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ORIGINAL ARTICLE
Phytochemical Analysis and Evaluation of the Antioxidant,
Anti-Inflammatory, and Antinociceptive Potential of Phlorotannin-Rich
Fractions from Three Mediterranean Brown Seaweeds
Amal Abdelhamid
1
&Meriem Jouini
2
&Haifa Bel Haj Amor
1
&Zeineb Mzoughi
3
&Mehdi Dridi
1
&Rafik Ben Said
4
&
Abderrahman Bouraoui
1
Received: 13 July 2017 /Accepted: 27 November 2017
#Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Phlorotannins, phenolic compounds produced exclusively by seaweeds, have been reported to possess various pharmacological prop-
erties. However, there have been few works on these compounds from Mediterranean seaweeds. In this study, we investigated the
phytochemical analysis and pharmacological potential of phlorotannin-rich fractions from three brown seaweeds collected along the
Tunisia coast: Cystoseira sedoides (PHT-SED), Cladostephus spongeosis (PHT-CLAD), and Padina pavonica (PHT-PAD).
Phytochemical determinations showed considerable differences in total phenolic content (TPC) and phlorotannin content (PHT). The
highest TPC level (26.45 mg PGE/g dry material (Dm)) and PHT level (873.14 μg PGE/g Dm) were observed in C. sedoides.The
antioxidant properties of these three fractions assessed by three different methods indicated that C. sedoides displayed the highest total
antioxidant activity among the three species (71.30 mg GAE/g Dm), as well as the free radical scavenging activity with the lowest IC
50
value in both DPPH (27.7 μg/mL) and ABTS (19.1 μg/mL) assays. Furthermore, the pharmacological screening of the anti-
inflammatory potential of these fractions using in vivo models, in comparison to reference drugs, established a remarkable activity of
PHT-SED at the dose of 100 mg/kg; the inhibition percentages of ear edema in mice model and paw edema in rats model were of 82.55
and 81.08%, respectively. The content of malondialdehyde (MDA) in liver tissues has been quantified, and PHT-SED was found to
remarkably increase the lipid peroxidation in rat liver tissues. In addition, in two pain mice models, PHT-SED displayed a profound
antinociceptive activity at 100 mg/kg and has proved a better analgesic activity when used in combination with the opioid drug, tramadol.
Keywords Phlorotannins .Brown seaweed .Phytochemical .Radical scavenging activity .Anti-inflammatory activity .
Antinociceptive activity
Abbreviations
SLE solid liquid extraction
PHT phlorotannins
RSA radical scavenging activity
PGE phloroglucinol equivalent
GAE gallic acid equivalent
Dm dry material
DPPH 2,2-diphenyl-1-picrylhydrazyl
ABTS 2,2′-azinobis-3-ethylbenzthiazoline-6-sulphonic acid
IC
50
half maximal inhibitory concentration
MDA malondialdehyde
Introduction
Marine seaweeds are considered as potentially new and valu-
able source of biomolecules for applications in the food
*Amal Abdelhamid
amal.abdelhamid87@gmail.com
1
Laboratory of Chemical, Galenic and Pharmacological Development
of Drugs, Faculty of Pharmacy of Monastir, University of Monastir,
5000 Monastir, Tunisia
2
Laboratory of Heterocyclic Chemistry, Natural Products and
Reactivity, Medicinal Chemistry and Natural Products Team, Faculty
of Sciences of Monastir, University of Monastir,
5019 Monastir, Tunisia
3
Laboratory of Interfaces and Advanced Materials, Faculty of
Sciences of Monastir, University of Monastir, 5000 Monastir, Tunisia
4
National Institute of Marine Sciences and Technologies,
Salambôo, Tunis, Tunisia
Marine Biotechnology
https://doi.org/10.1007/s10126-017-9787-z
industry as well as in the pharmaceutical and cosmetic sectors
(Lauritano et al. 2016).
Among them, marine brown seaweeds (Phaeophyceae) are
a rich source of natural bioactive compounds with potent bi-
ological capacities, mainly phlorotannins (Li et al. 2011).
Brown algae accumulate a variety of phloroglucinol-based
entities (phlorotannins), formed from the polymerization of
phloroglucinol (1,3,5-trihydroxybenzene) monomer units
generating compounds with different molecular weight
(Pádua et al. 2015). Phloroglucinol and its derivatives found
in brown seaweed have been demonstrated to possess antiox-
idant properties (Wijesekara et al. 2010; Kristinsson and
Miyashita 2014). Majority of investigations on phlorotannins
showed their potentiality as antioxidant, anti-inflammatory
(Sanjeewa et al. 2016; Murray et al. 2017), antidiabetic
(Kang et al. 2013a; Lopes et al. 2016), antiproliferative
(Nwosu et al. 2011;Lietal.2015), and other health benefits
especially neuroprotective potential (Jung et al. 2010;Barbosa
et al. 2014). Furthermore, phlorotannins were reported as anti-
allergic and anti-aging agents, owing to their strong inhibitory
effect on the activation of hyaluronidase (Ferreres et al. 2012).
Promising researches from the cell in vitro model and animal
models suggest that marine algal polyphenols may be effec-
tive in the prevention and management of such chronic dis-
eases (Murray et al. 2017).
Oxidative stress is a state present in all aerobic organisms,
and it has a key role in the originand the development of many
diseases connected with inflammation, such as neurodegener-
ation, cardiovascular diseases, and cancer. It occurs when the
production of reactive oxygen species (ROS) such as super-
oxide anion (O
2−
), hydroxyl radical (HO·), and hydrogen per-
oxide (H
2
O
2
) exceeds the limited capacity of the cellular an-
tioxidant defense system (Thomas and Kim 2011). These
small molecules, produced in radical reactions, have the ca-
pacity to quickly interact with cellular structures (Treml and
Šmejkal 2016). The radical scavenging potential of
phlorotannins has largely been assigned to their unique struc-
tural arrangements, which help in the scavenging of peroxyl,
superoxide anions, and hydroxyl radicals (Kirke et al. 2017).
Phlorotannins were previously reported as potent free radical
scavengers and about twice as effective as catechin, ascorbic
acid, and ɑ-tocopherol (Shibata et al. 2008).
