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A. Tanniou &L. Vandanjon &M. Incera &E. Serrano Leon &V. Husa &J. Le Grand &
J.-L. Nicolas &N. Poupart &N. Kervarec &A. Engelen &R. Walsh &F. Guerard &
N. Bourgougnon &V. Stiger-Pouvreau
Received: 9 June 2013 /Revised and accepted: 29 October 2013 /Published online: 26 November 2013
#Springer Science+Business Media Dordrecht 2013
Abstract Sargassum muticum, an invasive brown macroalga
presently distributed along European Atlantic coasts from
southern Portugal to the south coast of Norway, was studied
on a large geographical scale for its production of phenolic
compounds with potential industrial applications and their
chemical and biological activities. S. muticum can produce
high biomass in Europe, which could be exploited to supply
such compounds. S. muticum was collected in Portugal,
Spain, France, Ireland and Norway (three sites/country) to
examine the effect of the latitudinal cline and related environ-
mental factors. Assays focused particularly on polyphenols
and their activities. Crude acetone–water extracts were puri-
fied using solid phase extraction (SPE) and antioxidant and
antimicrobial activities of crude extracts and semi-purified
fractions measured. Total phenolic content was assessed by
colorimetric Folin–Ciocalteu assay and reactive oxygen spe-
cies activities by 2,2-diphenyl-1-picrylhydrazyl, reducing
power, β-carotene bleaching method and xanthine oxidase
assay. Antibacterial activities were tested on terrestrial and
marine strains to evaluate potential use in biomedical and
aquaculture fields. Purified active phlorotannins, isolated by
SPE, were identified using NMR. Phenolic contents differ
clearly among countries and among sites within countries.
Quality did not change between countries, however, although
there were some slight differences in phlorethol type. Addi-
tionally, some fractions, especially from the extreme north and
south, were very active. We discuss this in relation to envi-
ronmental conditions and the interest of these compounds. S.
A. Tanniou (*):E. Serrano Leon :N. Poupart :F. Guerard :
V. Stiger-Pouvreau
LEMAR (UMR 6539 CNRS UBO IRD Ifremer)–Institut
Universitaire Européen de la Mer (IUEM)Université de Bretagne
Occidentale (UBO), Technopôle Brest-Iroise, 29280 Plouzané,
France
e-mail: anaelle.tanniou@univ-brest.fr
L. Vandanjon
Laboratoire GEPEA, CRTT, GEPEA (UMR CNRS 6144)-Université
de Nantes, 44602 Saint-Nazaire, France
L. Vandanjon
Université de Bretagne-Sud, 56321 Lorient, France
M. Incera
CETMAR, 36208 Bouzas-Vigo, Spain
V. H u s a
Institute of Marine Research, 5817 Bergen, Norway
J. Le Grand :J.<L. Nicolas
LEMAR (UMR 6539 CNRS UBO Ifremer IRD)-Ifremer,
Centre de Brest, 29280 Plouzané, France
N. Kervarec
Service RMN-RPE, UFR Sciences et techniques, Université de
Bretagne Occidentale (UBO), Avenue Le Gorgeu, 29200 Brest,
France
A. Engelen
CCMAR-University of Algarve, Faro, Portugal
R. Walsh
Irish Seaweed Research Group, Ryan Institute, National University
of Ireland, Galway, Ireland
N. Bourgougnon
Laboratoire de Biotechnologie et Chimie Marines, EA3884, IUEM,
Université de Bretagne-Sud (UBS), 56000 Vannes, France
J Appl Phycol (2014) 26:1215–1230
DOI 10.1007/s10811-013-0198-x
Assessment of the spatial variability of phenolic contents
and associated bioactivities in the invasive alga Sargassum
muticum sampled along its European range from Norway
to Portugal
muticum represents a potential natural source of bioactive
compounds and its collection could offer an interesting op-
portunity for the future management of this species in Europe.
Keywords Antioxidant activities .Antibacterial activities .
Latitudinal gradient .Phenolic compounds .Solid phase
extraction .Sargassum muticum
Introduction
Seaweeds are used in many countries as a source of food and
molecules, with many industrial applications, mostly involv-
ing phycocolloid extraction and, to a lesser extent, isolation of
certain biomolecules with pharmaceutical, medicinal and oth-
er industrial uses (Bourgougnon and Stiger-Pouvreau 2011).
In the last few years, natural antioxidants from plant and
animal sources have been actively investigated as replace-
ments for synthetic antioxidants currently used as food addi-
tives. This recent interest in natural antioxidants as food
additives has increased partly because of the restriction in
synthetic antioxidant utilization in the food industry due to
their long-term toxicological effects, including carcinogenici-
ty (Ito et al. 1986;Aruomaetal.1997; Bandoniené et al.
2000). Brown algal species are known to naturally produce
antioxidant compounds in great quantities: up to 20 % DW in
Fucales (Ragan and Glombitza 1986; Targett et al. 1995).
Indeed, in coastal ecosystems, biotic and abiotic factors are
known to affect algae species. To defend themselves against
these stresses, seaweeds are known to produce a great variety
of defensive metabolites. Consequently, they can be a very
interesting source of new substances for industry. Among such
defensive substances, phenolic compounds (PC), also known
as phlorotannins, are secondary metabolites synthesized dur-
ing development as components of algal cell walls
(Schoenwaelder and Clayton 1998) or as a chemical defence
in response to abiotic or biotic stress conditions, such as UV
radiation, grazing, bacterial infection or epiphytism, as well as
for intra- and interspecific communication (Ragan and
Glombitza 1986; Connan et al. 2004; Stiger et al. 2004;
Koivikko et al. 2005;Plouguernéetal.2006; Bourgougnon
and Stiger-Pouvreau 2011). As these compounds are produced
in response to the production of reactive oxygen species
(ROS), PC exhibit anti-ROS, i.e. antioxidant, properties
(Nakai et al. 2006;Kudaetal.2007; Kumar Chandini et al.
2008). Structurally, phlorotannins are oligomers and polymers
of 1,3,5-trihydroxybenzene (phloroglucinol) (Ragan and
Glombitza 1986; Targett and Arnold 2001; Koivikko et al.
2007) and can be considered as a pool of PC with different
natures and/or polarity. Within a species, phenolic compounds
can also vary greatly both spatially (Steinberg 1986,1989,
1992; Van Alstyne and Paul 1990; Targett et al. 1992,1995;
Steinberg et al. 1995;PaviaandAberg1996; Van Alstyne
et al. 1999;Stigeretal.2004;LeLannetal.2012a)and
temporally (Stiger et al. 2004; Connan et al. 2004;2007;
Plouguerné et al. 2006). However, few studies have examined
this spatial variation in connection with the activities
displayed by these compounds though large-scale sampling
of a single species.
In Brittany, native marine algae species are already har-
vested for industrial applications (agri-food, cosmetics and
thalassotherapy). In some places, native species are in com-
petition with introduced species that have proliferated. Inva-
sive seaweeds are often very promising for industrial applica-
tions as the chemical defences they have developed that allow
them to overcome geographical barriers and colonize new
environments, making them a source of interesting active
molecules. In this context, Sargassum muticum (Yendo)
Fensholt, which is an invasive species in Europe living on
rocky shores, was chosen as a model organism to find appli-
cations for its phlorotannin pool. This brown macroalga,
native to Japan, has spread widely along the European
Atlantic coasts since its introduction on the Atlantic
coast (Plouguerné et al. 2006;Kraan2008; Engelen
et al. 2008;Inceraetal.2009; Olabarria et al. 2009;
Le Lann et al. 2012b); it is currently one of the most
readily available Sargassaceae species on European
shores. Its large sustainable biomass could represent a
viable biotechnological asset in European resource de-
velopment programs as it is known to produce
phlorotannins of interest (Tanniou et al. 2013).
The aim of this work was therefore to study the chemical
plasticity, i.e. the spatial variability, of Sargassum phenolic
compounds in Europe and to see how the quantity and quality
of the compounds vary according to country and, finally, to
see how S. muticum can be usefully exploited in Europe. We
assessed the antioxidant and antibacterial activities of extracts
obtained from specimens established in five countries along a
latitudinal gradient: Norway, Ireland, France, Spain and
Portugal, from north to south of the North-East Atlantic
coast. Antioxidant activities of extracts were character-
ized by four biochemical methods (2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging activity, re-
ducing activity, xanthine oxidase inhibition and β-
carotene–linoleic acid system), and their total phenolic
contents were quantified. This led us to further select
some crude extracts for fractionation by a solid phase
extraction (SPE) device in order to determine the anti-
oxidant activity and total phenolic content of each frac-
tion. Antibacterial assays were also conducted with six
bacterial strains, three marine and three terrestrial, to
determine the antibacterial activities of the crude and
purified extracts. Proton nuclear magnetic resonance (
1
H
NMR) analysis was carried out on active fractions to
determine compounds responsible for the activities
detected.
