Applied biochemistry and biotechnology mélody dutot, roxane fagon, marc hemon, patrice rat antioxidant, anti inflammatory, and anti senesce

Appl Biochem Biotechnol (2012) 167:2234–2240
DOI 10.1007/s12010-012-9761-1

Antioxidant, Anti-inflammatory, and Anti-senescence
Activities of a Phlorotannin-Rich Natural Extract
from Brown Seaweed Ascophyllum nodosum
Mélody Dutot & Roxane Fagon & Marc Hemon & Patrice Rat

Received: 28 September 2011 / Accepted: 29 May 2012 /
Published online: 13 June 2012
# Springer Science+Business Media, LLC 2012

Abstract Aging at the cellular level is characterized by oxidative stress, inflammation, and
cell senescence. An extract of the brown seaweed Ascophyllum nodosum rich in phlorotannins has been studied for its inhibitory activity against oxidative stress, inflammation, and
senescence. A. nodosum extract at 0.2 % prevented tBHP-induced reactive oxygen species
production (evaluated using the H2DCF-DA test in cytofluorometry) in epithelial cells and
LPS-induced TNF-α and IL-6 release (evaluated using ELISA technique) in macrophages.
A. nodosum extract also increased nuclear SIRT1 activity in epithelial cells. Altogether, these
beneficial cellular effects of phlorotannin-rich A. nodosum extract could be used in topical
therapeutic formulations against aging.
Keywords Oxidative stress . Inflammation . Sirtuin . Aging . Seaweed . Phlorotannins

Introduction
In almost every country, the proportion of people aged over 60 years is growing faster than
any other age group, as a result of both longer life expectancy and declining fertility rates. In
2008 in the USA, people aged 65 and over accounted for 13 % of the total population [1]. In
the European Union, the average life expectancy at birth increased over the last 50 years by
about 10 years [2].
Aging is characterized by functional declines that lead to morbidity and mortality. The
oxidative stress hypothesis for aging was first introduced in the 1980s and later explored in
depth. Degradation in aging results from a redox imbalance caused by incessant oxidative

stress and compromised antioxidant defense systems [3]. This redox imbalance induces
M. Dutot (*) : R. Fagon : M. Hemon
Yslab, 2 rue Félix Le Dantec, 29000 Quimper, France
e-mail:
P. Rat
Chimie-Toxicologie Analytique et Cellulaire (EA 4463), Sorbonne Paris Cité, Faculté de Pharmacie,
Université Paris Descartes, 75 006 Paris, France


Appl Biochem Biotechnol (2012) 167:2234–2240

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reactive species overproduction, including reactive oxygen species (ROS). Activation of
oxidative stress has numerous cellular consequences such as increased levels of proinflammatory molecules, a common phenomenon during aging [4]. Besides oxidative stress,
inflammation is directly linked with aging; indeed, the innate immune system is weakened
throughout life by antigenic stress leading to an age-dependent upregulation of the inflammatory response [5]. Another consequence of oxidative stress is the upregulation of SIRT1
[6]. SIRT1 is a NAD-dependent histone deacetylase. SIRT1 deacetylates p53 thereby
inhibiting apoptosis [7] and SIRT1 overexpression antagonizes cellular senescence [8].
To maintain redox balance, organisms require a network of antioxidant systems, as
well as a functioning antioxidant defense system. A hallmark of age-related dysfunction is the organism's inability to modulate redox homeostasis. Antioxidant enzymes
(catalase, superoxide dismutase, glutathione peroxidase…) work in several ways. For
one, they may reduce the energy of the free radical or give up some of their electrons
for its use, thereby causing it to become stable. Antioxidant enzymes may also stop
the free radical from forming in the first place. In addition, they may also interrupt an
oxidizing chain reaction to minimize the damage caused by free radicals. Cofactors of
antioxidant enzymes include manganese, zinc, copper, and selenium. In addition,
many vitamins such as vitamins C, E, A (beta-carotene) and nutrients such as lutein,
lycopene, vitamin B2, and coenzyme Q10 have antioxidant properties.
Diets containing an abundance of fruit and vegetables are protective against a variety of