Inflammation is the crucial first step in fighting in-
fection, healing wounds, and it is a defense mechanism
aimed to protect tissue, and thus, during this reaction,
various dynamic pathological changes take place. Many
adverse effects are related to the use of non-steroidal
anti-inflammatory drugs (NSAIDs) and opioids; there-
fore, it is an urgent need to develop new drugs with
lesser or no side effects. In this context, the focus has
been aimed towards natural products (Sengar et al.
2015). Thus, compounds derived from marine algae
are recently being explored since they are known to
possess anti-inflammatory benefits (D’Orazio et al.
2012; Fernando et al. 2016). A recent investigation on
the uptake and the systemic effect of phlorotannins is
available (Corona et al. 2016), but the bioavailability
and the metabolism of phlorotannins still remain a sub-
ject of debate. There are few studies in the implication
of phlorotannins on in vivo pathways and to determine
whether these in vitro effects occur in vivo and are
related to their antioxidant properties (Wells et al.
2017). To the best of our knowledge, there were no
studies available in the literature describing the extrac-
tion, the evaluation of the in vitro antioxidant activity,
and the in vivo pharmacological potential of
phlorotannin-rich fraction derived from brown seaweeds
from the Tunisian coast.
We reported in this work the phytochemical analysis and
the evaluation of antioxidant, anti-inflammatory, and
antinociceptive potential of phlorotannin-rich fractions from
three brown seaweeds collected along Mediterranean Tunisian
coasts: Cystoseira sedoides (Fucales), Cladostephus
spongeosis (Sphacelariales), and Padina pavonica
(Dictoyales).
Materials and Methods
Samples
Seaweed samples of Cystoseira sedoides,Cladostephus
spongeosis, and Padina pavonica were collected from the
northwestern coast of Tunisia, in July 2015. The taxonomic
identification was confirmed by the National Institute of
Marine Sciences and Technologies, Salambôo, Tunisia. The
fresh biomass was then cleaned, in order to remove epiphytes,
salt, and sand, afterward, it was repeatedly washed with dis-
tilled water prior to their shade drying. The dried material was
powdered (250–500 μm) and kept at 4 °C until use.
Chemicals
All reagents were purchased from Sigma Chemical Co (St.
Louis, MO, USA): standards (gallic acid, phloroglucinol, vi-
tamin C, quercetin, diclofenac, acetylsalicylate of lysine and
tramadol), carrageenan, Folin–Ciocalteu phenol reagent, free
stable radicals 2,2-diphenyl-1-picrylhydrazyl (DPPH) and
2,2′-azinobis-3-ethylbenzthiazoline-6-sulphonic acid
(ABTS), dimethoxy-benzaldehyde (DMBA), and
malondialdehyde (MDA).
Extraction of Phlorotannins
After optimizing the extraction conditions, solid-liquid extrac-
tion (SLE) was performed. One hundred grams of the fine-
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powdered material of each algae species were extracted with
500 mL of ethanol/water (50:50, v/v) under constant shaking
for30minat50°C;Kangetal.(2012a) previously found that
phlorotannins have been shown to be thermally stable at this
temperature. The supernatant was collected and the residue was
re-extracted three times under the same conditions as men-
tioned previously. Oxidation was prevented by adding 0.3%
(w/v) of the ascorbic acid to the aqueous phase (step 1) in order
to prevent oxidation, and it is not extracted with the ethyl ace-
tate (step 3). As previously reported by Koivikko et al. (2007),
the addition of the anti-oxidant agent is suggested to only in-
crease the stability of phlorotannins. The combined filtrate was
then concentrated under vacuum below 40 °C to a volume of
300 mL (Martínez et al. 2013). The purification process was
then carried out as reported by Stiger-Pouvreau et al. (2014)
with slight modifications. This procedure consists of three steps
as illustrated in Fig. 1. The aqueous phase was partitioned two
times with petroleum ether (PE) (1:1, v/v)(Shibataetal.2004).
Next, two dichloromethane (DCM) (1:1, v/v) washing were
applied. Finally, to increase the phlorotannin content, a liquid
partition was conducted three times with ethyl acetate (1:1, v/v)
and permitted to obtain an EtOAc fraction which has the char-
acteristics of the phlorotannin-richfractionasreportedbyLi
et al. (2009). The aqueous fraction served for the determination
of TPC values while the PHT fractions will be used for the
remaining tests.
The extraction yields were expressed as gram of dried res-
idue per 100 g of the dried material (Eq. 1):
Extraction yield %;w=wðÞ
¼Weight of the PHT fraction gðÞ
Weight of the dry material gðÞ100 ð1Þ
Phytochemical Analysis
Total Phenolic Content
The total phenolic content (TPC) was determined using the
Folin–Ciocalteu procedure (Alimi et al. 2011) with the adap-
tation that the absorbance was measured at 725 nm, using a
UV/Vis spectrophotometer (Thermo Scientific™Evolution
201). A calibration curve was prepared with a standard solu-
tion of phloroglucinol monomer (de Quirós et al. 2010;
Keyrouz et al. 2011), and results were given as milligrams
of phloroglucinol equivalents per gram of dry material
(mg PGE/g Dm).
Fig. 1 The SLE and the
purification procedure of
phlorotannins from three brown
seaweeds
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Dimethoxy-Benzaldehyde Assay
The DMBA assay was conducted as described previously
(Hagerman 2002; Montero et al. 2016) with the slight adapta-
tion that after 60 min, the absorbance was determined at
510 nm using a UV/Vis spectrophotometer (Thermo
Scientific™Evolution 201). Results were given as milligrams
of phloroglucinol equivalents per gram of dry material
(μg PGE/g Dm).
Antioxidant Activity of Seaweed Phlorotannins
The three seaweed species were tested for their in vitro anti-
oxidant activity by using three different assays. Different con-
centrations of PHT fractions were used, and standards were
also taken in the same concentration range.
Determination of Total Antioxidant Activity The total anti-
oxidant activity (TAA) of the seaweed phlorotannins
was evaluated by phosphomolybdenum method accord-
ing to the procedure described by Prieto et al. (1999).
The TAA was standardized against gallic acid (y=
0.00183x + 0.005, R
2
= 0.9997), and results were
expressed as milligrams of gallic acid equivalent per
gram of dry material (mg GAE/g Dm).
