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, 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 Appl Biochem Biotechnol (2012) 167:2234–2240 2235 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 2236 Appl Biochem Biotechnol (2012) 167:2234–2240 calf serum, mM L-glutamine, 50 IU/mL penicillin, and 50 IU/mL streptomycin The medium was changed every 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, 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 and % 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 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 ROS production induced by tBHP on epithelial cells Epithelial cells were incubated with A nodosum extract for 20 The cells were rinsed and then oxidative stress was induced by tBHP at 500 μM for 15 ROS production was quantified using H2DCFDA test **p