1216 J Appl Phycol (2014) 26:1215–1230
Materials and methods
Sampling: algal material and abiotic parameters
Thalli of S. muticum were collected between March and May
2011 in three sites in each of five countries along a latitudinal
gradient in Europe. Samples were collected, from South
(March) to North (May), in Portugal, Spain, France, Ireland
and, finally, Norway (Fig. 1). The sampling time was chosen
according to the physiological state of the algae in each
country. In these periods, S. muticum was still immature in
all sites. The sampled countries were also chosen according to
the chronology of expansion of S. muticum along Atlantic
coasts; of the countries our sampling, France was the first
colonized and Ireland was the most recently colonized.
Collection was at low tide on semi-exposed or exposed
sites in all countries except Norway, where one site was
qualified as “sheltered”. The hydrodynamic conditions of the
sites were determined according to their topography and the
flora present during sampling. Other environmental parame-
ters, such as seawater temperature, photosynthetically avail-
able radiation and water salinity, were determined from mea-
surements made by satellites as part of the AQUA Modis and
Aquarius missions of National Aeronautic and Space Admin-
istration (NASA). These data are presented as data ranges for
the sampling periods considered (Table 1) according to coun-
try (see Tanniou et al. 2013,inreview).
During collection, only the apical and median parts of the
thalli were taken and the holdfast was left in place to allow
regrowth and thus minimize collection impact. Immediately
after collection and epiphyte removal, the seaweeds were
washed first with filtered seawater then distilled water in order
to remove residual sediments and salts. The cleaned algal
materials were then surface dried with blotting paper towel,
chopped into fragments, pooled by site, freeze-dried, reduced
to powder with a Waring Blender and, finally, sieved at
250 μm.
Solid/liquid extraction of phenolic compounds
Two grams dry weight (DW) of finely ground algal material
was placed in 250 mL flasks and extracted using 150 mL of an
acetone−water mixture (50:50 v/vin distilled water). Each
preparation was left under stirring at 40 °C for 3 h in the dark.
The mixtures were then centrifuged using an Eppendorf 5810
R centrifuge (Eppendorf A.G., Germany) at 4,000×gfor
10 min at 4 °C. The supernatants were then filtered on cotton
wool and concentrated to dryness under reduced pressure at
40 °C using a rotary evaporator (R-3000, Büchi, Switzerland).
About 10 mL Milli-Q water were then added to each residue
4 Km
5 Km
1 Km
20 Km
1 Km
Fig. 1 Sampling sites along the Atlantic coasts of Europe where S. muticum were collected to examine variations in phenolic concentrations and
activities over a large scale (three sites per country: Norway, Ireland, France, Spain and Portugal)
J Appl Phycol (2014) 26:1215–1230 1217
to give the crude extract. All extracts were then freeze-dried
prior to further analyses (phenolic content quantification and
NMR analyses), apart from 4 mL that was used directly for the
purification. All the extracts were prepared in triplicate: three
extractions were made per site and then pooled for further
analyses.
Purification of crude extracts
Crude extracts were purified by SPE following a modified
protocol published by Zubia et al. (2009). The SPE cartridges
(HF Bond Elut C18, 5 mg, Varian) were used in combination
with an under partial vacuum system (Vac Elut SPS 24,
Varian) at a pressure of 0.52 kg cm
−2
. After conditioning,
successively, with methanol (20 mL) and distilled water
(20 mL), the SPE cartridge was loaded with the crude extracts
(4 mL). After adsorption, fractionation was performed by
stepwise elution with 40 mL of each of the following solvents:
distilled water, 50 % methanol (v/vin distilled water), 100 %
methanol, dichloromethane/methanol 50:50 (v/v)and100%
dichloromethane. Each crude extract was purified four times
to accumulate fractions in sufficient weight: the same eluted
fractions were pooled and evaporated under reduced pressure
at 40 °C using a rotary evaporator (R-3000, Büchi, Switzer-
land). Each fraction was redissolved in the appropriate solvent
and freeze-dried prior to further analyses.
Quantity and quality of extracted phenolics
Determination of the total phenolic contents and Folin–
Ciocalteu assay The total phenolic content of all extracts
was determined colorimetrically (Labsystems, Multiskan
MS, Finland) with a microplate-adapted Folin–Ciocalteu as-
say (Sanoner et al. 1999), which is known to be little affected
by interfering compounds. Interfering substances, however,
are thought to account for less than 5 % of the Folin–
Ciocalteu-reactive compounds in brown seaweeds (Toth and
Pavia 2001). Phloroglucinol (1,3,5-trihydroxybenzene,
Sigma, France) was used as a standard, and concentrations
were determined in each extract by freeze-drying three ali-
quots of 1 mL. Total phenolic contents (TPCs) were expressed
as percentages of phenolic compounds in the DW of the
aliquot or the algae.
NMR analysis of extracts The overall structural composition
of crude extracts was assessed by means of
1
H NMR analyses
on a Bruker Avance 400 using the standard pulse sequences
available in the Bruker software (Bruker, France). All spectra
were recorded in MeOD, at room temperature. Chemical shifts
were expressed in parts per million. NMR profiles unambig-
uously indicate the PC within the extracts, with peak(s) be-
tween 5.5 and 6.5 ppm. The nature of the phlorotannins
present in the MeOH–water fractions was established using
heteronuclear multiple quantum coherence (HMQC),
heteronuclear multiple bond correlation (HMBC) experiments
and comparison of the chemical shifts of the
1
H and
13
C
resonances with literature data (Cérantola et al. 2006). All
spectra were recorded in MeOD, at 25 °C.
Activity measurements on extracts
From a general point of view, the antioxidant/radical scaveng-
ing tests carried out on algal crude extract and/or semi-purified
fractions are based on both the determination of products
resulting from the oxidation and the measurement of the
efficiency of a substance to trap radicals (Huang et al. 2005).
We used several different tests in our study, with the aim of
revealing different types of mechanisms in the action of phe-
nolic compounds: DPPH radical scavenging assay, reducing
power, superoxide anion-scavenging capacity assay (xanthine
oxidase (XO) activity assay) and β-carotene bleaching meth-
od. Our assays included those based on electron-transfer re-
actions (DPPH and reducing power) together with an assay
involving hydrogen atom transfer reactions (β-carotene
bleaching method), as described by Huang et al. (2005). The
first two types measure the capacity of an antioxidant to
reduce an oxidant, which changes colour when reduced, while
the second type uses a competitive reaction scheme in which
antioxidant and substrate compete for thermally generated
peroxyl radicals through the decomposition of azo compounds
(Huang et al. 2005). DPPH and reducing power tests give
insight into the levels of the lipophilic and hydrophilic com-
pounds, whereas BCBM assesses the levels of lipophilic
compounds alone (Chew et al. 2008). The XO activity assay
measures another ROS scavenging capacity: the superoxide
anion (O
2
·−
) is generated by a xanthine oxidase/hypoxanthine
system and reduces nitroblue tetrazolium (NBT) to blue
formazon if no competitor, i.e. an antioxidant substance (the
Table 1 Environmental parameters according to country during the
sampling period (March to May 2011; see Tanniou et al. 2013 in review):
sea surface temperature, photosynthetically available radiation and sea
surface salinity. Data obtained from NASA satellites during the two
missions Aqua MODIS and Aquarius
Abiotic parameters Norway Ireland France Spain Portugal
Sea surface temperature
in °C
8–10 12–14 12–14 12–14 18–20
Photosynthetically
available radiation
in mol photons m
−2
day
−1
35–40 30–35 35–40 20–25 40–45
Sea surface salinity
PSU
34 35 35 36 37
Details presented in italics correspondto low values, in normal to average
values and bold to high values
1218 J Appl Phycol (2014) 26:1215–1230
sample), is added (Huang et al. 2005). Even though O
2
·−
is not
the most damaging ROS, it is an initiator of highly detrimental
ROS production such as singlet oxygen, hydroxyl radical
(HO·) and peroxynitrate (OONO–) and could thus increase
the oxidation risk indirectly (Huang et al. 2005).
DPPH radical scavenging assay The DPPH radical scaveng-
ing assay, modified according to Le Lann et al. (2008), was
used to determine the radical scavenging activities in extracts
and purified SPE fractions. The positive controls used were
ascorbic acid (Sigma, France), α-tocopherol (Sigma), butylat-
ed hydroxyl-anisole (BHA) and butylated hydroxyl-toluene
(BHT) (respectively, 2(3)-t-Butyl-4-hydroxyanisole, 2,6-Di-
tert-butyl-4-methylphenol; Sigma France). The protocol was
microplate-adapted for faster use and to decrease sample
preparation time. Briefly, five dilutions from 0.1 to
2mgmL
−1
of the extracts were prepared in triplicate before
addition to 12 μLaliquotsof150μM DPPH radical (222 μL).
