Absorption, Metabolism, and Effects at Transcriptome Level of A
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Article pubs.acs.org/JAFC Absorption, Metabolism, and Effects at Transcriptome Level of a Standardized French Oak Wood Extract, Robuvit, in Healthy Volunteers: Pilot Study † § † § † † † Fausta Natella, , Guido Leoni, , Mariateresa Maldini, Lucia Natarelli, Raffaella Comitato, ‡ † † Frank Schonlau, Fabio Virgili, and Raffaella Canali*, † Consiglio per la Ricerca e Sperimentazione in Agricoltura, Food and Nutrition Research Centre, via Ardeatina 546, 00178 Roma, Italy ‡ Horphag Research (U.K.) Ltd. 28 Old Brompton Road, Suite 393, South Kensington, London SW7 3SS, United Kingdom ABSTRACT: The consumption of wine and spirits, traditionally aged in oak barrels, exposes humans to roburin ingestion. These molecules belong to a class of ellagitannins (ETs), and their only known source is oak wood. Very little is currently known about roburin bioavailability and biological activity. We reported for the first time human absorption of roburins from a French oak wood (Quercus robur) water extract (Robuvit) by measuring the increase of total phenols (from 0.63 ± 0.06 to 1.26 ± 0.18 μg GAE equiv/mL plasma) and the appearance of roburin metabolites (three different glucoronidate urolithins and ellagic acid), in plasma, after 5 days of supplementation. Robuvit supplementation induced also the increase of plasma antioxidant capacity from 1.8 ± 0.05 to 1.9 ± 0.01 nmol Trolox equiv/mL plasma. Moreover, utilizing a combined ex vivo cell culture approach, we assessed the effect of Q. robur metabolites (present in human serum after supplementation) on gene expression modulation, utilizing an Affymetrix array matrix, in endothelial, neuronal, and keratinocyte cell lines. The functional analysis reveals that Robuvit metabolites affect ribosome, cell cycle, and spliceosome pathways KEYWORDS: ellagitannins, roburins, Quercus robur, human study, gene array ■ INTRODUCTION The French oak water extract Robuvit is a registered In the past decades, epidemiological surveys pointed out the proprietary water extract obtained from fresh Quercus robur fi wood, standardized to provide a specific profile of tannins and association between a signi cantly lower risk for cardiovascular, ≥ − metabolic, and cancer diseases, cognitive decline, and neuro- in particular a mixture of 20% roburins (A E) including degeneration and the consumption of diets rich in plant grandinin. The main ETs in Q. robur are two diastereoisomers, 1,2 fi vescalagin and castalagin, which were originally isolated and polyphenols. The bene cial role of polyphenols was initially 6 attributed to a “nonspecific” antioxidant capacity. After a first described by Mayer et al. Sugar derivatives of vescalagin with aldopentoses xylose and lyxose are defined as roburin E and enthusiasm, it became apparent that health contributions of 7 − polyphenols were not (only) associated with cellular protection grandinin, respectively (Figure 1). Roburins A D are dimers, from oxidation but are due to a more complex mechanism with roburin D consisting of vescalagin and castalagin and involving the induction of cellular response to potentially roburin A of two vescalagin subunits. Roburins B and C harmful stimuli finally resulting in a number of specific adaptive resemble the structure of roburin A with additional xylose and defensive responses. Despite a consensus about this (roburin C) or lyxose (roburin B) bound to one vescalagin unit “hormetic” (para-hormetic) activity,3 comprehensive knowl- (Figure 2). Further to the roburins, Robuvit contains edge about the absorption, metabolism, and specific biological monomeric vescalagin and castalagin, as well as ellagic acid activities of the different classes of polyphenols is still scarce. (EA) and gallic acid (GA). Oak wood is currently the only fi Tannins are a specific family of polyphenols having a known source of roburins, and according to this speci city, the relatively large molecular structure. Tannins are considered to major source of roburins in human diets results from the be “secondary” compounds of plant physiology, not functioning consumption of wine and spirits (cognac and whiskey) in “primary” metabolism such as biosynthesis or energy traditionally matured, aged, and stored in oak barrels. In fact, conversions, although they serve biological purposes such as oak wood constituents contribute to the improvement of the protecting plants from herbivores and microbial infections. aroma, taste, and color and to the building up of the sensation “ ” Tannins are divided into two groups on the basis of their of mouth-fullness . As a result of this technological practice, structural characteristics: hydrolyzable and condensed tannins. humans have been exposed to roburins for centuries. In wines In the hydrolyzable tannins, a carbohydrate is partially or totally and spirits, roburins (including grandinin and the monomers esterified with phenolic groups such as gallic acid (gallotannins, GTs) or ellagic acid (ellagitannins, ETs). These compounds are Received: August 7, 2013 present in significant amounts in many berries, nuts, Revised: December 16, 2013 