diseases, particularly cardiovascular disease and cancer. The primary nutrients thought to
provide the protection afforded by fruit and vegetables are the antioxidants [9]. Polyphenols
constitute one of the most numerous and widely distributed groups of substances in the plant
kingdom, with more than 8,000 phenolic structures currently known [10]. Phenolic compounds embrace a considerable range of substances that possess an aromatic ring bearing
one or more hydroxyl substituents. Phenolic compounds act as antioxidants with mechanisms involving both free radical scavenging and metal chelation. Phlorotannins are unique
polyphenolic compounds which are not found in terrestrial plants but found only in some
brown algal species. They result from the tridimensional polymerization of phloroglucinol
and possess potent antioxidant activity [11, 12]. Ascophyllum nodosum is one of the richest
sources of phlorotannins [13]. A. nodosum is a brown seaweed characteristic of the mild
intertidal zones of North Atlantic temperate rocky shores. In France, the distribution of A.
nodosum is concentrated along the coasts of Brittany, which is a major site along with
Norway for A. nodosum harvesting along the European rocky shores [14].
The aim of our study was to point up the antiaging properties of an A. nodosum extract
rich in phlorotannins on human epithelial cells.

Materials and Methods
Chemicals and Extract Chemicals for cellular culture were purchased from Eurobio (Les Ulis,
France). Tert-butyl hydroperoxide (tBHP) and LPS from Escherichia coli were purchased from
Sigma-Aldrich (Saint-Quentin-Fallavier, France). H2DCF-DA, ELISA, and SIRT1 kits were
purchased from Invitrogen (Villebon sur Yvette, France), R&D Systems (Abingdon, UK), and
Abnova (Taipei City, Taiwan), respectively. A. nodosum extract contains 18 % of phlorotannins
(Yslab, Quimper, France).
Cell Culture Human epithelial cells (ARPE-19 and WKD cell lines) were cultured under
standard conditions in Dulbecco's modified eagle's medium supplemented with 10 % fetal


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Appl Biochem Biotechnol (2012) 167:2234–2240

calf serum, 2 mM L-glutamine, 50 IU/mL penicillin, and 50 IU/mL streptomycin. The
medium was changed every 2 days. Confluent cultures were removed by trypsin incubation,
and then the cells were counted. They were seeded into 96-well culture microplates at a
density of 80,000 cells per well and into Petri culture dishes at a density of 200,000 cells per
dish and kept at 37 °C for 24 h.
Human leukemic monocytes (U937 cell line) were cultured in RPMI-1640 medium
supplemented with 10 % fetal calf serum, 2 mM L-glutamine, 50 IU/mL penicillin, and
50 IU/mL streptomycin. U937 cells were differentiated in macrophages using phorbol
myristate acetate at 16 ng/mL for 48 h. Once attached to the flask bottom, the cells were
scraped, counted, and seeded into 96-well culture microplates at a density of 106 cells/mL
and kept at 37 °C for 24 h. Cells were incubated with Calophyllum inophyllum oil at 1 and
2 % during 15 min; then, oil was removed and cells were washed, incubated with cell culture
medium at 2.5 % of FCS, and kept at 37 °C for 24 h
ROS Production Evaluation ROS were detected with the 2′,7′-dichlorofluorescein diacetate
probe. Once inside the cell, this probe is cleaved by endogenous esterases and can no longer
pass out of the cell. The de-esterified product becomes the fluorescent compound 2′,7′dichlorofluorescein after oxidation by reactive oxygen species. Cells were incubated for
20 min with a 20 μM DCFH-DA solution; fluorescence detection (λexc 0485 nm, λem 0535 nm)
was undertaken with a microplate fluorometer (Safire, Tecan, France).
Cytokine Release Measurement The release of TNF-α and IL-6 in cell supernatants was
determined by ELISA. After LPS incubation, cell supernatants were harvested and
stored at −20 °C until use for cytokine measurements. The quantity of released
cytokines was measured according to the manufacturer's instructions (R&D Systems
DTA00C for TNF-α with a minimum detectable dose that ranged from 0.5 to 5.5 pg/mL and
D6050 for IL-6 TNF-α with a minimum detectable dose typically less than 0.70 pg/mL).
SIRT1 Activity Assessment The SIRT1/Sir2 deacetylase fluorometric assay kit was used to
assess SIRT1 activity. After incubation with A. nodosum extract, the cells were lysed and
nuclear SIRT1 was isolated (sucrose gradient centrifugation, sonication followed by another
centrifugation). The SIRT1/Sir2 deacetylase fluorometric assay was performed as indicated
in the Abnova product sheets. Fluorescence detection (λexc 0340 nm, λem 0440 nm) was
undertaken with a microplate fluorometer (Safire, Tecan, France). Resveratrol served as a