The mixture was stored in the dark for 60 min prior to
absorbance measurement at 540 nm. Distilled water was used
as a negative control. All samples were assayed in triplicate.
Antioxidant activity was expressed as the IC50 (the concen-
tration of substrate that causes a 50 % loss of DPPH activity):
the lower the IC50 is, the stronger the antioxidant activity.
Extracts with an IC50 higher than 10 mg mL
−1
were consid-
ered as non-active extracts.
β-Carotene bleaching method The antioxidant activities of
extracts and controls were measured by the β-carotene
bleaching microplate-adapted method, modified in accor-
dance with Kaur and Kapoor (2002) and Koleva et al.
(2002). Two mL of a β-carotene solution in chloroform
(0.1 mg mL
−1
) were added to round-bottom flasks containing
20 mg of linoleic acid and 200 mg of Tween 40. After
evaporation with a rotavapor, oxygenated distilled water
(50 mL) was added, and the mixture was shaken to form a
liposome solution. This mixture was added to each of the
following: 12 μL of the extracts, positive controls
(α-tocopherol, BHA and BHT) and negative controls
(distilled water and ethanol). The absorbance of the solution
at 450 nm was measured immediately (t= 0 min) and after 2 h
at 50 °C (t=120 min). All samples were assayed in triplicate.
Antioxidant activity was expressed through the antioxidant
activity coefficient (AAC), calculated as follows (Eq. 1):
AAC ¼As 120ðÞ
−Ac 120ðÞ
Ac 0ðÞ
−Ac 120ðÞ
1;000 ð1Þ
where As
(120)
is the absorbance of the antioxidant mix at
t=120 min, Ac
(120)
is the absorbance of the control at
t=120 min and Ac
(0)
is the absorbance of the control at
t=0 min. Since the positive controls, BHA, BHT and vit E
had an average AAC of 700, this value was arbitrarily chosen
to express the antioxidant activity as AAC
700
, as in Le Lann
et al. (2008). So, AAC
700
was the concentration of substrate
needed to obtain an AAC value of 700 (Tanniou et al. 2013).
AhighAAC
700
was therefore considered as indicative of a
weak antioxidant activity.
Reducing power The reducing capacity of each extract was
assessed by the method adapted by Zubia et al. (2009)and
Kuda et al. (2005). In a 96-well microplate, aliquots of extracts
(25 μL) were mixed with phosphate buffer (25 μL, 0.2 M, pH
6.6) and potassium ferricyanide (K
3
Fe(CN)
6
)(25μL, 1 %).
After incubation at 50 °C for 20 min, the microplate was
cooled down prior to the addition of 25 μLtrichloroacetic
acid (10 %). Then, 25 μL 0.1 % FeCl
3
·6H
2
O and 100 μL
water were added to each well. The increase of absorbance,
which indicates an increase in reducing activity was read at
620 nm after 10 min at room temperature. Results were
expressed as the EC50 value (mg mL
−1
) (Oueslati et al.
2012), which is the effective concentration at which the ab-
sorbance was 0.5 for reducing power. This assay was carried
out in triplicate for each sample and the positive controls
(BHA, BHT, vit E and vit C).
Superoxide anion-scavenging activity Thesuperoxideanion-
scavenging assay was carried out according to the method of
Nagai et al. (2003). The reaction mixture consisted of 203 μL
0.05 M Tris–HCl buffer (pH 7.5), 57 μL 5 mM hypoxanthine,
30 μL 0.33 mM NBT and 13 μL of the sample. After incuba-
tion at 25 °C for 10 min, the reaction was started by adding
30 μL xanthine oxidase and keeping the temperature at 25 °C
for 21 min. The absorbance was measured every 3 min for
21 min at 560 nm. The inhibition ratio (%) was calculated
from the following equation (Chua et al. 2008):
%inhibition ¼rate of control−rate of testsample
rateof control
100
ð2Þ
Results were then expressed as IC50 (the concentration of
substrate that causes a 50 % inhibition).
Antibacterial tests
Algal extracts at five different concentrations, from 50 to
150 μgmL
−1
, were tested for inhibitory activity against three
marine bacterial strains obtained from the Ifremer collection:
Vibrio aestuarianus (S02-041), Vibrio anguillarum (S11-
054) and Vibrio parahaemolyticus (S12-011); and three ter-
restrial strains: Escherichia coli (T05-006, ATCC8739),
Staphylococcus aureus (T05-007, ATCC 65388) and Pseudo-
monas aeruginosa (T05-005, ATCC15442). Each treatment
J Appl Phycol (2014) 26:1215–1230 1219
and control was replicated four times. Extracts were incu-
bated for 24 h with the bacteria in exponential growth phase
(at 2 × 10
8
UFC mL
−1
) in 96-well plates (VWR) in LB
medium (Luria Hinton Broth, Sigma, UK), supplemented
with NaCl (35 g L
−1
) for the marine strains, at 21 or 37 °C
for marine or terrestrial strains, respectively. The antibiotic
chloramphenicol was used as a control, at the same concen-
tration as the tested extracts. Only the results for the smaller
concentration (50 μgmL
−1
) are presented here.
Statistics
All analyses were carried out in tri- or quadruplicate, and
their results are presented as mean values± standard devia-
Depending on the variable, means were calculated using
three or four values/measurements per extract and were used
for the statistical analysis. Homogeneity of variance was
tested with the Brown–Forsythe test at the 0.05 error risk.
Data that did not satisfy the criteria of normality and homo-
scedasticity for parametric tests were square root trans-
formed before further analyses. One-way nested ANOVAs,
with sites nested within countries, were performed on the data
concerning the crude extract. For the semi-purified extract,
simple one-way ANOVAs were performed to compare differ-
ences among sites in each country. When ANOVA demon-
strated significant difference, post hoc Tukey HSD tests were
carried out to identify which means contributed to the effect.
Chemicals
All reagents used in the experiments were of analytical grade
and most were obtained from Sigma. Solvents used for ex-
traction of algae samples were purchased from Fisher Scien-
tific. Water used was of Millipore quality.
Results
Distinction between countries regarding the crude extracts
Country of sampling had a significant effect on the TPCs
found in S. muticum (one-way ANOVA, p<0.001, Fig. 2).
S. muticum populations from the different sampled countries
were ranked by their level of phenolic content as follows:
Portugal>Norway>France=Ireland=Spain (post hoc Tukey).
The highest TPCs were observed at the extremes of the
latitudinal gradient, which correspond to the edges of the
distribution range of the species, i.e. Norway (1.79 ± 0.28 %
DW
algae
) and Portugal (3.40 ± 0.91 % DW
algae
). Phenolic con-
tent reached 1.26± 0.11 for France and the minimum concen-
tration is observed for Spain with 0.66±0.15 % DW
algae
.Inter-
site variations was also observed, especially in Portugal,
where phenolic content varied from 2.46±0.16 to
4.28± 0.26 % DW
algae
.
In addition to the algal phenolic concentration, it is inter-
esting to examine an extract containing the maximum PC.
Based on such extracts, the results are expressed in %DW
where the dry weight is that of the concentrated extract. As
above, great differences in maximum PC were observed
among the countries (one-way nested ANOVA, p< 0.001,
Fig. 3a). Thus, the maximum is observed for Portugal where
the total phenolic content reached more than 17 % of the
fraction dry weight (17.49±0.49 % DW
fraction
for site 2): in
Portugal phenolics represent more than 15.85 % on average,
whereas this content reached 13.06, 10.69, 8.25 and 7.29 % in
Norway, France, Ireland and Spain, respectively (Fig. 3a).