pomegranates, muscadine grapes, and edible dark-colored Accepted: December 19, 2013 fruits.4,5 Published: December 19, 2013 © 2013 American Chemical Society 443 dx.doi.org/10.1021/jf403493a | J. Agric. Food Chem. 2014, 62, 443−453 Journal of Agricultural and Food Chemistry Article Figure 1. Chemical structures of the monomers, castalgin, vescalgin, and their sugar derivatives roburin E and grandinin. Figure 2. Chemical structure of roburins A−D. castalagin and vescalagin) have been found to range between on gene expression in cultured endothelial, neuronal, and 1.2 mg/L (bourbon whiskey) and 9.4 mg/L (red wines).8 A keratinocyte cells to obtain a solid background for further number of indications suggest that, similarly to other studies addressing the understanding of possible beneficial polyphenols, ETs and their metabolites may exert beneficial effects of this family of molecules on human health. effects on human health.9 Despite the traditional consumption of roburins with wine and spirits, very little is currently known ■ MATERIALS AND METHODS about their bioavailability in humans. In general, ET from Chemicals. MeOH, HOCOOH, gallic acid, ellagic acid, Trolox, berries, pomegranate, walnuts, pecans, and pistachios are fluorescein, perchloric acid, and 2,2-azobis(2-amidinopropane) dihy- fl drochloride (AAPH) were purchased from Sigma-Aldrich (Milano, known to require the involvement of the colonic micro ora β 10 Italy); randomly methylated -cyclodextrin (RMCD) was purchased to be metabolized into dibenzopyranone species (urolithins). from Cyclolab (Budapest, Hungary). The consumption of oak-aged red wine was found to result in In Vivo Study. Experimental Design. Three healthy, nonsmoker, only trace amounts of urolithin-B aglycone and glucuronide in volunteers participated in this study, which was approved by the CRA- urine.11 Furthermore, several kinds of grapes have been found NUT ethical committee. The investigation conforms to the principles to contain EA, and therefore excreted urolithins may not outlined in the Declaration of Helsinki and its amendments. None of necessarily be attributed to oak constituents extracted following subjects was taking any medication or vitamin supplementation for the 2 weeks before the beginning of the experiment. The day before the wine aging in barrels. The aim of our study is to investigate the experiment subjects abstained from drinking coffee, tea, wine, and absorption and metabolism of Robuvit extract in healthy beer, and they ate no chocolate, fruits, and vegetables. The first day of volunteers and to evaluate the effect of Robuvit consumption the in vivo experimental protocol, blood was withdrawn in fasting 444 dx.doi.org/10.1021/jf403493a | J. Agric. Food Chem. 2014, 62, 443−453 Journal of Agricultural and Food Chemistry Article Figure 3. LC-MS total ion current (TIC) chromatograms of standards (top) and a representative plasma sample (bottom). Peaks: 1, gallic acid; 2, methyl gallate; 3, ellagic acid; 4, urolithin C diglucuronide; 5, urolithin A glucuronide; 6, urolithin B glucuronide. condition and serum isolated and stored at −80 °C until its utilization. with 1 mL of MeOH acidified with 0.1% HCOOH, conditioning with fi Volunteers were then supplemented with one capsule of Robuvit 1mLofH2O acidi ed with 0.1% HCOOH, loading of the sample, fi (containing 100 mg of Q. robur extract) three times a day for 5 days. washing with H2O acidi ed with 0.1% HCOOH (2 mL) and with During the 5 days of the supplementation, the subjects were water/methanol (95:5) (2 mL), and finally elution of metabolites with encouraged to reduce as much as possible the consumption of MeOH acidified with 0.1% HCOOH (2 mL). The eluted fraction was polyphenol-rich foods. The last day of the supplementation, subjects evaporated under nitrogen, and the residue was reconstituted with were administered 300 mg of Robuvit in fasting condition, about 1 h mobile phase to 0.3 mL and filtered through a 0.2 mm PVDF filter before blood withdrawal, and serum was collected and isolated again as (Whatman) into a vial insert for LC-MS/MS analyses. We determined described above. only the principal compounds that were extracted in MeOH/H2O. Q. robur Capsule Analyses. Robuvit capsule was extracted with 50 However, several ETs are insoluble and should be measured after mL of MeOH/H2O 80:20 (v/v) in a shaking bath overnight in hydrolysis (by detection of the produced EA). Moreover, our MS has a reduced light and temperature conditions. Subsequently, the extract maximum limit of m/z 1500; therefore, ETs that have higher was centrifuged for 10 min at 2000g and 4 °C. The supernatant was molecular weights (such as roburin dimers) cannot be measured. filtered through a 0.20 mm PVDF filter (Whatman, USA) and diluted ESI-MS, ESI-MS/MS, LC-ESI-MS, and LC-ESI- MS/MS Analyses. 1:1 with methanol before the analyses. Quantitative online HPLC-ESI-MS/MS analysis of the capsule extract Plasma Sample Preparation. Plasma samples were acidified and plasma sample were performed using a Perkin-Elmer series 200 immediately to pH 3 with 1 N HCl immediately after blood HPLC system interfaced to an Applied Biosystems (Foster City, CA, withdrawal and stored at −80 °C.