positive control for SIRT1 activation.
Statistics Results were obtained in fluorescence units and were expressed as percentage of
the control ± standard deviation of at least three experiments realized in triplicate. The mean
values for each concentration were analyzed with a one-way ANOVA test followed by
Dunnett's test (except for SIRT1 activity: Student's test) using Sigma Stat 2.0 software, and
the level of significance was fixed at 0.05.

Results
ROS Production Evaluation on Retinal Cells
tBHP was used at 500 μM as an inducer of oxidative stress. As shown in Fig. 1, tBHP
significantly increased ROS production, ×1.51 compared to control. The tBHP-increased


Fig. 1 ROS production induced by
tBHP on epithelial cells. Epithelial
cells were incubated with A. nodosum extract for 20 min. The cells
were rinsed and then oxidative
stress was induced by tBHP at
500 μM for 15 min. ROS production was quantified using H2DCFDA test. **p<0.005 compared to
negative control, $$p<0.005 compared to tBHP alone

Fold change compared to negative
control

Appl Biochem Biotechnol (2012) 167:2234–2240

2237

2.5
2.0

**
1.5

$$

1.0
0.5
1.51

1.17

1.14

1.57

0.0
0

0.1
0.2
A. nodosum extract concentrations (%)

0.5

ROS overproduction was prevented by A. nodosum extract 0.1 and 0.2 %. The most efficient
concentration was 0.2 % as ROS production (×1.14) was not different from negative control
(×1) and statistically different from positive control (×1.51, tBHP alone). A. nodosum extract
at 0.5 % had no effect on ROS overproduction.
Proinflammatory Cytokine Release on Macrophages

LPS from E. coli was used at 0.5 μg/mL as an inducer of TNF-α (Fig. 2) and IL-6 (Fig. 3)
release by macrophages. LPS increased TNF-α and IL-6 release, ×16.8 and ×1.9, respectively, compared to negative control. A. nodosum extract at 0.05 % decreased TNF-α release
but had no effect on IL-6 release. A. nodosum extract at 0.2 % fully prevented LPS-induced
TNF-α and IL-6 release.
Evaluation of SIRT1 Activity on Conjunctival Cells

Fig. 2 TNF-α release induced by
LPS on macrophages. Macrophages were incubated with A.
nodosum extract for 2 h before
LPS 0.5 μg/mL incubation for
24 h. TNF-α release was quantified
using ELISA. *p<0.01 compared
to negative control

Fold change compared tonegative
control

A. nodosum extract 0.2 % stimulated SIRT1 activation. When it was incubated for 20 min, A.
nodosum extract induced a 1.65-fold change in SIRT1 activity, and when it was incubated
for 24 h, it induced a 2.33-fold change in SIRT1 activity (Fig. 4). Resveratrol was used as a
positive control for SIRT1 activity.

25
20

*
*

15
10

*
5
0
0

0.05

0.1

A. nodosum extract concentrations (%)

0.2


Fig. 3 IL-6 release induced by
LPS on macrophages. Macrophages were incubated with A.
nodosum extract for 2 h before
LPS 0.5 μg/mL incubation for
24 h. IL-6 release was quantified
using ELISA

Appl Biochem Biotechnol (2012) 167:2234–2240
Fold change compared to negative
control

2238
2.5
2.0
1.5

1.0
0.5
0.0
0

0.05

0.1

0.2

A. nodosum extract concentrations (%)

Discussion

Fig. 4 Effect of Ascop extract on
SIRT1 activity in epithelial cells.
Epithelial cells were incubated
with A. nodosum extract for
20 min or 24 h. SIRT1 was
extracted from the nucleus and its
activity was quantified using a
fluorescent substrate. *p<0.01,
**p<0.05 compared to negative
control

Fold change compared to negative
control

Elucidating the mechanisms controlling aging has significant clinical implications because aging reduces the function of organs and increases the risk of diseases [15].