Radical Scavenging Activity Radical scavenging activities
were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging activity assay and 2,2′-Azino-bis-3-ethyl-
benzothiazoline-6-sulfonic acid (ABTS) cation decolorization
method. Quercetin and ascorbic acid were used as positive
control, and results were expressed as half maximal inhibitory
concentration (IC
50-DPPH
and IC
50-ABTS
,μg/mL).
DPPH free radical scavenging activity (RSA %)
The free radical scavenging activity of PHT fractions was
measured by the DPPH assay following the method of Brand-
Williams et al.(1995)modifiedbySharmaandBhat(2009).
The absorbance was measured at 517 nm on a UV/Vis spec-
trophotometer (Thermo Scientific™Evolution 201). The abil-
ity to scavenge the DPPH radical was calculated using the
following equation (Eq. 2):
RSA%¼1−
Abssample−Abssample blank
Abscontrol
100 ð2Þ
Where the Abs
control
is the absorbance of the control (DPPH
solution without sample), the Abs
sample
is the absorbance of
the test sample (DPPH solution plus test sample), and the
Abs
sample blank
is the absorbance of the sample only (sample
without the DPPH solution).
ABTS radical cation decolorization assay
The ABTS test was carried out as described by Re et al.
(1999). The absorbance was read at 734 nm using a UV/Vis
spectrophotometer (Thermo Scientific™Evolution 201), and
results were expressed as scavenging activity (RSA %) using
Eq. (2).
Pharmacological Evaluation
Animals
Experiments were conducted using adult Wistar rats (150–
170 g) and Swiss mice (20–25 g) of both sex and of approx-
imately 6–8 weeks old. Animals were obtained from the
Pasteur Institute (Tunis, Tunisia). They were kept in polypro-
pylene cages at 25± 2 °C. Balanced pellet diet and water were
supplied ad libitum.All animals were treated in accordance
with guidelines established by the European Union regarding
the Use and the Animal Care (CCE Council 86/609).
Evaluation of the Anti-Inflammatory Activity
Xylene-Induced Ear Edema in Mice Ear edema was induced
according to the procedure previously described by Kou et al.
(2005), with slight modifications. Swiss mice were assigned
into groups of six mice each. Group 1 which served as the
control group received 2.5 mL/kg of the vehicle, while the
reference group (group 2) received dexamethasone subcuta-
neously (s.c) at the dose of 5 mg/kg. Groups 3–8received50
and 100 mg/kg (s.c) of PHT-SED, PHT-CLAD, and PHT-
PAD, respectively. Thirty minutes after, 30 μLofxylenewere
applied to the anterior and posterior surfaces of the right ear
while the left ear was used as the control. Three hours after
xylene application, the thickness of the ear was determined
using a Digital Metric Micrometer (Blue-Point®). The mean
of the difference between the right and the left ears (ΔE)was
determined for each group, and the inhibition level (%) was
calculated according to the following equation (Eq. 3):
Inhibition %ðÞ¼1−
ΔEt
ΔEc
100 ð3Þ
Where
E
t
average edema in the treated groups (2–8, and
E
c
average edema in the untreated group (control group).
Carrageenan-Induced Paw Edema in Rats Effects of PHT frac-
tions on carrageenan-induced paw edema in rats were per-
formed according to Winter et al. (1962). Rat paw edema
was induced in the left hind by subplantar injection of
0.05 mL of carrageenan suspension (1% w/v,in0.9%saline
solution) and these 30 min after intraperitoneal (i.p)adminis-
tration of reference drug or samples. Rats were arbitrarily al-
located into eight treatment groups of six (n=6)asfollows:
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(i). Group 1: negative control, received the vehicle (2.5 mL/
kg, i.p)
(ii). Group 2: positive control, received the reference drug
(diclofenac, 25 mg/kg, i.p)
(iii). Group 3–4: pretreated with PHT-SED (50 and 100 mg/kg,
i.p)
(iv). Group 5–6: pretreated with PHT-CLAD (50 and 100 mg/kg,
i.p)
(v). Group 7–8: pretreated with PHT-PAD (50 and 100 mg/
kg, i.p).
We assessed the intensity of the edema development of
each animal by means of a volume-displacement technique,
using a plethysmometer (model 7150, Ugo Basile, Italy) im-
mediately before carrageenan injections and was then mea-
sured hourly during 5 h after injections. The percentage of
inhibition of paw edema for each group was calculated as
follows (Eq. 4):
%Inhibition of edema ¼Vt−V0
ðÞ
control−Vt−V0
ðÞ
treated
Vt−V0
ðÞ
control
100 ð4Þ
Where
V
t
is the average volume for each group at the indicated t
time, after carrageenan injection and
V
o
is the average volume obtained for each group at t
0,
before the beginning of the challenge.
Determination of Malondialdehyde in Liver Tissues For the
MDA assay, liver tissues were collected 5 h after carrageenan
injection. The formation of MDA was evaluated by the thio-
barbituric acid-reacting substance (TBARS) as described by
Zaoualí et al. (2011). Results were expressed as nanomoles
per milligram protein.
Evaluation of the Antinociceptive Activity
Abdominal Constriction Test The abdominal constriction test
induced by acetic acid stimulation in mice was conducted as
described by Koster et al. (1959). Adult Swiss mice used for
this study were randomized into groups of six mice per cage.
In brief, samples of phlorotannins or the vehicle were subcu-
taneously (s.c) administered 30 min prior to acetic acid injec-
tion. Acetylsalicylate of lysine (ASL) used as a reference drug
at the dose of 200 mg/kg was administered, by the same route,
to mice. Immediately after intraperitoneal injection of 10 mL/
kg of acetic acid (1%, v/vin normal saline 0.9%), animals
were isolated in a transparent observation box.Following that,
the number of abdominal constrictions episodes which
consisted of contraction of the abdominal area, with the
extension of the hind limb, was recorded during 30 min. The
antinociceptive activity (reduction in the number of writhes)
was expressed as percentage of pain inhibition in treated mice
(Eq. 5):
Inhibition %ðÞ¼Mean of writhesðÞ
control−Mean of writhesðÞ
treated
Mean of writhesðÞ
control
100 ð5Þ
Hot-Plate Test The central antinociceptive activity was evalu-
ated by the hot-plate test in mice following the method of
Özkay and Can (2013). The mice were placed on a hot-plate
analgesiameter (Ugo Basile, Italy, no. 35100), which was
maintained at 55 ± 0.5 °C. A sensitivity test was carried out
before the experimental assays, and only the animals reacting
within 15 s were selected (Wilson et al. 2003). Maximum
cutoff time was chosen as 50 s to prevent tissue damage.