The antioxidant activity of S. muticum extracts differed
among countries (one-way ANOVA, p<0.001). According
to the ANOVA post hoc Tukey test (p<0.05), the AAC700
following the β-carotene bleaching method (AAC
700
)inthe
extracts was superior to those of all the positive controls,
showing that those extracts have very low antioxidant activity
(p<0.001, Fig. 3b). In the same way, radical scavenging
activity differed among countries (one-way ANOVA,
p<0.001). Activity of crude extracts differed from the positive
controls (post hoc Tukey test, p<0.001). However, some
extracts showed activities close to those of the positive con-
trols: the radical scavenging activity determined by DPPH
method (IC50) was the highest in extracts from Norway and
Portugal, with 0.44±0.03, 0.41±0.03 and
0.46± 0.01 mg mL
−1
for the sites 2 and 3 of Norway and the
site 1 of Portugal, respectively (Fig. 3c). As previously seen
for the phenolic content, the most active extracts are those of
the extreme countries, followed by France, Spain and finally
b
c
c
c
a
Norway Ireland France Spain Portu
g
al
Total phenolic content (%DWalgae)
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Fig. 2 Spatial variability of phenolic content of crude extracts from S.
muticum collected along the Atlantic coasts of Europe from Portugal to
Norway. Data (mean values per country±standard deviation) were analysed
using one-way ANOVA followed by post hoc Tukey tests. (a–c) Different
letters indicate significant differences
1220 J Appl Phycol (2014) 26:1215–1230
tion (SD), using Statistica 8 (StatSoft ®) software for PC.
the less active extracts, those from algae collected in Ireland
(0.75±0.01, 0.94± 0.02 and 0.84± 0.02 mg mL
−1
for sites 1, 2
and 3, respectively). This tendency is confirmed by the results
of the reducing activity assay (Fig. 3d) showing that the
highest reducing activity was displayed by the extracts from
Norway and Portugal (0.079±0.01, 0.074± 0.01, 0.082± 0.01
and 0.086±0.01, 0.082±0.01, 0.079±0.01 mg mL
−1
for the
sites 1, 2 and 3 of Norway and Portugal, respectively).
Moreover, according to the statistical analysis, this reducing
activity is equivalent to those displayed by the positive con-
trols (0.10±0.01; 0.086±0.01; 0.092±0.01; 0.13±
0.01 mg mL
−1
for BHA, BHT, vit E and vit C, respectively).
Finally, XO inhibition also depends on the country of origin of
the samples (Fig. 3e). Only extracts from Norway and Portu-
gal displayed activities that can be compared to the positive
controls (0.20 ± 0.01, 0.19± 0.01, 0.17±0.01 and 0.29±0.01,
c
a
a,b,c
g
e,f,g
g
d,e
d,e,f
d
d,e,f,g
g
f,g
g
b,c
a,b
0 5 10 15 20
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Portugal Spain France Ireland Norway
Phenolic content (%DWfraction)
a
a
a
a
c
d
d,e
g
f
g
e
f,g
f,g
f,g
i
h
h
b,c
b
BHA
BHT
Vit E
Vit C
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Positive controls Portugal Spain France Ireland Norway
EC50 (mg.mL-1) - DPPH
a
a
a,b
e,f
g,h
h
c,d,e
e,f
c,d,e
c,d,e
f,g
d,e
a,b,c
e,f
i
e
a,b,c,d
b,c,d
0123456
BHA
BHT
Vit E
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Positive
controls Portugal Spain France Ireland Norway
AAC700 (mg.mL-1) – caroten Bleaching method
a
bc
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Fig. 3 Phenolic content (a) and screening of several antioxidant activi-
ties measured on phenolic extracts from S. muticum collected along the
Atlantic coasts of Europe from Norway to Portugal, using the β-carotene
bleaching test (b), radical scavenging activity (c), reducing power (d)
and XO activity (e). Data were analyzed using one-way nested ANOVA
followed by post hoc Tukey tests. (a–k) Different letters indicate signif-
icant differences
J Appl Phycol (2014) 26:1215–1230 1221
0.21± 0.02, 0.19± 0.01 mg mL
−1
for sites 1, 2 and 3 of Norway
and Portugal, respectively).
Inter-country differences in the purified extracts
Figure 4shows the phenolic polarity repartition in fractions
obtained after an SPE procedure. Depending on the country of
origin, the majority of the phenolics were found in the water,
methanol–water or methanol fraction. Thus, for Norway,
France and Portugal, compounds were present in the water
fraction in small quantity (means: 0.53±0.11, 0.095±0.03 and
0.56± 0.25 % DW
algae
for Norway, France and Portugal, re-
spectively) but with the majority in the MeOH–water fraction
(means: 0.82±0.19, 0.44±0.06 and 1.45±0.37 % DW
algae
for
Norway, France and Portugal, respectively). For Ireland, apart
from the site 1, the great majority of the phenolic compounds
were found in the MeOH fraction. For Spanish extracts, the
same quantities were present in the water and MeOH–water
fractions, representing approximately 0.15 % DW
algae
.
Only the results of antioxidant assays and radical scaveng-
ing activities will be presented for the purified fractions, other
anti-ROS activities were not measured on purified extracts.
Only S. muticum from Norway and Portugal contained high
amounts of active phenolics, so only the results for these two
countries are presented in Table 2. Radical scavenging activ-
ities (determined by the DPPH method and using IC50 to
calculate the activity) were always better in purified fractions
than in the crude extracts and were close to those of the
positive controls. In addition, the MeOH–water fraction was
more active than the MeOH one in almost all cases. Thus, the
best radical scavenging activities were displayed by MeOH–
water fraction from sites 2 and 3 in Norway (0.28±0.02; 0.27
±0.02 mg mL
−1
, respectively), by the MeOH fraction from
site 2 in Portugal (0.25±0.03 mg mL
−1
) and by the MeOH–
water fraction from site 3 in Portugal (0.25± 0.01 mg mL
−1
).
As for the crude extracts, for both these countries, antioxidant
activities determined by the β-carotene bleaching method
(ACC
700
) were very low and dissimilar to the positive con-
trols. From here on, we will focus on the most active fractions
(MeOH–water and MeOH), which contain sufficient PC.
Antibacterial activities were dependent on country and varied
strongly among sites within country (Table 3). Thus, if we focus
on marine strains, almost all extracts, i.e. both crude and purified,
possessed activities against V. aestuarianus and V. anguillarum .
Conversely, few extracts were active against V.
parahaemolyticus : only crude extracts from Ireland (all sites)
and from sites 2 and 3 in Portugal displayed good activities
(>50 % bacterial growth inhibition) against this strain. In many
cases, the purified extracts were less active than the crude
extracts, depending both on the country and on the studied strain.
When purified extracts showed higher activity, it was always
from the MeOH fractions. For the terrestrial strains, in the case of
a
a
a
b
a
a
a
e
b
d
a
c
a
b
d
b
a
a
a
BHA
BHT
Vit E
Vit C
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Positive controls Portugal Spain France Ireland Norway
IC50 (mg.mL-1) – Reducing Power
b,c
b,c,d
c,d,e
e,f,g
b
a
a
e,f
d,e,f
f,g
g
i
e,f,g
k
h
j
a
a
a
BHA
Trolox
Vit E
Vit C
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Positive controls Portugal Spain France Ireland Norway
IC50 (mg.mL-1) – XO activity assay
de
0.0 0.2 0.4 0.6 0.8 1.0
0.0 0.1 0.2 0.3 0.4
Fig. 3 continued.
Fig. 4 Spatial variability of phenolic content determined on crude and
SPE-purified extracts from S. muticum collected along the Atlantic coasts
of Norway (a), Ireland (b), France (c), Spain (d) and Portugal (e). For
each country, data were analysed using one-way ANOVA followed by
post hoc Tukey tests. (a–j) Different letters indicate significant differences
1222 J Appl Phycol (2014) 26:1215–1230
e
b
c
a
a
a
e
b,c
c
a
a
a
f
b,c
d
a
a
a
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
1
2
3
Phenolic content (%DWalgae)
Phenolic content (%DWalgae)
d
a
b
a
a
a
e
a
a
c
a
a
d
a
a
b,c
a
a
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
1
2
3
e
a,b
d
a,b
a,b
a
e
b
c,d
a,b
a,b
a,b
e
a,b
c
a,b
a,b
a
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
1
2
3
c
a,b
a,b
a
a
a
d
a,b
a,b
a
a
a
c
a,b
b
a
a
a
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
1
2
3
h
c
g
a,b,c
a,b a
i
e
g
a,b
a,b a
j
d
f
b,c
a,b a
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
Crude
Water
MeOH-Water
MeOH
DCM-MeOH
DCM
123
Norway Ireland
France Spain
Portugal
a
c
b
d
e
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0.0
0.2
0.4
0.6
0.8
1.0
2.5
2.0
1.5
1.0
0.5
0.0 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Phenolic content (%DWalgae)
Phenolic content (%DWalgae)
Phenolic content (%DWalgae)
J Appl Phycol (2014) 26:1215–1230 1223
S. aureus strains, the purified extracts were more active than the
crude ones for all the countries tested, except Ireland. Norwe-
gian and Irish extracts showed very high activities
against E. coli. Interestingly, all the crude extracts were
active against P. aeruginosa .
Identification of active phlorotannins from Norway, France
and Portugal
Figure 5shows the spectrum obtained using
1
H NMR to analyse
active fractions from Norway, France and Portugal. In crude and
purified extracts, PCs are visible between 5.5 and 6.5 ppm. In
both purified extracts, the two phlorotannin profiles seem to be
very similar (Fig. 5b). In the chosen active purified fractions, i.e.
for the MeOH–water ones, phenolics were always present and
the two profiles were always quite similar. In addition, the
separation by polarity allows the removal of a great amount of
mannitol, which is prevalent in crude extracts.
