5 ml of lysis buffer 50 mM Tris, 150 mM NaCl
adjusted to pH (7.4) and centrifuged at 8000 g for 10 min at 4  C.
The supernatant was collected and used for the measurement of
malondialdehyde (MDA), superoxide dismutase (SOD) and catalase
(CAT) activity.
2.8.3.1. Malondialdehyde (MDA) level. The MDA level in tissue
homogenate was determined according to the method of Draper
and Hadley [21]. An amount of 0.5 ml of organ extract supernatant
was mixed with 1 ml of trichloroacetic acid solution (20%) and
centrifuged at 2500 g for 10 min. A 1-ml solution containing
0.67% thiobarbituric acid (TBA) and 0.5 ml of supernatant were
incubated for 15 min at 80  C and cooled. Absorbance of TBA–MDA
complex was determined at 530 nm using a spectrophotometer
(Jenway UV-6305; Essex, England). Lipid peroxidation was
expressed as nanomoles of TBA reactive substances using
1,1,3,3-tetraethoxypropane as standard.
2.8.3.2. Superoxide dismutase (SOD) activity. SOD activity was
estimated according to Beyer and Fridovich [22]. The reaction
mixture contained 50 mM of tissue homogenates in potassium
phosphate buffer (pH 7.8), 0.1 mM ethylene diamine tetra-acetic
acid (EDTA), 13 mM L-methionine, 2 mM riboflavin and 75 mM nitro
blue tetrazolium (NBT). The developed blue color in the reaction
was measured at 560 nm. Units of SOD activity were expressed as
the amount of enzyme required to inhibit the reduction of NBT by
50%, and the activity was expressed as units per milligram of
protein.
2.8.3.3. Catalase (CAT) activity. CAT activity was assayed by the
decomposition of hydrogen peroxide according to the method of
Aebi [23]. A decrease in absorbance due to H2O2 degradation was
monitored at 240 nm for 1 min and the enzyme activity was
expressed as mmol H2O2 consumed/min/mg protein.
118 Z. Ghlissi et al. / Biomedicine & Pharmacotherapy 84 (2016) 115–122

Table 1
Total Plyphenolic, flavonoid and tannins contents of ACAE.

Polyphenols (mg GAE/g extract) Flavonoid (mg RE/g extract) Tannins (mg CE/g extract)
ACAE 313.20
4.35 28.30
1.45 8.30
0.45

ACAE: Artemisia campestris aqueous extract; GAE: gallic acid equivalents; RE: rutin equivalent; CE: catechin equivalents.

2.8.4. Histological analysis

At day 12, animals were sacrificed and the skin from the healed
wound area was removed. Skin samples were fixed in 10% neutral
buffered formalin solution, embedded in paraffin wax, cut into
5 mm-thick sections, and stained with hematoxylin–eosin for
evaluation under light microscopy.

2.9. Statistical analysis

Data are expressed as mean
SD (standard deviation). The

statistical significance between experimental groups was assessed
by one-way analysis of variance (ANOVA) followed by Tukey post-
hoc test. Statistical significance was set at p < 0.05.

3. Results and discussion

3.1. Polyphenols, flavonoids and tannins contents

The amounts of the total polyphenolics (TP), total flavonoids

(TF) and total tannins (TT) in the aqueous extract of AC are listed in
Table 1. The highest amount of global chemical composition
detected is that of TP. Our findings are in agreement with previous
work of Akrout et al. [7]. The amount of TF in ACAE found in our
investigation is 28.30
1.45 mg RE/g. In preceding studies on AC
[24,25], the amount of TF in the methanolic extracts obtained was
higher than that we found. The result showed that extraction mode
that they adopted yielded prominent amounts of flavonoids as
compared to our extracts. Flavonoids have a higher solubility in
methanol than in water. We observed equally a considerable
amount of TT in ACAE which is comparable to some earlier works.
It was apparent that the TP were the dominant compounds of the
aqueous extract of AC. The results we encourage to study its
potential biological activities, however further studies should be
performed for isolation and identification of the compounds of the
extracts.