Response latencies were recorded and were defined as the
time between the placement and the response, i.e., mice per-
formed either licking or shaking the hind paws or eventually
jumping. Mice were divided into nine groups, six mice each
(n= 6). The control group was pretreated by s.c injection with
10 mL/kg of 0.9% saline. Two groups received the reference
drug: tramadol at the dose of 25 and 50 mg/kg by the same
route. The remaining groups were treated with PHT fractions
at the dose of 50 and 100 mg/kg, respectively. The latency
time was barely recorded before challenge and at various time
points after injection of saline or the tested compounds (30,
60, 90, and 120 min). The elongation in the response latencies
is considered as an antinociception parameter.
Statistical Analysis
All experiments of phytochemical analysis and of the antiox-
idant activity were performed in triplicate. The results of these
experiments and those of pharmacological evaluation were
expressed as mean ± SEM. Data were analyzed by one-way
or two-way analysis of variance (ANOVA) followed by the
post hoc Tukey’s test or Fisher’s LSD test as appropriate for
multiple comparisons. The pvalues less than 0.05 (p<0.05)
were considered statistically significant. The IC
50
values were
calculated with polynomial curve fit between the sample con-
centrations and the percentage of scavenging using GraphPad
Prism version 6.0 (GraphPad Software, CA, USA).
Results
Evaluation of Phenolic Contents
Results of extraction yields, TPC, and phlorotannin content
(PHT content) are summarized in Table 1. Significant differ-
ences were found in the extraction yield among the three sea-
weed species, ranging from1 to 6.8%. The quantification of
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TPC and PHT in the three species was based on the standard
curve of phloroglucinol. A strong and positive correlation (r=
0.9997, p<0.0001) was established between the absorbance
and the concentrations ranging from 0.015 to 0.500 mg/mL.
As shown in Table 1, the total phenolic content varied among
the three species: C. sedoides had the highest mean of TPC
(26.45 mg PGE/g Dm) followed by C. spongeosis
(10.91 mg PGE/g Dm) and P. pavonica (7.06 mg PGE/g
Dm). Similar trends were observed in PHT content: the
highest phlorotannins level occurred in the same species,
and it was ranged from 873.14 μg PGE/g Dm in
C. sedoidesto 56.68 μg PGE/g Dm in P. pavonica.
Antioxidant Properties of PHT Fractions
The antioxidant activity of PHT fractions was assessed by
three different methods. The correlation analysis using the
Pearson’s correlation coefficients (r) indicated that the antiox-
idant activity, as measured bythe phosphomolybdenum assay,
positively correlated with the PHT content at r=0.943. The
TAA of C. sedoides was found to be the highest among the
three species of brown algae (71.30 mg GAE/g Dm),followed
by C. spongeosis (26.13 mg GAE/g Dm) as shown in Table 2.
In order to confirm the potent antioxidant activity of
C. sedoides, we investigated the radical scavenging activity
on two types of free radicals.
Free Radical Scavenging Activity
The scavenging activity is the ability of the compound to be
reacted preferentially with free radicals. Phlorotannin-rich
fractions play here the role of scavenger against these radicals.
DPPH and ABTS scavenging activities of the three fractions
expressed as IC
50
values (concentration of the fraction re-
quired for 50% inhibition) are illustrated in Fig. 2. Lower
IC
50
concentrations indicate higher scavenging activity. The
antioxidant effect by DPPH assay was strongly correlated with
the results detected by ABTS assay (r=0.952,p<0.05).As
shown in Table 2, the three fractions have different reactivity
towards free radicals. C. sedoides is more reactive than
C. spongeosis which is more reactive than P. pavonica.The
highest scavenging activity, with relatively low IC
50
values,
occurred for C. sedoides in both DPPH and ABTS assay
(27.7 μg/mL for the DPPH assay and 19.19 μg/mL for the
ABTS assay). In the ABTS assay, this PHT fraction had a
relatively higher scavenging ability similar to quercetin and
vitamin C used as standard antioxidant in this study (19.21
and 18.06 μg/mL, respectively). The radical scavenging ac-
tivity was correlated negatively with the PHT content of the
three fractions, whatever the radical used, suggesting that
Fig. 2 Radical scavenging activity (RSA-IC
50
) of PHT fractions from the
three seaweeds by DPPH and ABTS assays in comparison to quercetin
and vitamin C
Table 2 The total antioxidant activity and radical scavenging activity
on DPPH and ABTS of the phlorotannin-rich fractions from C. sedoides,
C. spongeosis,andP. pavonica in comparison to standards (quercetin and
vitamin C)
Samples Antioxidant assays
TAA
a
RSA
b
,IC
50-DPPH
RSA
b
,IC
50-ABTS
PHT-SED 71.30 ± 1.68 27.71 ± 1.59 19.19 ± 2.36
PHT-CLAD 26.13 ± 2.32 41.92 ± 2.13 36.83 ± 1.98
PHT-PAD 14.69 ± 1.82 91.78 ± 1.98 75.55 ± 2.55
Standards
Quercetin 4.36 ± 2 19.21 ± 1.5
Vitamin C 11.30 ± 0.5 18.06 ± 0.23
Values are expressed as mean ± SEM of three parallel measurements
a
Total antioxidant activity (mg GAE/g dry material)
b
Radical scavenging activity—IC
50
(μg/mL)
Table 1 Yields obtained, total phenolic content, and phlorotannins
content of the three brown algae: C. sedoides,C. spongeosis,and
P. p av o n i c a
Algal species Yields
a
TPC
b
PHT content
c
C. sedoides 1 ± 0.32 26.45 ± 1.25 873.14 ± 3.5
C. spongeosis 2.65 ± 0.75 10.91 ± 1.57 112.23 ± 2.86
P. p av o n i c a 6.8 ± 0.5 7.06 ± 2.52 56.68 ± 3.37
Values are expressed as mean ± SEM of three parallel measurements
a
Yields of extraction (% of the dry material)
b
Total phenolic content (mg PGE/g dry material)
c
Phlorotannin content (μg PGE/g dry material)
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most of the antioxidant capacity was directly proportional to
the amount of phlorotannins.