Results from the two dimensional NMR analysis (HMBC)
showed that the purified extracts from S. muticum collected in
Norway, France and Portugal were all rich in phlorethol(s)
(Fig. 6) and that these compounds seem not to be linear
(Cérantola et al. 2006).
For these two types of analyses (
1
HNMRand2DNMR),the
small quantity of compound present in extracts of samples from
Ireland and Spain did not allow clear signals to be obtained in the
aromatic area. These minor signals are masked by other more
abundant molecules. Despite the low anti-ROS activities of the
extracts from France, these results are presented here for infor-
mation purposes, to see whether phenolic compounds produced
by French populations are of the same type as from Portuguese
and Norwegian populations. Some small differences were visible
between spectra obtained from French, Portuguese and Norwe-
gian populations (Fig. 6). Using HMBC, Norwegian and French
populations can be seen to be similar and separated from the
Portuguese population; using HMQC, the three populations
appear quite similar, but with differences remaining in the gen-
eral phlorotannin profiles.
Discussion
Activities of the crude extracts
This study examined the effect of the country and site of
sampling on the TPC, antioxidant and antibacterial activities
Tabl e 2 Antioxidant activities measured using solid phase extraction (SPE) purified polar extracts from S. muticum collected along the European
Atlantic coasts, from Portugal to Norway. Data were analysed using one-way nested ANOVA followed by post hoc Tukey tests
Variable Countries Fractions Means values±SD Tukey HSD (p<0.05)
Site 1 Site 2 Site 3
DPPH
IC 50 (mg mL
-1
)
Norway Crude 0.84±0.02 (h) 0.44± 0.03 (d,e) 0.41±0.03 (d,e)
MeOH–water 0.39±0.02 (c,d) 0.28± 0.02 (a,b) 0.27±0.02 (a,b)
MeOH 0.51±0.03 (f) 0.54±0.03 (f) 0.41±0.03 (d,e)
Portugal Crude 0.46±0.01 (e,f) 0.58±0.01 (g) 0.62±0.01 (g)
MeOH–water 0.33± 0.01 (b,c) 0.33± 0.01 (b,c) 0.25±0.01 (a)
MeOH 0.43±0.03 (d,e) 0.25±0.03 (a) 0.46±0.03 (e,f)
Positive controls BHA 0.27±0.01 (a,b)
BHT 0.27 ±0.01 (a,b)
Vit C 0.26±0.01 (a,b)
Vit E 0.26±0.01 (a,b)
β-carotene
AAC
700
(mg mL
-1
)
Norway Crude 1.53±0.01 (e) 0.79± 0.01 (b,c) 0.94±0.02 (b,c)
MeOH–water 1.65±0.01 (e,f) 1.33±0.01 (e) 1.74±0.01 (e,f,g)
MeOH 1.10±0.01 (c,d) 1.33±0.01 (e) 1.07± 0.01 (c,d)
Portugal Crude 1.63±0.01 (e,f) 2.80±0.14 (h) 3.20±0.55 (i)
MeOH–water 2.06±0.01 (g) 1.63± 0.01 (e,f) 1.41±0.01 (d,e)
MeOH 1.93±0.01 (f,g) 0.93±0.01 (b,c) 1.48± 0.01 (e)
Positive controls BHA 0.25±0.01 (a)
BHT 0.26 ±0.01 (a)
Vit C 0.26±0.01 (a)
Vit E 0.26±0.01 (a)
The most active extracts/fractions compared with the positive controls are presented in italics. Different letters indicate significant differences among
extracts for each variable (i.e. activity test)
1224 J Appl Phycol (2014) 26:1215–1230
of S. muticum extracts. The screening procedure also provided
us with a way to identify the best extracts for later application,
and also to know whether exploitation of this species would
be viable all the countries in the study.
The anti-ROS tests showed that at the selected sampling
period the extracts were quite active. Crude extracts had very
good reducing activities and anti-XO comparable to activities
measured for positive controls used in industry (BHA, BHT,
vit E and vit C for the reducing power and Trolox for the XO
test). The best activities were shown by the extracts from the
extremities of the gradient, i.e., Norway and Portugal; this was
also where the phenolic compound concentrations were
highest, showing the important role of these compounds in
the detected activities. Moreover, extracts from samples taken
in France also showed good reducing activities, in accordance
with the phenolic concentrations in the extracts. Even though
the PC concentration was lower in France than in these two
other countries, it was still more than 1 % of the dry weight of
the seaweed. Here, only a small quantity of phenolics was
extracted from seaweeds compared with what has been shown
to be possible in other studies on the same species (Connan
et al. 2004; Plouguerné et al. 2006; Parys et al. 2009), it can be
understandable by the chosen sampling period. Indeed it has
been known for a long time that the quantity of phenolics
contained in seaweeds is largely dependent on the season and
environmental parameters in general (Jormalainen and
Honkanen 2001; Hemmi and Jormalainen 2004; Fairhead
et al. 2005;Connanetal.2007). Here, the collection period
was chosen to sample individuals of S. muticum in a same
physiological state along the European gradient studied. This
approach was taken to enable the comparison of results and to
avoid bias caused by reproduction; indeed, only immature
individuals were collected. However, this period is not the
most convenient for the production of a large quantity of PC
by seaweeds, as shown by numerous authors (Connan et al.
2004;Plouguernéetal.2006;LeLannetal.2012a). Never-
theless, crude extracts showed interesting antioxidant
and antibacterial activities. Some extracts inhibited the
growth of five bacterial strains by more than 50 % and
some have activities equivalent to the one detected by
the antibiotic, the chloramphenicol at the same concen-
tration (50 μgmL
−1
).
Tabl e 3 Bioassays for the antibacterial activities of crude or SPE-purified extracts (fractions MeOH-water and methanol) for the three sites in each of the
five studied countries along the Atlantic coasts. SPE: solid phase extraction. ns: no activity
Norway-site 1 Norway-site 2 Norway-site 3 Ireland-site 1 Ireland-site 2 Ireland-site 3
Crude M-W M Crude M-W M Crude M-W M Crude M-W M Crude M-W M Crude M-W M
02-041 +++ ++++++ ++++++ ++++++ +++++++++ ++
11-054 ++ ++ + + ++ ++ ++ –++ ++ ++ + ++ ++ ++ ++ ++ –
12-011 + ––+––+––+++ ––++ ––++ ––
T007 –++ + –+++ +++++++ +++++ +++++ ++
T006 ++ +++ +++ +++++ ++ +++ +++ –+++ +++ –++ +++ –+++ +++
T005 ++ ns Ns +++ ns ns ++ ns ns ++ ns ns +++ ns ns +++ ns ns
France-site 1 France-site 2 France-site 3 Spain-site 1 Spain-site 2 Spain-site 3
Crude M-W M Crude M-W M Crude M-W M Crude M-W M Crude M-W M Crude M-W M
02-041 –++++ +++++ ++++++ +++++ +++–++
11-054 + + + ++ + + + + + ++ ++ + –+++++++
12-011 ––+––+–––––+––+–––
T007 –++ ++ –++ +++ –++ + –+++ ++ –++ +++ +++++
T006 –++ –– +–– – –++++ –+++ ++ –+++ +–
T005 ++ ns Ns +++ ns ns + ns ns +++ ns ns ++ ns ns ++ ns ns
Portugal-Site 1 Portugal-Site 2 Portugal-Site 3
Crude M-W M Crude M-W M Crude M-W M
02-041 +++ +++ +++ +++ ++ ++ ++ ++ ++
11-054 ++ + + ++ + + ++ –+
12-011 + ––++ ––++ + –
T007 + + + + + ++ –++
T006 ++ ++ ++ +++ +++++ +–
T005 ++ ns Ns +++ ns ns ++ ns ns
Marine bacterial strains 02–041, V. aestuarianus ;11–054, V. anguillarum;12–011, V. parahaemolyticus; terrestrial bacterial strains T007, S. aureus;
T006, E. coli; T005, P. aeruginosa; M-W and M, methanol–water and methanol fractions, respectively, after SPE purification. Antibiotic chloram-
phenicol at 50 μgmL
−1
inhibited growth by >75 % for all strains (marine and terrestrial)
+++ >75 %, ++ 50–75 %, +25–50 %, −<25 % bacterial growth
J Appl Phycol (2014) 26:1215–1230 1225
Norway Portugal
Mannitol
Mannitol
SPEMeOH/Water
Mannitol
France
Semi-Purification
Crude extract
a
N
P
F
SPEMeOH/Water
Crude extract
N
P
F
Aromatic compounds
b
Fig. 5
1
H NMR analysis of active crude and SPE purified extracts from S. muticum collected in Norway, France and Portugal (a) and enlargement of the
aromatic area between 5.5 and 6.5 ppm for the crude and purified extracts (b). NNorway, FFrance and PPortugal
Norway France Portugal
HMBC HMQC
Fig. 6 The nature of the phlorotannins present in the MeOH–water fractions was established using heteronuclear multiple bond correlation (HMBC) and
heteronuclear multiple quantum coherence (HMQC) experiments
1226 J Appl Phycol (2014) 26:1215–1230
Activities of the purified extracts
The compound distribution by polarity seems rather similar
from one country to another. The PCs were found for the
greater part in the polar or quite apolar fractions, such as
aqueous, methanol–water and methanolic fractions. This dis-
tribution was not identical among samples, which seems to
indicate that the pool of extracted compounds does not consist
of a single phenolic type. Indeed, as we have already shown
for S. muticum, the pool of compounds can vary according to
different parameters (Tanniou et al. 2013). The SPE is used as
an assay here to separate molecules contained in the extract by
polarity but also, therefore, to obtain one or several “cleansed”
fractions concentrating the compounds of interests (here the
PCs). One can note that there is an increase of the radical
scavenging activities after purification, showing that phenolic
compounds are responsible for the measured activities. These
activities are comparable to those obtained with the positive
controls, which are molecules used in industry. For the anti-
oxidant activity results measured by the β-carotene bleaching
method, no activity was measured after purification. As this
test measures the activity of lipophilic molecules (Koleva et al.
