Physicochemical characterization and
pharmacological evaluation of marine polyphenols
from the brown algae Padina pavonica
Mohammed Alshaikheid1*, Amal Abdelhamid1 and Abderrahman Bouraoui1
1Laboratoire de Développement des Médicaments, Equipe de Pharmacologie marine, Faculté de Pharmacie, Université de Monastir,
5000 Monastir, Tunisie
Mohammed Alshaikheid, Laboratoire de Développement des Médicaments, Equipe de Pharmacologie marine,
Faculté de Pharmacie, Université de Monastir, 5000 Monastir, Tunisie ; Tel: +21673461000; Fax: +21673461830; E-mail:
Received: February 4, 2019; Accepted: March 18, 2019; Published: March 20, 2019
Alshaikheid MD, Abdelhamid A, Bouraoui A (2019) Physicochemical characterization and pharmacological evaluation of marine polyphenols from the brown algae Padina pavonica
J Adv Res Biotech 4(1):1-6. DOI: http://dx.doi.org/10.15226/2475-4714/4/1/00141
Background and Aim: Natural products continue to be a primary
resource in biomedicine and biotechnology. The marine environment is
highly reserve for novel pharmaceutical and medical compounds. The
aim of this work was to identify bioactive components from the brown
seaweed Padina pavonica with specific pharmacological potential.
Methods: In the present study, we investigated the efficacy of
polyphenol fraction from Padina pavonica for in vivo anti-inflammatory
activity using the carrageen an-induced paw edema model in rats and
there in vitro antioxidant activity using two methods: DPPH radical
scavenging assay and ferric reducing antioxidant power (FRAP) .
Results: The polyphenol-rich fraction from the brown seaweed
Padina pavonica exhibits a significant anti-inflammatory activity at the
dose of 100 and 200 mg/kg and the maximum reduction of the edema
was observed at the third hour with 53.49% and 58.6% of inhibition,
respectively. Along with, we were interested in the investigation of
the antioxidant activity. The DPPH radical-scavenging assay shows
that the polyphenol fractions have an interesting cavenging activity
at a low concentration (0.25 mg/mL). In addition, the Ferric reducing
antioxidant power (FRAP) method reveals an antioxidant activity with
IC50 = 0.4 mg/mL.
Conclusions: These findings indicate that the polyphenol fraction
of Padina pavonica is a promising bio source of compounds with
anti-inflammatory and antioxidant potential; this may be useful as a
candidate for the developing of potential therapeutic products.
Keywords: Brown Seaweeds; Padina pavonica; Anti-Inflammatory
Activity; Antioxidant Activity
New trends in the search for active natural compounds with
potential health benefits, focused on some unexplored habitats
especially marine environment. Recently, a huge number of
new compounds have been isolated from marine sources. The
use of these metabolites steadily increased in food, cosmetics,
biotechnology and pharmacy .
Marine brown algae have been investigated for their
promising bioactive components including polyphenols, sulfated
polysaccharides, sterol, pigments and peptides with beneficial
bioactivities such as anti-inflammatory, anti-diabetic, anti-UV
and inhibitory effect on hyaluronidase enzyme . Brown
algae (Phaeophyceae) synthesize unique phenolic compounds
called phlorotannins, through the acetate-malonate pathway.
Phlorotannins are polymers of phloroglucinol monomer units (1,
3, 5-trihydroxybenzene) of different size and composition. These
predominant polyphenols may reach a high percentage of the
algae dry mass (up to 15%) .
In the current study, we first extract polyphenols from
Tunisian brown algae Padinapavonica. Padina pavonica belongs
to the class of Phaeophyceae, the order of Dictyotales, the family
of Dictyotaceae and the genus Padina . Further, the antioxidant
activity of the polyphenol fraction has been evaluated by two
different assays. Along with, the anti-inflammatory potential was
studied using the carrageen an-induced paw edema in rats.
Reagents and chemicals
Organic solvent and standards (Gallic acid, Phloroglucinol≥99%,
Vitamin C, Quercetin and Acetylsalicylate of lysine), Carrageen an,
Folin–Ciocalteu phenol reagent, free stable radicals 2,2-diphenyl-
1-picrylhydrazyl (DPPH) and Dimethoxy-benzaldehyde (DMBA)
were purchased from Sigma Chemical Co (Sigma, St.Louis, MO,
All the organic solvents used in extraction and antioxidant
assays are of analytical grade.