3.2. Antioxidant activity of AC aqueous extract

ACAE exhibited a significant radical-scavenging activity at all

tested concentrations. The strongest value (86%) was observed at a
concentration of 50 mg/ml (Fig. 1A). Similarly, the reducing power
of AC increased with increasing concentrations (Fig. 1B) and
reached a maximum of 1.46
0.10 at a dose of 50 mg/ml. Same
results were observed for b-carotene activity that increased with
the increase of ACAE concentrations. Indeed, at 50 mg/ml the%
inhibition of extracted AC and BHA (positive control) were 64.81%
and 93%, respectively. Our results are in concordance with several
studies that demonstrated the antioxidant action of AC extracts
[26,27]. Antioxidant properties of the aqueous extracts could be
attributed to the phenolic and flavonoid contents of the Artemisia
species. Despite ACAE showed a lower in vitro antioxidant activity
than the synthetic antioxidant BHA at tested concentrations, this
plant extract exhibited a good antioxidant capacity. It is interesting
then to increase the concentrations of ACAE in our future
investigations.
Fig. 1. DPPH, reducing power and b-carotene activities of ACAE.
 Z. Ghlissi et al. / Biomedicine & Pharmacotherapy 84 (2016) 115–122 119

Table 2 Table 3
Antibacterial activity of ACAE (inhibition diameter in mm). Effect of pre-treatment with ACAE on carragenan induced paw edema in rats.

Diameter of inhibition zone (mm
SD) Groups 0h 3h 7h

Bacteria ACAE (50 mg/ml) Ampicillin (50 mg/ml) Carragenan 3.25
0.25 6.28
0.5** 6.76
0.55**
 Carragenan + AA 3.45
0.21 6.12
0.45** 5.38
0.36**
Gram
Carragenan + ACAE 3.32
0.13 5.45
0.33* 3.86
0.24
Klebsiella pneumoniae 9
0.2 14
0.5
Salmonella enterica 6
0.1 13
0.3 Values are expressed as mean
SD. ACAE: Artemisia campestris aqueous extract; AA:
Escherichia coli 10
0.1 17
0.6 acetylsalicylic acid.
*
p  0.05.
**
Gram+ p  0.01 compared with zero time.
Listeria monocytogenes R 15
0.4
Staphylococcus aureus 13
0.2 19
0.5
Micrococcus luteus 8
0.3 13
0.4

Values represent averages
standard deviations for triplicate experiments. R:

resistant; ACAE: Artemisia campestris aqueous extract.

Fig. 2. (A) Histological sections of edema paws of the control showed normal tissue (a), the carrageenan displayed massive signs of inflammation (^) (b), carrageenan + AA
showed moderate density of inflammatory cells (R) (c) and carrageenan + ACAE groups exhibited a slight density of inflammatory cells (") (d). Tissues were stained with
hematoxylin–eosin and visualized at 400  magnifications. (B) Number of inflammatory cells in the paw muscle tissue. Scale bar = 200 mm. Data are the mean
SD values.
***
p < 0.001, significant difference compared with the control. #p < 0.05; ##p < 0.01 and ###p < 0.001, significant difference compared with the carrageenan group.
120 Z. Ghlissi et al. / Biomedicine & Pharmacotherapy 84 (2016) 115–122