2002;LeLannetal.2008), then the active compounds here
would thus tend to be polar, or slightly apolar.
Extracts from all five countries showed evidence of bacte-
rial inhibition. Extracts were active against three marine bac-
terial strains (V. aestuarianus ,V. anguillarum and V.
parahaemolyticus) and three terrestrial strains (E. coli,S.
aureus and P. aeruginosa). In this test, the most active ex-
tracts were no longer those from the gradient extremities.
Indeed, if we look at the crude extracts, it was especially in
those from Ireland and Portugal that we found the highest
activities. For the purified extracts, only those with high anti-
ROS activities and a large phenolic content were tested here.
Yet, in the course of our experiments we found that many
extracts were less active after purification. This would suggest
that the compound(s) active against bacteria is/are not neces-
sarily phenolic. Preliminary studies have described antibacte-
rial activities for S. muticum extracts (Hellio et al. 2002,
2004a,b). For the same genus, some authors also demonstrat-
ed antibacterial activity of chloroform extracts (Sastry and
Rao 1994). Plouguerné et al. (2008,2010)alsoshowedthat
non-polar extracts of S. muticum were the most active against
bacterial strains. In our study, apolar extracts were not tested.
The goal was to determine whether fractions containing PCs
had antibacterial activities. In the literature, we find that
phlorotannins isolated from Sargassum vestitum and Sargas-
sum natans also have antifouling activity (Sieburth and
Conover 1965; Jennings and Steinberg 1997). Our results
showed that only a few extracts keep their antibacterial
activities after purification. This is the case of the site 1
Portuguese extract tested against V. aestuarianus and E.
coli, for example.
Taking into account all results, Norway and Portugal are
the two countries where active phenolic compounds could be
obtained in great quantity after separation. One could hypoth-
esize that “extreme”environmental conditions along the At-
lantic coasts could stress S. muticum, thus forcing it to pro-
duce more phenolic compounds to act as a defence against UV
radiation and seawater temperature changes, for example.
Indeed, if we take into account the tendencies observed during
the study of the environmental parameters, we can propose
some hypotheses regarding the factors that could influence
phenolic content. In the chosen sampling periods, we noticed
that the quantity of available radiation for photosynthesis was
higher where we found the largest quantities of active CP, i.e.
in the extremities of the gradient and in France. Other authors
observed an effect of season, and thus probably of light levels
(Connan et al.2004;Plouguernéetal.2006), on the produc-
tion of CP as photoprotective molecules (De la Coba et al.
2009). Other parameters seem less correlated with the ob-
served variations, although salinity has been cited by other
authors as a factor that can influence and reduce phenolic
content (Ragan and Glombitza 1986; Connan and Stengel
2011). Here, salinity was not correlated with lowered PC
concentration, although the smallest content was for Spain,
where salinity is the highest (up to 37 psu). The dates of
colonization of S. muticum could also be taken in consider-
ation. Indeed, this species spread northward and southward
from the south of England starting in 1973. It has been post
recently found on the north Portuguese coast (by 2002–2010)
and in Norway around 2000. It is thus possible that at first,
early in their arrival in new area, these seaweeds produce more
defensive compounds to colonize their novel environment. In
any case, it seems a little dangerous to draw general conclu-
sions on the effect of geographical position and thus environ-
mental parameters on the production of PC by S. muticum;
indeed, numerous authors having demonstrated the existence
of very small scale sources of variation, such as day/night or
seasonal variation (Connan et al. 2004,2007). Also, the
expression of genes involved in PC biosynthesis occurs only
a few hours after a light stimulation (E. Creis, personal com-
munication). It would thus be necessary to repeat this study at
another period of the year and/or make several samplings per
day, to be able to identify the sources of variation in total
phenolic content.
Identification of active phlorotannins from Norway, France
and Portugal
The
1
H-NMR profiles show a great similarity between the
spectra of Norway, France and Portugal. The 2D spectra
allowedustoidentifythephlorotannins produced by S.
muticum as being of the phlorethol type, and HMBC revealed
that they are rather not linear (Cérantola et al. 2006). It seems
that S. muticum does not produce different compounds
J Appl Phycol (2014) 26:1215–1230 1227
according to geographical zones studied. According to quan-
titative analyses, only the amount varies. It is possible, con-
sidering phenolic content as a pool, that the relative propor-
tions of the different compounds vary. This is easiest to see on
the
1
H NMR spectra: the French PC profile (with visible
peaks) is different from that of Portuguese and Norvegian
populations in the aromatic area. Phenolic compounds have
already beenisolated and identified in certain brown seaweeds
(Kang et al. 2005; Cérantola et al. 2006;SinghandBharate
2006). Here, even if we know the overall structure of the
isolated compounds, we still need to find out the number of
repeated units within the molecules to really discern the struc-
ture of the present compounds. Mass spectrometry analyses are
therefore currently in progress at the laboratory, which should
provide us with answers regarding the various sizes of
phlorethols present in extracts and also give an idea of the
category of compounds varying according to the environmental
conditions and thus the geographical position of S. muticum .
Spatial variation in levels of brown algal phenolic com-
pounds has been examined at very different scales (Table 4).
Van Alstyne et al. (1999) highlighted this by presenting num-
ber of various studies made in the field. Among these studies
on the brown algae, Sargassum is a model classically studied
for the variability of its phenolic content (Plouguerné et al.
2006; Le Lann et al.2012), but, to our knowledge, no previ-
ous study has focused on the spatial variability of both the
quantity and the quality of phlorotannins on a large scale
(Atlantic coasts). Therefore, the present study allowed us to
gain a first idea of the variability of this type of compound
produced by a species on a large geographical scale. Studies
on the variability of macromolecules (carbohydrates, lipids
and proteins) of S. muticum along to the same gradient are
now in progress. The results should provide an overview of
compartments subject to variation for the same species
according to its geographical position, especially in a context
of global change.
Future research
Phenolics or phlorotannins can have many applications in
industry, correlated with their numerous properties: anti-
diabetic, anti-cancer, anti-oxidation, antibacterial, radioprotec-
tive and anti-HIV (Kohen and Nyska 2002; Nakai et al. 2006;
Kuda et al. 2007; Kumar Chandini et al. 2008; Gupta and
Abu-Ghannam 2011; Yong-Xin et al. 2011). Phloroglucinol
can be used as a coupling agent in printing, in explosives and
for the industrial synthesis of pharmaceuticals (Flopropione).
The interest of this molecule makes it important to try to find
ways of exploiting natural marine resources like seaweeds.
However, numerous studies show that these compounds can
vary a great deal in terms of polarity and structure, according
to species of seaweed and even within the same species
according to the environmental conditions (Jormalainen and
Honkanen 2001; Hemmi and Jormalainen 2004; Fairhead
et al.2005;Connanetal.2007). It is thus rather difficult to
find a standard extraction protocol for all these compounds.