Sample collection and preparation of polyphenol fraction
Padinapavonica was collected from the Mediterranean Sea
in various area of the coastal region of Tabarka, Tunisia in May
2014, at a depth between 1 and 2 m. The taxonomic identification
was confirmed by the National Institute of Marine Sciences and
Technologies, Salambôo, Tunisia.400 g of finely powdered algae
material of Padina pavonica were packed in small bags (5x10
cm) of what man filter paper No1. All bags were sealed and
soaked in Ethanol in water (30% v/v) bath, steeping for 24hr at
room temperature. This was repeated for three times. The 30%
ethanol extract was concentrated in a rotating evaporator (Buchi,
B-480) at low temperature (< 40°C). The crude extract was then
lyophilized to obtain powdered extract, which was stored at
2-8°C until use .
Total phenolic content (TPC)
The total phenolic content of the polyphenol fraction
was determined by the Folin-Ciocalteu method as previously
described by Aroa and co-workers . The absorbance was
measured at 725 nm using a UV/Vis spectrophotometer (Thermo
Scientific™ Evolution 201). A calibration curve of Gallic acid (0.03
to 1 mg/mL) was prepared (y = 0,0014x - 0,0152; R² = 0,9981)
and TPC was expressed as milligram Gallic acid equivalent per
gram of dry material (mg GA/g Dm). All experiments were carried
out in triplicate.
Dimethoxy-Benzaldehyde assay for Phlorotannins
For the DMBA assay, the procedure followed the method
developed . The absorbance was determined at510 nm using a
UV/Vis spectrophotometer (Thermo Scientific™ Evolution 201).
Phloroglucinol (PG) was used as a standard at a concentration
scale between 0.03 to 1mg/mL and a calibration curve was
obtained (y = 0,4968x - 0, 5433 R² = 0, 9533) Results were
given as microgram of phloroglucinol equivalents per gram of
dry material(μg PGE/g Dm).All experiments were performed in
Evaluation of the antioxidant activity
DPPH• radical-scavenging assay
The DPPH scavenging assay was performed using to the
method described by Goulas and Manganaris  with slight
modification. Briefly, Different concentrations (0.03-1 mg/mL)
of the polyphenol fraction were prepared in distilled water. The
reaction started by adding an equal volume of a 2 mM solution
of DPPH in methanol, to each tested samples, positive control
and blank. 30 minutes after incubation in dark, the absorbance
was measured at 517 nm using a UV/Vis spectrophotometer
(Thermo Scientific™ Evolution 201).All assays were performed
in triplicate. The radical scavenging activity of the tested sample,
expressed as percentage of inhibition of DPPH, was calculated
according to the formula:
Where Abs Blank is the absorbance of the DPPH solution
without any test compounds and the Abs sample is the absorbance
of the tested samples or positive control after the reaction takes
Quercetin and Ascorbic acid were used as positive control, and
results were expressed as half maximal inhibitory concentration
Ferric reducing antioxidant power (FRAP)
The ferric reducing power of the polyphenol fraction was
studied following the method described by Oyaizu and coworkers
. Serial dilutions ranging from 0.03 to 1 mg/mL of the
tested samples as well as the standard Gallic acid were prepared
in ethanol. 200μL of each concentration were mixed with 500
μL of sodium phosphate buffer (0.2 M, pH=6.6) and 500 μL of
potassium ferricyanide (1%, v/v). The mixture was incubated in
water bath at 50°C for 20 minutes. Then 500 μL oftrichloro acetic
acid (10%, v/v)were added. The mixtures were centrifuged at
3000 rpm for 10 min. A 500 μL of the obtained supernatant was
mixed with 500 μL of distilled water and 100 μL of ferric chloride
(0.1%, m/v).Samples were incubated in the dark for 30 minutes
and the absorbance was measured at 700 nm using a UV/Vis
spectrophotometer (Thermo Scientific™ Evolution 201).Three
independent assays were performed in triplicate.
The FRAP values were expressed as half maximal inhibitory
concentration (IC50). The lower the IC50, the stronger is the
Hlila Anti-Inflammatory test using carrageen an-induced
paw edema model
Wistar rats (150–170 g) of both sex and of approximately
6–8 weeks old were procured from the Pasteur Institute (Tunis,
Tunisia). Animals were housed in polypropylene cages and
maintained under standard conditions (12h light and dark cycles
at an ambient temperature of 25 ± 2 °C and 45% RH). Balanced
pellet diet and water were supplied ad libitum. All experimental
tests were conducted in compliance with standard guidelines of
the European Union regarding the Use and the Animal Care (CCE
Carrageen an-Induced Paw Edema in Rats
The effect of the polyphenol extract on carrageen an-induced
paw edema in rat model was evaluated as reported before .
The animals were divided into four groups of 6 rats each (n=6).