The Anti-Inflammatory Potential of PHT Fractions
In order to investigate the in vivo anti-inflammatory efficacy
of the three PHT fractions, two different models were
employed: xylene-induced ear edema in mice and
carrageenan-induced paw edema in rats.
Effect of Phlorotannin Fractions on Xylene-Induced Ear
Edema in Mice
The acute anti-inflammatory activity of the seaweeds was
evaluated by xylene-induced ear edema in mice model. As
shown in Fig. 3, the topical application of xylene on the right
ear of the control group produced an increase in the average of
the ear thickness. The administration of PHT fractions, 30 min
before xylene application, significantly suppressed the ear
edema when compared to the control group (p< 0.001).
PHT-SED and PHT-CLAD exerted a dose-dependent anti-in-
flammatory effect with the highest inhibition percentage at the
dose of 100 mg/kg (82.55 and 68.60%, respectively). Both
also showed a high anti-inflammatory effect compared with
those of the reference drug dexamethasone which the effect
did not exceed53.49%. However, PHT-PAD (50 and 100 mg/
kg) did not exhibit a considerable anti-inflammatory potential
in this model. Differences in the anti-inflammatory activity
can be explained, at least partially, by the total amount of
phlorotannins present in samples.
Effect of PHT Fractions on Carrageenan-Induced Paw Edema
in Rats
Results of the carrageenan-induced edema are summarized in
Table 3. The subplantar injection of carrageenan suspension
(0.05 mL/paw) to the control group induced an increase of the
paw volume within 1 h, and the inflammatory edema reaches
its maximum level at the third hour, and after that, it starts
declining. A similar observation concerning the paw edema
development was previously reported by Posadas et al. (2004)
and Cong et al. (2015). In the other hand, diclofenac used as a
standard anti-inflammatory drug showed, at the dose of
25 mg/kg, a significant reduction in paw thickness, which
begins to decrease 3 h after, but it has a maximum effect at
5h(p< 0.001) by a suppression of the edema up to 54.51%.
When looking at the percentage of inhibition of inflammation
in the experimental groups and the positive control (Table 3),
it was clear that 5 h after initiation of the experiment, the
percentage of inhibition increased in a dose-dependent man-
ner with a maximum effect at the dose of 100 mg/kg. PHT-
SED has a higher effect than the standard drug by preventing
edema at all intervals when compared to the control group:
pre-treatment with PHT-SED at the dose of 50 and 100 mg/kg
significantly prevented the carrageenan effect by 65.09 and
81.08% reduction in edema, respectively, and this 5 h after
injection. Although C. spongeosis and P. pavonica have sup-
pressed the increase in paw thickness at all intervals, a low
dose of 50 mg/kg showed less efficacy with inhibition of
edema of 41.55 and 32.09%, respectively. However, at the
dose of 100 mg/kg, the two fractions displayed higher activity
when compared to diclofenac. As shown in Table 3,PHT-SED
exhibited the higher anti-inflammatory properties with
Fig. 3 Anti-inflammatory effect
of PHT fractions on xylene-
induced ear edema in mice in
comparison to the reference group
dexamethasone (Dexa). Data are
expressed as mean ± SEM (n=6).
*p<0.01,**p<0.001when
compared with the control group
(vehicle). Statistical analysis was
performed using one-way analy-
sis of variance followed by
Tukey’s post hoc test
Mar Biotechnol
maximal inhibition effect at the dose of 100 mg/kg. Therefore,
this dose was selected to examine possible involvement of
MDA in preventing the inflammatory response towards carra-
geenan injection.
Effect of PHT-SED Fraction on MDA Production in Liver
Tissues
To confirm the antioxidant effect of PHT-SED, we set
out to measure the MDA level by the means of TBARS
method in liver tissues. As shown in Fig. 4,the
subplantar paw injection of carrageenan induced a
marked increase of MDA concentration. As expected,
the MDA level remarkably increased (3.16 ±
0.19 nmol/mg protein) in the carrageenan group, com-
pared to the control group (0.26 ± 0.06 nmol/mg pro-
tein). However, pre-treatment with both PHT-SED
(100 mg/kg) and diclofenac (25 mg/kg) significantly de-
creased the MDA production (1.32 ± 0.3 and 0.94 ±
0.2 nmol/mg protein, respectively).
Table 3 Effect of phlorotannin-rich fractions from C. sedoides,C. spongeosis,andP. pavonica on carrageenan-induced paw edema in rats in comparison to the reference drug, diclofenac
Samples Dose (mg/kg) Edema (10
−2
mL) Edema inhibition (%)
1h 3h 5h 1h 3h 5h
Control –45 ± 2.45 71.33 ± 1.81 74 ± 0.95 –––
Diclofenac 25 24.66 ± 1.03* 35.83 ± 1.72** 33.66 ± 2.6** 45.20 49.77 54.51
PHT fractions
PHT-SED 50 19.83 ± 1.33** 28.5 ± 1.04** 25.83 ± 1.16** 55.93 60.04 65.09
100 9.5 ± 1.64** 16.75 ± 0.89** 14 ± 1.21** 78.89 76.52 81.08
PHT-CLAD 50 21.25 ± 2.43** 42.75 ± 1.05** 43.25 ± 3** 52.78 40.07 41.55
100 15.75 ± 1.78** 22.75 ± 4.2** 21 ± 2.34** 65 68.11 71.62
PHT-PAD 50 36.25 ± 2* 49.25 ± 237* 50.25 ± 1.89* 19.44 30.95 32.09
100 20 ± 3.4* 32.33 ± 2.56* 30 ± 2.63* 55.56 54.68 58.11
Data are expressed as mean ± SEM; n=6animals.*p< 0.01, **p< 0.001 when compared with the control group
Fig. 4 Effect of PHT-SED (100 mg/kg; s.c) and diclofenac (Diclo,
25 mg/kg; s.c) on carrageenan-induced MDA production in rat paw
edema model. The concentration of MDA was measured in liver
samples 5 h after carrageenan injection. Each point represents the
mean ± SEM values obtained from six animals. ####p< 0.0001
compared with the control group (vehicle); ****p<0.0001
compared with the carrageenan group (vehicle + carrageenan).
