An extract of the brown seaweed Ascophyllum nodosum rich in phlorotan-nins has been studied for its inhibitory activity against oxidative stress, inflammation, and senescence.. nodosum e
Trang 1Antioxidant, Anti-inflammatory, and Anti-senescence
Activities of a Phlorotannin-Rich Natural Extract
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 phlorotan-nins 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 DOI 10.1007/s12010-012-9761-1
M Dutot ( *):R Fagon:M Hemon
Yslab, 2 rue Félix Le Dantec, 29000 Quimper, France
e-mail: melody.dutot@yslab.fr
P Rat
Chimie-Toxicologie Analytique et Cellulaire (EA 4463), Sorbonne Paris Cité, Faculté de Pharmacie, Université Paris Descartes, 75 006 Paris, France
Trang 2reactive species overproduction, including reactive oxygen species (ROS) Activation of oxidative stress has numerous cellular consequences such as increased levels of proinflam-matory 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 inflam-matory 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 dysfunc-tion 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 com-pounds embrace a considerable range of substances that possess an aromatic ring bearing one or more hydroxyl substituents Phenolic compounds act as antioxidants with mecha-nisms 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
Trang 3calf 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 106cells/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 20μM DCFH-DA solution; fluorescence detection (λexc0485 nm, λem0535 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 (λexc0340 nm, λem0440 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
Trang 4ROS 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 0.5μg/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, respec-tively, 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
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
0.0 0.5 1.0 1.5 2.0 2.5
A nodosum extract concentrations (%)
**
$$
Fig 1 ROS production induced by
tBHP on epithelial cells Epithelial
cells were incubated with A
nodo-sum extract for 20 min The cells
were rinsed and then oxidative
stress was induced by tBHP at
500 μM for 15 min ROS
produc-tion was quantified using
H2DCF-DA test **p<0.005 compared to
negative control, $$p<0.005
com-pared to tBHP alone
0 5 10 15 20 25
A nodosum extract concentrations (%)
*
*
*
Fig 2 TNF- α release induced by
LPS on macrophages
Macro-phages 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
Trang 5Elucidating the mechanisms controlling aging has significant clinical implications be-cause 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
0.0 0.5 1.0 1.5 2.0 2.5
A nodosum extract concentrations (%)
Fig 3 IL-6 release induced by
LPS on macrophages
Macro-phages 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
1.20
1.65 1.65
2.33
0 0.5 1 1.5 2 2.5 3
Positive control (resveratrol 100µM)
A nodosum extract 0.2%
20 minutes 24 hours
*
*
* *
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
Trang 6inflammatory 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 fluorophore-containing 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 non-physiological 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 sirtuin-activating compounds (STAC) The A ring of the most powerful STAC features a meta-positioning 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, anti-inflammatory, 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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