During the past decades, a series of scientific studies demonstrate that the progression
of severe diseases, even coronary heart diseases, often can be reversed simply by
making comprehensive changes in diet and lifestyle [16–18]. Seaweed as a staple item
of the diet has been used in Japan, Korea, and China since prehistoric times. We
focused our attention on the preventive beneficial effects of a phlorotannin-rich extract
of A. nodosum, a brown seaweed. We observed that the tested A. nodosum extract has
antioxidant properties when incubated at 0.1 and 0.2 %. At higher concentration
(0.5 %), A. nodosum extract has no more antioxidant properties on our model. The
dual antioxidant/prooxidant effect of polyphenols is well known [19–21]. They can
either scavenge or generate radicals depending on their concentrations. Their prooxidant
effect has been related to their iron- and copper-reducing activities. For the following
experiments, the concentration of A. nodosum extract did not exceed 0.2 %. According
to the molecular inflammatory theory of aging, age-related oxidative stress causes the
activation of inflammatory system molecules [22]. The tested A. nodosum extract
showed potent anti-inflammatory effects based on their ability to inhibit cytokines
release. TNF-α and IL-6 are both multifunctional cytokines with important regulatory
roles in numerous processes. TNF-α, with IL-1ß, is the first cytokine in the

20 minutes

24 hours

3

**
2.5

*

2

*

1.5
2.33

1
0.5

1.65

1.65

1.20

0
Positive control (resveratrol
100µM)

A. nodosum extract 0.2%


Appl Biochem Biotechnol (2012) 167:2234–2240

2239

inflammatory cascade. TNF-α constitutes a direct and important stimulator of IL-6
production. Circulating levels of TNF-α and IL-6 increase with aging [23]. Thereby,
the tested A. nodosum extract, inhibiting TNF-α and IL-6 release, is of interest in
antiaging therapies.

Genetic overexpression of protein deacetylase Sir2 increases longevity in a variety of
lower organisms [24, 25], and this has prompted interest in the effects of its closest
mammalian homologue, SIRT1 on aging. SIRT1 downregulates p53-dependent apoptosis
[7] and increases FOXO-dependent stress resistance [26]. Assays using fluorophorecontaining peptides as substrate for recombinant SIRT1 to reveal SIRT1 activators have
been criticized. Indeed, resveratrol, a well-known SIRT1 activator, has been shown to
enhance binding and deacetylation of peptide substrates that contain Fluo de Lys, a nonphysiological fluorescent molecule, but had no effect on binding and deacetylation of
acetylated peptides lacking the fluorophore [27]. For this reason, we did not incubate the
tested A. nodosum extract directly with recombinant SIRT1 but with living epithelial cells. In
this way, we took into account the metabolism of A. nodosum extract by epithelial cells and
possible targets that indirectly affect SIRT1. The tested A. nodosum extract increased SIRT1
activity after a short incubation time (20 min) and a longer incubation time (24 h). The increase
in SIRT1 activity was more important when A. nodosum extract was incubated 24 h compared
to 20 min. Plant polyphenols are well-known SIRT1 activators; they are referred to as sirtuinactivating compounds (STAC). The A ring of the most powerful STAC features a metapositioning of its phenolic hydroxyl groups [28], which is the case of phlorotannins.
Therefore, the way the tested A. nodosum extract enhances SIRT1 activity may be similar to
the way polyphenolic STAC does. The tested A. nodosum extract could act directly on SIRT1
like resveratrol, the most potent STAC, which lowers the Michaelis constant of SIRT1 through a
direct allosteric effect [29]. The tested A. nodosum extract may also be a STAC via its
antioxidant activity. Indeed, authors recently reported that oxidative stress decreases SIRT1
activity by a redox-dependent mechanism [30]. The tested phlorotannin-rich A. nodosum
extract that showed antioxidant activity in our model modulates the redox status of cells and
may indirectly act on SIRT1 activity.

Conclusion
The tested A. nodosum extract, rich in phlorotannins, showed potent antioxidant, antiinflammatory, and pro-SIRT1 activities. Such an extract, able to prevent ROS production
and cytokine release and to stimulate SIRT1, is very promising in antiaging therapies. It
opens new perspectives not only in dermatology and cosmetology as a skin protector but
also in ophthalmology; both fields have largely recourse to topical drugs that could include
the tested A. nodosum extract.
Acknowledgments The authors would like to thank Adebiopharm ER67.

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