The control group received the vehicle intraperitoneally (2.5 mL/
kg of saline solution, i.p.). Animals of the control group received
the reference drug (Acetyl salicylate of lysine (ASL), 300 mg/
kg i.p.). The same route with the polyphenol fraction of Padina pavonica
(EtOH-PAD, 100 and 200 mg/kg) pretreated the test
groups. 30 minutes after administration of different substances,
each animal received a sub-cutaneous injection of 0.05 mL of
freshly prepared carrageen an suspension (1%, w/v in 0.9%
saline solution) into the sub-plantar side of the left hind paw. The
paw volume was measured using Plethysmometer (model 7150,
UgoBasile, Italy) before carrageen an injection (V0
) and 1, 2, 3, 4
and 5 h after injection (VT
The percentages of inhibition were then calculated as follows:
Where Vt: is the mean edema volume obtained for each group
at the indicated t time, after carrageen an injection and Vo: is the
mean edema volume obtained for each group at t0before the
phlogistic stimulus injection.
All experimental results were expressed as mean ± SEM. Data
were analyzed by two-way analysis of variance (ANOVA) followed
by the post hoc Turkey’s test or Fisher’s LSD test as appropriate for
multiple comparisons. The p values less than 0.05 (p< 0.05) were
considered statistically significant. Graph Pad Prism (version 6.0
software, CA, USA) was used for all statistical analysis.
Results and DiscussionTop
The extraction of polyphenols from brown algae involves the
use of one or several organic solvent, which can be used alone
or in combination with water. In most cases, alcoholic solvents
have been the most preferred for the extraction of bioactive
compounds from marine algae . However, the extraction
method have shown a marked influence on the yield of different
preserving compounds . In the actual study, a solution of 30%
ethanol was used to extract polyphenols from Padinapavonica
and a total of 23.2 g was obtained with a yield ratio of 5.42 %
of dry material.Similar finding have been reported for the brown
algae Cystoseiracompressa  .Another Tunisian team working
in polyphenols from the Padina pavonica reported a yield of 9%
for the acetonic extract .
Phenolic compounds, including phenols and flavonoids,
are secondary metabolites that have an important role in the
maintenance of the human body. Phlorotannins and fucoxanthin
have been reported to be the major compounds in seaweed
Brown seaweed generally contains higher amounts of
polyphenols in comparison with red and green algae . The
total phenolic content (TPC) was estimated using Folin-ciocalteu
method. As results, the amount of phenolic compound quantified
in a pavonica was 123.33 mg GAE/ g dry material. Chiboub and
co-workers  reported that the TPC of the brown algae Padina pavonica is closely related to the solvent used and the EtOAc
-fraction had significantly higher content of phenolic component
(17.4 mgGAE/g of dried algae).A similar study performed with
three species belonging to brown algae revealed high values of
TPC ranged from 3 to 10% . In addition, algae of the same
genus (Padinaantillarum) contained 24.30 mg GAE/g of phenolic
Phlorotannins derived from brown seaweeds are polymers
of phloroglucinol often extracted in aqueous organic mixture and
are generally quantified using colorimetric assays such as DMBAassay
. In general, phlorotannin content varies from 1 to 10%
of the algal dry mass . In this study, the phlorotannins content
in Padina pavonica was determined using the DMBA-assay with a
phloroglucinol. Padina pavonica collected from the Tunisian coast
in July was analyzed earlier and it was reported that it exhibited
much more phlorotannins with 56.68 μg PGE/g of the dried algae
. This variation confirms the influence of different factors in
phlorotannin levels: higher content of phlorotannin seems to be
influenced mainly by higher temperatures and light exposure, the
solvent used and the extraction method.
The antioxidant activity
Phlorotannins derived from brown seaweed have shown
protective effect against free endogenous radicals responsible for
oxidation damage and were reported as potent antioxidant agent
. Earlierreports on the antioxidant potential of brown algae
used different in vitro method. Herein, the ethanolic extract (30%,
v/v) of Padina pavonica revealed good ferric reducing antioxidant
power (FRAP) and DPPH• scavenging activity. EtOH-PAD showed
a remarkable dose‐dependent free radical scavenging activity
and antioxidant effect towards both assay performed .
The radical scavenging activity of EtOH-PAD was tested using
an ethanolic solution of the stable free radical DPPH•. Fig. 1 &
Fig. 2 showed that the radical-scavenging activity of EtOH-PAD, of
Quercetin and Gallic acid increased in a dose-dependent manner.
Interestingly, the 30% ethanolic extract exhibited a strong
antioxidant activity at a low concentration (0.25 mg/mL) with an
inhibition percent of 72%. The maximum reduction percentage
was reached at 1 mg/mL (77.6%) with an IC50 value of 0.026
mg/mL (Table 1). However, standards Quercetin and Gallic acid
showed the most important antioxidant activity with 95.33% and
99.66% of inhibition at 1 mg/mL, respectively (fig. 3).In another
study, Pinteus and co-workers  reported lower values of IC50
value in the acetonic extract of P.pavonica (0.338 mg/mL) which
can be explained by the difference of the collection season and
the geographic location.