3.3. Antibacterial activity
The antibacterial activity of ACAE was evaluated against three
Gram-negative bacteria species (E. coli, S. enterica and K. pneumo-
niae) and three Gram-positive bacteria species (L. Monocytogenes,
S. aureus and M. luteus). The inhibition zones relative to the
antimicrobial activity of ACAE and positive control (Ampicillin) are
represented in Table 2. Although ACAE showed a lower antibacte-
rial activity than Ampicillin, it exhibited a good inhibition zone
with the strongest value (13 mm) against S. aureus (the medici-
nally important pathogen) at only a concentration of 50 mg/ml.
While, ACAE was not active against L. monocytogenes; this may
means that the extract should be applied at higher concentration
against this species. According to the literature, this is the first
study to investigate the antibacterial activity of the aqueous
extract of the AC leaves. However several extracts such as
methanolic and ethanolic extracts were previously studied.
Karabegovi’c et al. [24] showed that methanolic extracts of this
plant possesses relatively high antibacterial activity. Besides, our
results confirmed the previously reported inhibition of S. aureus
and E. coli growth by an AC extract at a concentration of
125 mg ml1 [28]. The differences in antimicrobial activities of
AC extracts can be attributed to differences in their qualitative and
quantitative active compound compositions.
3.4. Anti-inflammatory activity
Table 3 illustrates the time-dependent increase of paw edema
after carrageenan injection. After 3 h of carrageenan injection, paw
edema increased to approximately 93% in the carrageenan group,
71% in the carrageenan + Acetylsalicylic acid and 61% in the
carrageenan + ACAE. However after 7 h of treatment, ACAE showed
26% inhibition of edema respectively, compared to values of 3 h of
treatment in the same group. The percentage inhibition of
standard anti-inflammatory drug was (55%, 10 mg/kg bw). The
macroscopic results of the paw edema were confirmed through the

Fig. 3. Effects of ACAE on wound’s evolution. (A) Representative photographs of excised wound of rat treated with saline, glycerol, CICAFLORA and ACAE. The photographs of
the wound at 0–12 days are representative of six rats in each group. (B) Reduction of wound diameter (0–12 days). Each data point represents the mean
SD of six rats.
 Z. Ghlissi et al. / Biomedicine & Pharmacotherapy 84 (2016) 115–122 121

histological examination and conducted quantitative analysis
(Fig. 2). The paw tissue in the saline control group showed normal
histological sections (Fig. 2A-a), while the carrageenan-treated
group displayed massive signs of inflammation, with edema
disruption of tissue structure (Fig. 2A-b) and an increased number
of inflammatory cells, particularly neutrophils and macrophages
(Fig. 2(B)). Neutrophils and macrophages mediate inflammatory
responses in many diseases by expressing pro-inflammatory
proteins that promote tissue inflammation [29]. The treatment
with ACAE from leaves reduced significantly the inflammations in
rat paw tissues (fig. 2A-c); a significant decrease in the
inflammatory cells number (Fig. 2(B)) was detected, however,
moderate improvements in edema tissue were observed in the
reference drug-treated group (Fig. 2d). These results clearly
demonstrate that AC leaves has an anti-inflammatory effect on
edema tissue following injection of carrageenan. To our knowl-
edge, this is the first study to investigate the anti-inflammatory
effect of AC. However, it was reported that methanol/water extract
of A. herba-alba, another species of Artemisia possess anti-
inflammatory activity [30]. Anti-inflammatory effect of AC can
also be related to flavonoid and phenolic contents of AC as
demonstrated above. This result is encouraging to prompt us to try
to identify the molecules responsible for this activity. Further
studies should be therefore carried on to identify the anti-
inflammatory active components from this extract.
3.5. Wound healing effect
As it is illustrated in Fig. 3, each group revealed different rates of
wound contraction. Treated group with ACAE displayed a
significant wound healing progression, compared to other groups.
Approximately 50% of the wounds contraction was achieved on
days 6, 8, 10 and 12 respectively for the ACAE, CICAFLORA, glycerol
and untreated groups. After 12 days, the macroscopic assessment
revealed that the wound area of the AC-treated group was
completely healed. However the untreated, glycerol and CICA-
FLORA groups still show an open wound (23%, 21% and 11%,
respectively) (Fig. 3A). The visual observations are confirmed with
microscopic results. Histopathological survey of wound tissues on
12th day is illustrated in Fig. 4. Tissue sections of wound areas in
ACAE-treated rats revealed a normal histology sections. Layers of
skin were clearly identified; epidermis and dermis were normal
(Fig. 4D). A complete re-epithelialization and well formed
granulation tissue of epidermis with remarkable neo-vasculariza-
tion and fewer inflammatory cell infiltrations. However, in the
histological sections of the wound tissue of the control and
glycerol-treated group (Fig. 4A/B), the epidermal layers were
poorly structured, the collagen fibers were unorganized, the
dermis showed the presence of massive number of inflammatory
cells and a prominent hyperemia of capillary blood vessels.
These results clearly indicate the potential effect of ACAE in
accelerating wound healing, which may be due to various effects.
Generally, the natural wound healing process is carried out in three
phases: the inflammatory phase, the granulation phase (or
proliferative stage) and the maturation phase, giving the final
appearance of the scar. We demonstrated in this study that AC
exhibited an anti-inflammatory effect in carragenan-induced rat
paw edema. The enhanced wound healing progression may be
therefore due to the reduction of the inflammatory phase and its
rapid transition to the proliferative phase. Additionally, we showed
in this study that AC revealed a better antimicrobial activity against
several medicinally important pathogens such S. Aureus, because
the presence of this infectious agents may extended the anti-
inflammatory phase. Many others factors are also mentioned in the