Statistical analysis was performed using one-way analysis of
variance followed by Tukey’s post hoc test
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Antinociceptive Potential of PHT Fractions
Visceral Antinociceptive Effect
Table 4shows that pre-treatment of animals with PHT frac-
tions, by the s.c. route, significantly reduced the number of
writhing at two different doses when compared to that of the
control group (p< 0.001). The maximum effect was observed
at the dose of 100 mg/kg: the percentages of inhibition of
writhing were 65.15, 74.18, and 90.16% for PHT-PAD,
PHT-CLAD, and PHT-SED, respectively. Likewise, ASL
was used as the reference drug, reduced the writhing up to
57.79%.
Thermal Antinociceptive Effect
The thermal antinociceptive potential of samples was evaluat-
ed by the hot-plate test in mice. Tramadol used as reference
drug showed a maximum latency time of 23.43 and 35.16 s
after 30 min at the dose of 25 and 50 mg/kg, respectively. In
the other hand, PHT-SED and PHT-CLAD showed a maxi-
mum reaction 90 min after and this at the dose of 100 mg/kg
while PHT-PAD did not act in this test (Fig. 5). The observed
effect was found to be more pronounced (p<0.001)inmice
treated with the PHT-SED fraction (50 and 100 mg/kg) which
was quite comparable with the standard, tramadol. This effect
was long lasting and still observable 120 min after the treat-
ment, while tramadol showed a rapid action but it is short
compared to PHT-SED groups. So, a combination between
tramadol (25 mg/kg) and PHT-SED (50 and 100 mg/kg) was
investigated (25T+ 50S and 25T+100S). Figure 5illustrates a
long-lasting antinociceptive effect after the co-administration
of tramadol and PHT-SED with maximum prolongation of
latency 60 min following the treatment (37.35 ± 1.78 and
46.7 ± 1.14 s, for 25T+ 50S and 25T+ 100S, respectively).
Moreover, the combination of both agents showed better an-
algesic potential than that of each single treatment.
Discussion
In addition to nutritional values of seaweeds, they are being
increasingly marketed as functional foods or nutraceuticals:
terms which describe foods that contain bioactive compounds
(phytochemicals) that may benefit health (Wells et al. 2017).
The type of the solvent used as well as the extraction approach
Table 4 Antinociceptive effects of phlorotannin-rich fractions from
C. sedoides,C. spongeosis,andP. p a v o n i c a using the writhing test in
mice in comparison with the reference drug, acetylsalicylate of lysine
(ASL)
Samples Dose
(mg/kg)
Number of writhing
behavior
Inhibition of
writhing (%)
Control –61 ± 1.09 –
ASL 200 25.75 ± 3.09*** 57.79
PHT fractions
PHT-SED 50 21.25 ± 2.06*** 65.16
100 6 ± 2.64*** 90.16
PHT-CLAD 50 31.7 ± 3.5*** 48.03
100 15.75 ± 0.5*** 74.18
PHT-PAD 50 42.33 ± 2.51*** 30.6
100 21.25 ± 1.5*** 65.16
Data are expressed as mean ± SEM; n= 6 animals. ***p<0.0001when
compared with the control group
Fig. 5 Antinociceptive effect of
PHT fractions and tramadol (25
and 50 mg/kg) on a hot-plate test
in mice. Different groups (n=6
mice) were compared to the con-
trol (vehicle). Data were
expressed as mean ± SEM:
*p<0.05,**p<0.001,
***p< 0.0001. Statistical analysis
was performed using one-way
analysis of variance followed by
Tukey’s post hoc test
Mar Biotechnol
have an important impact on the extraction yield and the
resulting antioxidant potencies. This is due to the presence
of different antioxidant compounds of varied chemical char-
acteristics and polarities that may or may not be soluble in a
particular solvent (Sultana et al. 2009). In our study, an extrac-
tion of phlorotannins from three Mediterranean seaweeds was
made using an aqueous-ethanol mixture. Ethanol and water
were chosen mainly because of their lower toxicity, high ex-
traction yield, and in advances their polarity (Sultana et al.
2007; Norra et al. 2016).
The results in Table 1show that the extraction yields
ranged from 1 to 6.8% are comparable to the results obtained
in previous reports of Phaeophyceae species. In fact,
phlorotannins have a wide range of molecular mass (400–
400,000 Da) and can occur in variable concentrations (0.5–
20% Dm) in brown seaweed (Balboa et al. 2013). Variation in
the phenolic content among the three PHT fractions was quite
large (Table 1) and is probably attributed to different parame-
ters such as algae size, age, tissue type, salinity, nutrient levels,
and intensity of herbivory (Lopes et al. 2012). Although the
quantification of the polyphenol content using Folin–
Ciocalteu’s reagent is a well-known and documented assay,
this method is unspecific for polyphenols since it is based on
the reductive potential of compounds which may react with
the reagent, and for that reason, it does not always reflect the
biologicalactivity or the reactivity of a phlorotannin-rich sam-
ple (Parys et al. 2007; Heimler et al. 2009;Crucesetal.2016).
As it can be seen in Table 1, TPC values were larger than
extraction yields. In fact, TPC values were obtained from the
Folin–Ciocalteu assay which was assigned using the aqueous
fraction while the extraction yields were calculated in the base
of the EtOAc fraction obtained at the end of the extraction
procedures. This fraction contains only phlorotannins, while
in the aqueous phase, other phenols may be present (e.g.,
flavonoids).
The2,4-dimethoxy-benzaldehyde (DMBA) assay is based
on the ability of DMBA to react specifically with 1,3- and
1,3,5-substituted phenols. Our results showed that the PHT
content is quite large between the species and Cystoseira
sedoides had the higher value (Table 1). Similar to our find-
ings, Lopes etal. (2012) pointed out a significant difference in
terms of PHT content among brown seaweeds and reported
values ranged from 288.2 to 815.82 mg PGE/kg Dm for the
Cystoseira genus.