Also, if we wanted to use them in the food or pharmaceutical
industries, extraction and purification processes would need to
comply with European requirements. In a previous study,
Tanniou et al. (2013) demonstrated for the first time the
unquestionable usefulness of using modern, solvent-free and
environmentally friendly methods for the non-denaturing ex-
traction of brown algal polyphenols, using S. muticum as a
model. In particular, CPE and PLE seemed to be the most
promising methods for the extraction of polyphenols with
useful antioxidant potential. It will thus be necessary to re-
place the acetone–water extraction by one of these processes,
for example. Finally, large biomasses of S. muticum are pres-
ent at the northern and southern extremities of Europe. This
available biomass contains a large amount of phenolics, which
can be purified to a point where phenolic structure can be
determined. The purified extracts have good anti-ROS and
antibacterial activities and, consequently, could be very useful
for future industrial applications. As we find S. muticum in
great quantities on the Atlantic coasts, algal harvesting could
be possible in Europe. However, the particular weather con-
ditions of each country could represent an additional barrier.
For example, it is difficult to have access to seaweeds all year
long in Norway. The exploitation of this species would thus be
easier in southern Europe, e.g. in Portugal. Besides being
more easily accessible, our study showed seaweeds in this
country are richer in phenolics. However, in countries such as
Ireland, France and Spain,where the algae did not produce the
highest amount of phenolics in our results, such exploitation
would be a compromise. A solution could be the cultivation of
S. muticum under controlled conditions which mimic natural
stresses conditions (light intensity, temperature, etc) to force S.
Tabl e 4 Different scales of spatial variation in levels of brown algal
phenolic compounds according to published reports
Scale Examples Authors
Large Interspecific variations
between hemispheres
Steinberg (1989,1992);
Steinberg et al. (1995)
Interspecific variations
between temperate and
tropical habitats
Steinberg (1986); Van Alstyne
and Paul (1990); Targett
et al. (1992)
Average Inter- and intraspecific
variations within a large
geographical area (more
than hundred km)
Pavia and Aberg (1996); Van
Alstyne et al. (1999); Le
Lann et al. (2011)
Small Inter- and intraspecific
variations between sites in
the same country or small
geographical area (ten to
hundred km)
Steinberg (1989); Targett et al.
(1992,1995); Plouguerné
et al. (2006); Connan et al.
(2007); Le Lann et al.
(2012a)
1228 J Appl Phycol (2014) 26:1215–1230
muticum to produce phenolics in large quantities (metabolic
forcing), thus making the exploitation of Irish, French and
Spanish biomasses possible.
Conclusion
Our paper presents the first study taking into account the
spatial variability of the phenolic compounds for one species
along a large spatial scale and the potential of this species for a
future use in industry as anti-ROS or anti-bacterial molecules.
The most interesting extracts were those from Portuguese
populations, which display the best anti-ROS activities after
purification and contain a very large amount of PCs. S.
muticum biomass is available in considerable quantities on
Portuguese coasts where the environmental conditions (espe-
cially weather) allow it to be harvested easily for a large part of
the year. This brown invasive macroalga, which caused envi-
ronmental disturbance when it arrived, could finally become a
valuable marine resource for European countries.
Acknowledgments This study is part of the PhD. thesis work carried
out by the first author at the Laboratoire des Sciences de l’Environnement
Marin (LEMAR UMR 6539), which is part of the IUEM (UBO-UEB). It
was supported by the Ministère de l’Education Nationale, de
l’Enseignement Supérieur et de la Recherche (UBO grant to the first
author). We would like to thank Helen McCombie-Boudry of the Bureau
de Traduction de l'Université (BTU) of the University of Western Britta-
ny, Brest, France for her fruitful assistance with the improvement of the
English in this article. This study is related to two research projects:
BIOTECMAR (Interreg IVB, 2009-2011) and INVASIVES (Era-net,
2012-2016).
References
Aruoma OI, Spencer JPE, Warren D, Jenner P, Butler J, Halliwell B
(1997) Characterization of food antioxidants, illustrated using com-
mercial garlic and ginger preparations. Food Chem 60:149–156
Bandoniené D, Pulaskas A, Veskutonis PR, Gruzdiené D (2000)
Preliminary screening of antioxidant activity of some plant extracts
in rapeseed oil. Food Res Int 33:785–791
Bourgougnon N, Stiger-Pouvreau, V (2011) Chapter 4: Chemodiversity
and bioactivity within red and brown macroalgae along the French
coasts, metropole and overseas departments and territories. In: Kim
S.-K. (ed) Handbook of marine macroalgae: biotechnology and
applied phycology. Wiley, New York, pp. 58–105
Cérantola S, Breton F, Ar Gall E, Deslandes E (2006) Co-occurrence and
antioxidant activity of fucol and fucophlorethol classes of polymeric
phenols in Fucus spiralis . Bot Mar 49:347–351
Chew YL, Lim YY, Omar M, Khoo KS (2008) Antioxidant activity of
three edible seaweeds from two areas in South East Asia. LWT-Food
Sci Technol 41:1067–1072
Chua MT, Tung YT, Chang ST (2008) Antioxidant activities of ethanolic
extracts from the twigs of Cinnamomum osmophloeum.Bioresour
Technol 99:1918–1925
Connan S, Stengel DB (2011) Impacts of ambient salinity and copper on
brown algae: 2. interactive effects on phenolic pool and assessment
of metal binding capacity of phlorotannin. Aquat Toxicol 14:1–13
Connan S, Goulard F, Stiger V, Deslandes E, Ar Gall E (2004)
Interspecific and temporal variation in phlorotannin levels in an
assemblage of brown algae. Bot Mar 47:410–416
Connan S, Deslandes E, Ar Gall E (2007) Influence of day–night and
tidal cycles on phenol content and antioxidant capacity in three
temperate intertidal brown seaweeds. J Exp Mar Biol Ecol 349:
359–369
De la Coba F, Aguilera J, Figueroa FL, de Galvez MV, Herrera E (2009)
Antioxidant activity of mycosporine-like amino acids isolated from
three red macroalgae and one marine lichen. J Appl Phycol 21:161–9
Engelen A, Espirito-Santo C, Simoes T, Monteiro C, Serrao EA, Pearson
GA, Santos ROP (2008) Periodicity of propagule expulsion and
settlement in the competing native and invasive brown seaweeds,
Cystoseira humilis and Sargassum muticum (Phaeophyta). Eur J
Phycol 43:275–282
Fairhead VA, Amsler CD, McClintock JB, Baker BJ (2005) Variation in
phlorotannin content within two species of brown macroalgae
(Desmarestia anceps and D. menziesii ) from the Western
Antarctic Peninsula. Polar Biol 28:680–686
Gupta S, Abu-Ghannam N (2011) Bioactive potential and possible health
effects of edible brown seaweeds. Trends Food Sci Technol 22:315–326
Hellio C, Berge JP, Beaupoil C, Le Gal Y, Bourgougnon N (2002)
Screening of marine algal extracts for antisettlement activities
against microalgae and macroalgae. Biofouling 18:205–215
Hellio C, Marechal JP, Véron B, Bremer G, Clare A, Le Gal Y (2004a)
Seasonal variation of antifouling activities of marine algae from the
Brittany coast (France). Mar Biotechnol 6:67–82
Hellio C, Simon Colin C, Clare AS, Deslandes E (2004b) Anti-settlement
activities of Grateloupia turuturu hydroalcoholic extracts on
cyprids of Balanus amphitrite. Biofouling 20:139–145
Hemmi A, Jormalainen V (2004) Geographic covariation of chemical
quality of the host alga Fucus vesiculosus with fitness of the her-
bivorous isopod Idotea baltica. Mar Biol 145:759–768
Huang D, Ou B, Prior RL (2005) The chemistry behind antioxidant
capacity assays. J Agric Food Chem 53:1841–56
Incera M, Olabarria C, Troncoso JS, Lopez J (2009) Response of the
invader Sargassum muticum to variability in nutrient supply. Mar
Ecol Prog Ser 377:91–101
Ito N, Hirose M, Fukushima H, Tsuda T, Shirai T, Tatenatsu M (1986)
Studies on antioxidants: their carcinogenic and modifying effects on
chemical carcinogens. Food Chem Toxicol 24:1071–1092
Jennings JK, Steinberg PD (1997) Phlorotannins versus other factors
affecting epiphyte abundance on the kelp Ecklonia radiata.