Fig. 4. Representative photomicrographs of epidermal and dermal architecture of wounds on the 12th day. Control group treated with serum physiologic (A); group treated
with glycerol (B); group treated with “CICAFLORA cream” (C) and group treated with ACAE (D). Tissues were stained with hematoxylin–eosin and visualized at
400  magnifications. e: epidermis, d: dermis, o: inflammatory cells, v: blood vessels, collagen.
122 Z. Ghlissi et al. / Biomedicine & Pharmacotherapy 84 (2016) 115–122
Table 4
Effects of ACAE on the MDA, SOD and CAT activities in the healed wound area in
different groups of rats.
Groups Control CICAFLORA ACAE
MDA (nmol/mg protein) 16.53
2.23 11.21
2.11* 7.61
1.05**
SOD (U/mg protein) 17.45
3.22 21.32
4.15* 35.68
5.04**
CAT (mmol H2O2/min/mg protein) 6.12
0.73 10.66
2.33* 15.66
3.23**
Data expressed as mean
SD, 6 rats/group. ACAE: Artemisia campestris aqueous
extract. SOD: superoxide dismutase; CAT: catalase; MDA: malondialdehyde (MDA).
*
p  0.05.
**
p  0.01 compared with Control.
process of wound healing such as the excessive production of
reactive oxygen species and oxidative stress which slows the
healing. In the present study, the CAT and SOD activities and MDA
levels in the homogenate tissues from wounds were calculated
(Table 4). Our results showed that the level of MDA, lipid
peroxidation marker, increased significantly in untreated group.
Lipid peroxidation is found to be an important pathophysiological
event in a variety of diseases including wound healing. It promotes
the generation of reactive oxygen species (ROS) in the tissue site,
which damage cell structures. The decrease in CAT and SOD
antioxidant activities in wound tissue homogenates of the control
rats may be also attributed to the increased production of reactive
oxygen species (ROS). Therefore, the deficiency of the wound
healing process may be attributed to the oxidative stress defect. In
contrast the activities of these antioxidant enzymes were
significantly restored (p < 0.01) in rats treated with AC compared
to those of the untreated group (Table 4). As already shown at the
beginning of our findings, AC leaves contain a high content of
antioxidants (Phenols, flavonoids and tannins). Antioxidants are
known to scavenge free radical- activity and inhibit its reaction
propagation [31]. The free radical scavenging properties of these
compounds could protect wound tissues from oxidative damage
and reduce the risk of inflammation. Therefore, the antioxidant
property of AC may be among the most efficient contributing
factors for improved wound healing.
4. Conclusion
Our results indicate that AC accelerates wound healing and
increases wound contraction. Several factors could be involved in
the enhancement of the wound healing process. The presence of
the antioxidant, anti-inflammatory and antimicrobials properties
may be among the factors that contributed to the potential wound-
healing effect of ACAE. Further studies are required to establish the
principal bioactive compound responsible for the wound healing
effect and the optimal conditions for the production and
application of the ACAE.
Conflict of interest
The authors declare that they have no conflict of interest.
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