Most of the studies on the antioxidant capacities of
phlorotannins involved so far either a single dosing technique
or more to accurately reflect all the antioxidants. The use of
several methods is recommended to initiate a more or less
complete antioxidant profile and in order to better understand
the mechanism of the anti-oxidative potential of the seaweed
extract(Blancetal.2011; Peinado et al. 2014). Furthermore,
varieties of in vitro techniques have been developed because
antioxidant mechanisms are diverse. The response of
antioxidants depends on factors such as the solvent and sub-
strate used inthe test and the affinity between the substrate and
the antioxidant (Sun et al. 2011). The antioxidant ability of the
three fractions was assessed by three different methods. The
total antioxidant capacity data reported in Table 2show that
the PHT-SED possessed a greater antioxidant ability than the
other seaweeds which is probably due to the highest TPC data
of this fraction. Moreover, there was a significant correlation
between antioxidant activity and phenolic content of these
three species and this finding is in agreement with previous
studies (Matanjun et al. 2008).
The three PHT fractions were assayed as scavenger com-
pounds against DPPH and ABTS radicals. The DPPH assay is
routinely practiced for assessment of free radical scavenging
potential of an antioxidant molecule and is considered as one
of the standards and easy colorimetric methods for the evalu-
ation of antioxidant properties of pure compounds and natural
antioxidants (de Alencar et al. 2016). On the other hand, the
ABTS decolorization assay is used for both hydrophilic and
lipophilic antioxidants (Alam et al. 2013). Phlorotannins are
the unique polyphenols of brown algae to whom the antioxi-
dant potential is mainly attributed, since they are acting as
hydrogen and electron donating agent (Ahn et al. 2007).
Other abundant compound such as sulfated polysaccharides
and carotenoids could possibly be co-extracted and might also
support its antioxidant activity (Tenorio-Rodriguez et al.
2017). However, there are many literatures on the strong cor-
relation between the antioxidant properties and the
phlorotannins content of Phaeophyceae (Connan et al. 2006,
2007; Breton et al. 2011; Heffernan et al. 2015). Phlorotannins
exhibit defensive or protective functions against oxidative
stress, and thus, Ecklonia cava, a brown seaweed, is actually
known for its protective effects against intracellular ROS ac-
cumulation as reported by Kang et al. (2012b).
Antioxidants are known to exhibit anti-inflammatory ac-
tion. To date, various in vitro studies have been assessed to
the anti-inflammatory activities of phlorotannins but few
in vivo studies were conducted (Sugiura et al. 2013). In the
current work, two in vivo models have been used to evaluate
the eventual anti-inflammatory effect of the three PHT frac-
tions. The xylene-induced ear edema in mice is one of the
appropriate models for acute inflammation. The application
of xylene induces neurogenous edema, which is partially as-
sociated with the substance P that acts as a neurotransmitter or
neuromodulator in several physiological processes. In the pe-
riphery, the release of substance P from sensory neurons leads
to vasodilatation and plasma extravasations, which causes ear
swelling in mice (Bai et al. 2017). The inhibitory effect of the
three brown seaweeds, tested on xylene-induced mouse ear
edema thickness, shows that C. sedoides and
C. cladostephus attenuated the inflammation in a dose-
dependent manner, which is comparable to dexamethasone
(Fig. 3). Similarly, the effect of four phlorotannins earlier
Mar Biotechnol
isolated from Eisenia arborea on mouse ear edema was eval-
uated by several inflammatory inducers and they exerted the
strongest inhibition of ear damage up to 79.8% (Sugiura et al.
2013). This inflammatory effect is in part due to the structure
of phlorotannin components as well as their remarkable anti-
oxidant abilities. Thus, Kim and Kim (2010) reported that
phloroglucinol, a monomer of phlorotannins, derived from
Ecklonia cava has an anti-inflammatory effect in addition to
its radical scavenging activity. The antioxidant mechanism of
phloroglucinol can be attributed to three hydroxyl groups
existed in phloroglucinol that can react with ROS. It may be
suggested that the structure–activity relationships mainly de-
pend on the number of phenolic ring substituents and phenyl
ether linkages. In addition, evidences are obtained from liter-
ature and state that phlorotannins are recognized as potent
anti-inflammatory agents (Wijesekara et al. 2010; Kim et al.
2012;Yangetal.2016).
The administration of PHT fractions prevented significant-
ly carrageenan-induced paw edema in a dose-related manner
with a long-lasting effect (Table 3). However, the PHT-PAD
has a more significant action when administered at the dose of
100 mg/kg. The carrageenan-induced paw edema was per-
formed in rats. Carrageenan is a phlogistic agent not known
to possess systemic effect. It is used as a standard experimen-
tal model because of its high degree of reproducibility in acute
inflammation (Ananthi et al. 2011). Carrageenan-induced paw
edema involves several chemical mediators, including hista-
mine, serotonin, bradykinin, and prostaglandins. In this mod-
el, the edema isbelieved to be biphasic: The first phase begins
immediately after injection of carrageenan and lasts for 2 h,
while the second phase begins at the end of the first phase and
remains through three to 5 h. The earlier phase being mediated
by the release of histamine, serotonin, and similar substances,
followed by the subsequent release of kinin-like substances,
i.e., prostaglandins (mainly prostaglandin E2), proteases, bra-
dykinin, and nitric oxide (NO) pathway. The late edema phase
is dependent on cytokine production by resident cells and
neutrophil infiltration (Eddouks et al. 2012; Brito et al.
2013; Sengar et al. 2015). Several in vitro researches suggest
that phlorotannin-rich fractions exhibited anti-inflammatory
activity in lipopolysaccharide (LPS) stimulated RAW-264.7
macrophages through a variety of mechanisms, including the
inhibition of LPS-stimulated NO production, the suppression
of inducible NO synthase (iNOS) and cyclooxygenase 2 ex-
pression, (COX-2), and the reduction of tumor necrosis factor
alpha and interleukin-6 secretion levels (Jung et al. 2013;
Murray et al. 2017). Likewise, previous studies have demon-
strated that Eckol, a phlorotannin compound isolated from
Eiseniabicyclis, presents a protective effect against LPS-
stimulated inflammation in HepG2 cells (Kang et al. 2013b).
Our results revealed that PHT fractions possess potent activity
on the acute phase of inflammation, possibly due to the inhi-
bition of release of inflammatory mediators.