Oecologia 109:461–473
Jormalainen V, Honkanen T (2001) Multiple cues for phenotypic plastic-
ity in phlorotannin production of the bladder wrack Fucus
vesiculosus. Phycologia 40:59–60
Kang KA, Lee KH, Chae S, ZhangR, Jung MS, LeeY, Kim SY, Kim HS,
Joo HG, Park JW, Ham YM, Lee NH, Hyun JW (2005) Eckol
isolated from Ecklonia cava attenuates oxidative stress induced cell
damage in lung fibroblast cells. FEBS Lett 579:6295–6304
Kaur C, Kapoor HC (2002) Anti-oxidant activity and total phenolic
content of some Asian vegetables. Int J Food Sci Tech 37:153–161
Kohen R, Nyska A (2002) Oxidation of biological systems: oxidative
stress phenomena, antioxidants, redox reactions and methods for
their quantification. Toxicol Pathol 30:620–650
Koivikko R, Loponen J, Honkanen T, Jormalainen V (2005) Contents of
soluble, cell-wall-bound and exuded phlorotannins in the brown
alga Fucus vesiculosus, with implications on their ecological func-
tions. J Chem Ecol 31:195–212
Koivikko R, Loponen J, Pihlaja K, Jormalainen V (2007) High-
performance liquid chromatographic analysis of phlorotannins from
the brown alga Fucus vesiculosus. Phytochem Anal 18:326–332
Koleva II, Van Beek TA, Linssen JPH (2002) Screening of plant extracts
for antioxidant activity: a comparative study on three testing
methods. Phytochem Anal 13:8–17
J Appl Phycol (2014) 26:1215–1230 1229
Kraan S (2008) Sargassum muticum (Yendo)FensholtinIreland:an
invasive species on the move. J Appl Phycol 20:825–832
Kuda T, Tsunekawa M, Hishi T, Araki Y (2005) Antioxidant
properties of dried ‘kayamo-nori’,abrownalgaScytosiphon
lomentaria (Scytosiphonales, Phaeophyceae). Food Chem 89:
617–622
Kuda T, Kunii T, Goto H (2007) Varieties of antioxidant and antibacterial
properties of Ecklonia stolonifera and Ecklonia kurome products
harvested and processed in the Noto Peninsula, Japan. Food Chem
103:900–905
Kumar Chandini S, Ganesan P, Bhaskar N (2008) In vitro antioxidant
activities of three selected brown seaweeds of India. Food Chem
107:707–713
Le Lann K, Jégou C, Stiger-Pouvreau V (2008) Effect of different
conditioning treatments on total phenolic content and antioxidant
activities in two Sargassacean species: comparison of the frondose
Sargassum muticum (Yendo) Fensholt and the cylindrical Bifurcaria
bifurcata R Ross. Phycol Res 56:238–245
Le Lann K, Ferret C, VanMee E, Spagnol C, Lhuillery M, Stiger-
Pouvreau V (2012a) Total phenolic, size-fractionated phenolics
and fucoxanthin content of tropical Sargassaceae (Fucales,
Phaeophyceae) from the South Pacific Ocean: spatial and specific
variability. Phycol Res 60:37–50
Le Lann K, Connan S, Stiger-Pouvreau V (2012b) Phenology, TPC and
size-fractioning phenolics variability in temperate Sargassaceae
(Phaeophyceae, Fucales) from Western Brittany: Native versus in-
troduced species. Mar Environ Res 80:1–11
Nagai T, Inoue R, Inoue H, Suzuki N (2003) Preparation and antioxidant
properties of water extracts of propolis. Food Chem 80:29–33
Nakai M, Kagayama N, Nakahara K, Miki W (2006) Phlorotannins as
radical scavengers from the extract of Sargassum ringgoldianum.
Mar Biotechnol 8:409–414
Olabarria C, Rodil IF, Incera M, Troncoso JS (2009) Limited impact of
Sargassum muticum on native algal assemblages from rocky inter-
tidal shores. Mar Environ Res 67:153–158
Oueslati S, Trabelsi N, Boulaaba M, Legault J, Abdelly C, Ksouri R
(2012) Evaluation of antioxidant activities of the edible and medic-
inal Suaeda species and related phenolic compounds. Ind Crop Prod
36:513–518
Parys S, Kehraus S, Pete R, Kupper FC, Glombitza KW, Konig GM
(2009) Seasonal variation of polyphenolics in Ascophyllum
nodosum (Phaeophyceae). Eur J Phycol 44:331–338
Pavia H, Aberg P (1996) Spatial variation in polyphenolic content of
Ascophyllum nodosum (Fucales, Phaeophyta). Hydrobiologia 326/
327:199–203
Plouguerné E, Le Lann K, Connan S, Jechoux G, Deslandes E, Stiger-
Pouvreau V (2006) Spatial and seasonal variation in density, repro-
ductive status, length and phenolic content of the invasive brown
macroalga Sargassum muticum (Yendo) Fensholt along the coast of
Western Brittany (France). Aquat Bot 85:337–344
Plouguerné E, Hellio C, Deslandes E, Véron B, Stiger-Pouvreau V (2008)
Anti-microfouling activities in extracts of two invasive algae:
Grateloupia turuturu and Sargassum muticum .BotMar51:
202–208
Plouguerné E, Georgantea P, Ioannou E, Vagias C, Roussis V, Hellio C,
Kraffe E, Stiger-Pouvreau V (2010) Anti-microfouling activity of
lipidic metabolites from the invasive brown alga Sargassum
muticum (Yendo) Fensholt. Mar Biotechnol 12:52–61
Ragan MA, Glombitza KW (1986) Phlorotannins, brown algal polyphe-
nols. Prog Phycol Res 4:129–241
Sanoner P, Guyot S, Marnet N (1999) Polyphenol profiles of French cider
apple varieties (Malus domestica sp.). J Agric Food Chem
47:4847–4853
Sastry VMVS, Rao GRK (1994) Antibacterial substances from marine
algae: successive extraction using benzene, chloroform and metha-
nol. Bot Mar 37:357–360
Schoenwaelder MEA, Clayton MN (1998) Secretion of phenolic sub-
stances into the zygote wall and cell plate in embryos of Hormosira
and Acrocarpia (Fucales, Phaeophyceae). J Phycol 34:969–980
Sieburth JM, Conover JT (1965) Sargassum tannin, an anti-biotic which
retards fouling. Nature 208:52–53
Singh IP, Bharate SB (2006) Phloroglucinol compounds of natural origin.
Nat Prod Rep 23:558–591
Steinberg PD (1986) Chemical defenses and the susceptibility of tropical
brown algae to herbivores. Oecologia 69:628–630
Steinberg PD (1989) Biogeographical variation in brown algal polyphe-
nolics and other secondary metabolites: comparison between tem-
perate Australasia and North America. Oecologia 78:374–383
Steinberg PD (1992) Geographical variation in the interaction between
marine herbivores and brown algal secondary metabolites. In: Paul
VJ (ed) Ecological roles of marine natural products. Comstock,
Ithaca, pp 51–92
Steinberg PD, Estes JA, Winter FC (1995) Evolutionary consequences of
food chain length in kelp forest communities. Proc Natl Acad Sci
USA 92:8145–8148
Stiger V, Deslandes E, Payri CE (2004) Phenolic contents of two brown
algae, Turbinaria ornata and Sargassum mangarevense on Tahiti
(French Polynesia): interspecific, ontogenic and spatio-temporal
variations. Bot Mar 47:402–409
Tanniou A, SerranoLeon E, Vandanjon L, Ibanez E, Mendiola JA,
Cerantola S, Kervarec N, LaBarre S, Marchal L, Stiger-Pouvreau
V (2013) Green improved processes to extract bioactive phenolic
compounds from brown macroalgae using Sargassum muticum as
model. Talanta 104:44–52
Targett NM, Arnold TM (2001) Effects of secondary metabolites on
digestion in marine herbivores. In: McClintock JB, Baker BJ (eds)
Marine chemical ecology. CRC, Boca Raton, pp 391–411
Targett NM, Coen LD, Boettcher AA, Tanner CE (1992) Biogeographic
comparisons of marine algal phenolics: evidence against a latitudi-
nal trend. Oecologia 89:464–470
Targett NM, Boettcher AA, Targett TE, Vrolijk NH (1995) Tropical marine
herbivore assimilation of phenolic-rich plants. Oecologia 103:170–179
Toth GB, Pavia H (2001) Removal of dissolved brown algal
phlorotannins using insoluble polyvinylpolypyrrolidone (PVPP). J
Chem Ecol 27:1899–1910
Van Alstyne KL, Paul VJ (1990) The biogeography of polyphenolic
compounds in marine macroalgae: temperate brown algal defenses
deter feeding by tropical herbivorous fishes. Oecologia 84:158–163
Van Alstyne KL, McCarthy JJ, Hustead CL, Duggins DO (1999)
Geographic variation in polyphenolic levels of Northeastern
Pacific kelps and rockweeds. Mar Biol 133:371–379
Yong-Xin L, Isuru W, Yong L, Se-Kwon K (2011) Phlorotannins as
bioactive agents from brown algae. Process Biochem 46:2219–2224
Zubia M, Fabre MS, Kerjean V, Le Lann K, Stiger-Pouvreau V, Fauchon
M, Deslandes E (2009) Antioxidant and antitumoural activities of
some Phaeophyta from Brittany coasts. Food Chem 116:693–701
1230 J Appl Phycol (2014) 26:1215–1230
