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Neuroprotective Effects of Bioavailable PolyphenolDerived Metabolites against Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells Antonio Gonzalez-Sarrias, María Ángeles Núñez-Sánchez, Francisco A. Tomas-Barberan, and Juan Carlos Espín J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04538 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 21, 2016
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Journal of Agricultural and Food Chemistry
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Neuroprotective Effects of Bioavailable Polyphenol-Derived Metabolites against
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Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells
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Antonio González-Sarrías*, María Ángeles Núñez-Sánchez, Francisco A. Tomás-Barberán, Juan
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Carlos Espín.
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Research Group on Quality, Safety, and Bioactivity of Plant Foods, Dept. Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain
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*Correspondence: Dr. Antonio González-Sarrías, Research Group on Quality, Safety and
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Bioactivity of Plant Foods, Dept. Food Science and Technology, CEBAS-CSIC, P.O. Box 164,
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30100 Campus de Espinardo, Murcia, Spain. Tel: +34 968 396200; Ext: 6249, Fax: +34 968
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396213.
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E-mail:
[email protected] 17 18
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ABSTRACT
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Oxidative stress is involved in cell death in neurodegenerative diseases. Dietary polyphenols can
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exert health benefits, but their direct effects on neuronal cells are debatable since most phenolics are
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metabolized and do not reach the brain as they occur in the dietary sources. Herein, we evaluate the
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effects of a panel of bioavailable polyphenols and derived metabolites at physiologically relevant
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conditions against H2O2-induced apoptosis in human neuroblastoma SH-SY5Y cells. Among the 19
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metabolites tested, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, gallic acid,
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ellagic acid and urolithins prevented neuronal apoptosis via attenuation of ROS levels, increased
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REDOX activity and decreased oxidative stress-induced apoptosis by preventing the caspase-3
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activation via the mitochondrial apoptotic pathway in SH-SY5Y cells. This suggests that dietary
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sources containing the polyphenols precursors of these molecules such as cocoa, berries, walnuts,
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and tea could be potential functional foods to reduce oxidative stress associated with the onset and
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progress of neurodegenerative diseases.
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KEYWORDS: Gallic acid; Ellagic acid; Neuroprotective effects; Phenolic acids; Polyphenol;
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Urolithins.
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Oxidative stress is involved in neuronal cell death, which is one of the leading causes of
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neurodegenerative diseases such as Alzheimer's disease, Parkison's disease, or cerebral ischemia
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reperfusion after stroke.1 Oxidative stress is characterized by the imbalance between antioxidants
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and accumulation of reactive oxygen species (ROS) and reactive nitrogen species that can be
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spontaneously generated, such as superoxide anion and hydrogen peroxide, or made due to
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exogenous factors, such as drug exposure or radiation.2 ROS generally are found in the course of
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cell death, mainly by apoptosis caused by intracellular microenvironmental changes, and in the brain
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leading to neurodegeneration and cognitive decline.3
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There is much evidence showing that naturally occurring antioxidants such as vitamins, minerals
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and phenolic compounds present in high amounts in fruits, vegetables, and natural products
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scavenge the free radicals and their intake acts as an upstream preventive measure to
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neurodegeneration.4,5 In fact, dietary polyphenols have been reported to display potential
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neuroprotective effects as antioxidants preventing oxidation of proteins, lipid peroxidation, and
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generation of ROS as well as anti-inflammatory and anti-apoptotic properties in several in vitro
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models of toxicity and animal models of neurological disorders.6,7 In this regard, previous studies
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have indicated the neuroprotective effect of red wine, grapes, pomegranate, cocoa and tea-derived
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polyphenols as sources of flavan-3-ols, stilbenes such as resveratrol and ellagitannins (ETs) using
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animal models of neurodegenerative diseases.8-11 However, polyphenols are poorly bioavailable and
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cannot reach systemic tissues as they occur in the dietary sources. On the contrary, they are
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extensively metabolized and converted by colonic microbiota into other metabolic forms.6,12
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Therefore, the actual metabolites responsible for the in vivo neuroprotective effects of polyphenols
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are not entirely known.
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To date, only low concentrations of a few polyphenol-derived metabolites have been detected in
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the brain of animals, i.e. gallic acid (GA), ellagic acid (EA) and its gut microbial derived
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metabolites urolithins,13-15 resveratrol and its microbial metabolites,16 and also anthocyanins.17,18
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Nevertheless, many derived metabolites have been detected in the human blood stream, so that they
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should be taken into account as potential candidates to exert the neuroprotective effects attributed to
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the polyphenolic dietary precursors.19,20
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Whereas dietary polyphenols such as ETs, procyanidins and stilbenes are poorly bioavailable,
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we hypothesize here that their derived metabolites, which can target the brain, could protect
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neuronal cells from oxidative stress-induced cell death. To check this hypothesis, we aimed at
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determining the anti-apoptotic and antioxidant effects of a panel of 19 polyphenols-derived
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metabolites (Figure 1), at physiologically relevant concentrations, on H2O2-induced neurotoxicity in
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human neuroblastoma SH-SY5Y cells.
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MATERIALS AND METHODS
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Materials and Reagents. Trypan blue, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
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bromide (MTT), hydrogen peroxide solution, gallic acid (GA), ellagic acid (EA), 3,4-
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dihydroxyphenylpropionic acid (DHPPA), 3,4-dihydroxyphenylacetic acid (DHPAA) and trans-
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resveratrol (RSV) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The microbial EA-
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derived urolithins were chemically synthetized and purified by Villapharma Research S.L. (Parque
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Tecnológico de Fuente Alamo, Murcia, Spain). Urolithin D (Uro-D) and urolithin C (Uro-C) were
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purchased from Dalton Pharma Services (Toronto, Canada). RES-sulfate, RES-glucuronide and the
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microbial RES-derived metabolites dihydroresveratrol (DHR) and DHR-glucuronide were obtained
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as described in Azorín-Ortuño et al. (2011).16 Phosphate Buffered Saline (PBS) was obtained from
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Fisher Scientific (USA). Dimethylsulfoxide (DMSO) was obtained from Panreac (Barcelona,
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Spain). Milli-Q system (Millipore Corp., USA) ultra-pure water was used throughout the study.
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Cell Culture. The SH-SY5Y neuroblastoma cell line was purchased from the European Collection
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of Authenticated Cell Cultures (ECACC) (Salisbury, UK). Cells were cultured as recommended by
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the ECACC in Ham's F12:EMEM (EBSS) (1:1) + 2 mM Glutamine + 1% non-essential amino acids
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+ 15% fetal bovine serum (FBS). Cells were incubated at 37 °C in a humidified atmosphere of 95 %
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air/5 % CO2. Test compounds were solubilized in DMSO (