Figure 1:Representative morphology image of the carrageenan-induced
paw edema in rats.
Figure 2:DPPH radical radical-scavenging activity of the polyphenol
fraction of Padina pavonica, in comparison to Quercetin and Gallic acid.
Table 1: IC50 values of DPPH• radical-scavenging activity and FRAP
assay of the polyphenol fraction of Padina pavonica, in comparison
with the standard Quercetin and Gallic acid.
DPPH, IC50 (mg/mL)
FRAP, IC50 (mg/mL)
Figure 3:Reductive potential of the polyphenol fraction of Padina pavonica
and the standard Gallic acid using spectrophotometric detection
of Fe+3-Fe+2 transformations (FRAP assay).
The FRAP assay was employed to evaluate the ability of
EtOH-PAD extract and Gallic acid likewise to reduce Fe+3into Fe+2 at concentration ranged from 0.03 to 1 mg/mL. Fig. 3
showed that both polyphenol fraction and Gallic acid exerted a
dose-dependent reducing effect. Gallic acid used as standard
had the highest reducing power with an IC50value of 0.033 mg/
mL followed by the polyphenol fraction (IC50 = 0.4 mg/mL).It
is worth noting that the antioxidant effect as measured by the
FRAP assay is important to evaluate the contribution of different
compounds (phenolic and non-phenolic) present in the extract
which may have a synergistic interaction that could enhance its
potential . This fact suggests that the nature of compounds is
more important than the total phenolic contents of extracts from
brown seaweeds .
Effect of EtOH-PAD on Carrageenan-Induced Rat Paw Edema
Anti-inflammatory activity of the polyphenol fraction of the
brown algae Padinapavonica was assessed by carrageen aninduced
edema in rat model. Carrageenan-induced inflammation
assay is one of the most commonly used assays to assess antiinflammatory
activity of natural products . The percentage of
edema inhibition by the polyphenol fraction of Padinapavonica
with two different dosesis shown in table 2. All treated groups
showed a statistically significant inhibition of inflammatory
activity in comparison to control group (p< 0.05). Intraperitoneal
administration of EtOH-PAD reduced the edema volume during
the entire period of observation when compared to the carrageen
an-treated group (control). In addition, EtOH-PAD dosedependently
reduce the paw edema with a maximum effect three
hours following the sub plantar injection of carrageen an. As
shown in Table 2, a significant inhibition of edema was obtained
at the dose of 100 and 200mg/kg by reducing the edema by 53.48
% and 586%, respectively. This effect was higher than that of the
reference drug LSA with an inhibition of edema that does not
exceeded 45.73 %.Several studies have also reported that organic
fractions purified from the brown algae Cystoseiracompressa
exhibited anti-inflammatory activity .
Table 2: Effect of the intraperitoneal administration of polyphenol fraction from Padina pavonica on carrageenan-induced paw edema in rats in comparison with the reference drug Acetyl salicylate of lysine (ASL)
Edema volume (10-2mL)
Edema inhibition (%)
Data are expressed as mean ±SD; n=6 animals. * p< 0.01, ** p< 0.001 when compared to the carrageenan treated group
Carrageenan is a phlogogen agent that can produce an acute
inflammation resulting from the sequential action of several
mediators mainly Histamine, serotonin, kin in and prostaglandins
. Acute inflammation is also accompanied by an early release of
pro-inflammatory cytokine in the hind paw which such as TNF-α
and IL-6. IL-6 in turn promotes the release of the cyclooxygenase
involved in the arachidonic acid (AA)cascade and AA is converted
into prostaglandins PGE2 . Basing on earlier reports, the
anti-inflammatory effect of brown seaweeds may be due to
the inhibition of inflammatory mediator’s (NO, iNOS, COX‐2,
and PGE2) production in a dose-dependent manner . This
interesting effect observed with P.pavonica could be due to the
high amount of antioxidant compounds present in the ethanolic
The present study aimed to valorize natural resources present
in the coastline of Tunisia. The polyphenol fraction of Padina pavonica exhibited a significant anti-inflammatory activity
associated with antioxidant potential. These activities are likely
related to the presence active substances including phenolic
and non-phenolic compounds acting synergistically. Further
investigations will focus on other valuable pharmacological effect
and on their molecular mechanism in order to be exploited as
active ingredients for preparation of nutraceutical, cosmeceutical
and pharmaceutical products.
This study was financially supported by the Ministry of
Higher Education and Scientific Research of Tunisia (Grant N°.
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