On the other hand, a positive correlation between the PHT
content and the antioxidant activity was found (r=0.943)
which suggests that phlorotannins may possess a strong
ROS scavenging ability. Cruces and co-workers (2016)previ-
ously reported that polyphenols contain hydroxyl groups
which may act as reducing agents, hydrogen donors, and sin-
glet oxygen. An overproduction of ROS is involved in many
pathologies such as Crohn’s disease, asthma, and chronic pan-
creatic highlighted a cross talk between inflammation and ox-
idative stress (Silva et al. 2017). Lipid peroxidation response
is an indicator of oxidative damage which is mainly due to the
attack of plasma membranes by free radicals (Chiu et al.
2012). One of the most widely used methods to evaluate cell
damage by oxidation is to quantify the MDA concentration,
since MDA is the final product of the lipid peroxidation re-
sponse (Jung et al. 2014). In fact, earlier studies reported that
polyphenols belonging to brown algae are inhibitors of lipid
peroxidation (Jung et al. 2014). Likewise, Wei et al. (2003)
found that phlorotannins isolated from Sargassum
kjellmanianum had a strong antioxidative effect in inhibiting
mouse liver lipid peroxidation. According to Fig. 4, a signif-
icant decrease in MDA level was observed after the PHT-SED
treatment. The MDA values were lower in the diclofenac-
treated group; however, this was not statistically significant
(p> 0.05). In this regard, PHT-SAD may protect against
ROS damage by decreasing endogenous biomarker levels in-
volved in carrageenan-induced paw edema, and thus reducing
the inflammatory response.
Most anti-inflammatory agents are also expected to possess
analgesic and antipyretic activities (Sengar et al. 2015).
Therefore, the last issue of this work consisted in the evalua-
tion of a possible analgesic effect of phlorotannins derivatives
from brown algae. The peripheral and central analgesic effects
of the phlorotannin-rich fractions were screened on in vivo
models. The acetic acid-induced abdominal writhing response
in mice was examined. The intraperitoneal injection of acetic
acid, as a chemical noxious agent, causes a response charac-
terized by the contraction of abdominal muscles accompany-
ing an extension of the hind limbs and elongation of the body.
This irritation triggers the release of several mediators espe-
cially prostaglandins (Özkay and Can 2013). ASL, PHT-SED,
PHT-CLAD, and PHT-PAD pretreatment produced a dose-
dependent and significant decrease in acetic acid writhing
(p< 0.0001) (Table 4), plausibly suggesting that the
antinociceptive effect is related to the inhibition of prostaglan-
dins released in response to acetic acid. In order to distinguish
between peripheral and central effect of the different fractions,
the hot-plate test was performed. The hot-plate test is a model
of heat nociception, which is only sensitive to centrally acting
analgesic drugs. PHT-SED, at the dose of 50 and 100 mg/kg,
exhibited significant (p< 0.001) ability to prolong the latency
of response to thermal-induced nociception during the exper-
imental challenge (Fig. 5). PHT-CLAD and PHT-PAD
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showed stronger effect only at the dose of 100 mg/kg. The
evidence obtained in this study further suggests that the
phlorotannin-rich fraction acted through peripheral and central
pathway as antinociceptive agent. Overall antinociceptive
tests, PHT-SED displayed an important potential when com-
pared to the standard drug. From this point, a combination of
tramadol (25 mg/kg) with PHT-SED (50 and 100 mg/kg) was
examined. The obtained results (Fig. 6) showed that this com-
bination increases (p< 0.01) the latency time in comparison to
a single administration of tramadol. This co-administration
can be qualified as an additional effect. Unfortunately, the
exact mechanisms of these effects could not be elucidated
from this observation. Structurally related to codeine and mor-
phine, tramadol belongs to the opiate agonist class with rela-
tively mild side effects possessing both opioid-like and non-
opioid-like properties. Tramadol provides pain therapy at mul-
tiple levels due to its dual mechanism of action: it binds to the
μ-opioid receptor and it inhibits the reuptake of serotonin and
norepinephrine. Tramadol is also a non-competitive NMDA
(N-methyl-D-aspartate) receptor antagonist (Cannon et al.
2010;Vadiveluetal.2017). Previous researches on the reac-
tivity of phlorotannin-rich fractions suggested that the action
of phlorotannins on both COX-2 and NO pathway may rep-
resent a potential central mechanism of action of these com-
ponents, highlighting a possible role in the dopaminergic sys-
tem. In addition, the involvement of the opioid and NMDA
receptors is possible. On the other hand, this effect may result
in enhancing the activity of GABA. According to previous
reports on Ecklonia cava (Cho et al. 2012), phlorotannins
derived from this brown algae possess a sedative effect via
Fig. 6 Antinociceptive effect of
the combination tramadol (25 mg/
kg, s.c.) aPHT-SED (50 mg/kg,
s.c.) and bPHT-SED (100 mg/kg;
s.c.), in the hot-plate test.
*p<0.01,**p< 0.001 compared
to the tramadol group, #p<0.001
compared to the PHT-SED treated
group. Data were expressed as
mean ± SEM (n= 6). Statistical
analysis was performed using
one-way analysis of variance
followed by Tukey’s post hoc test
Mar Biotechnol
positive allosteric modulation of the GABA
A
receptor, but its
mode of action is still controversial and not yet fully
elucidated.
Conclusion
In summary, phlorotannin-rich fractions of the three
Mediterranean brown seaweeds exhibited an interesting phar-
macological potential. They effectively react in the anti-
inflammatory assays by reducing paw edema and ear thick-
ness in a dose-dependent fashion. Moreover, these com-
pounds exhibited potent peripheral and central antinociceptive
effect when studied in mice. This study demonstrated that
phlorotannins have a strong antioxidant activity towards rad-
icals and this may explain in part their pharmacological bio-
activities. The obtained results from the PHT-SED fraction
prove that C. sedoides possesses greater effect than the two
other brown seaweeds and should be a source of new anti-
inflammatory and analgesic drugs. The anti-inflammatory po-
tential of this fraction is in part due to the inhibition of oxida-
tive stress by decreasing the production of MDA. The co-
administration of this fraction with a known opioid drug
(tramadol) showed encouraging results which need to be fur-
ther studied for elucidating its appropriate mechanism of ac-
tion. As a consequence, it is crucial to purify samples, charac-
terize them, and find the individual bioactive phlorotannin
derived from C. sedoides involved in its reactivity.
Funding Information This study was financially supported by the
Ministry of Higher Education and Scientific Research of Tunisia (Grant
no. LR12ES09).
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
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