Absorption, Metabolism, and Effects at Transcriptome Level of a

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

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INTRODUCTION In the past decades, epidemiological surveys pointed out the association between a significantly lower risk for cardiovascular, metabolic, and cancer diseases, cognitive decline, and neurodegeneration and the consumption of diets rich in plant polyphenols.1,2 The beneficial role of polyphenols was initially attributed to a “nonspecific” antioxidant capacity. After a first enthusiasm, it became apparent that health contributions of polyphenols were not (only) associated with cellular protection from oxidation but are due to a more complex mechanism involving the induction of cellular response to potentially harmful stimuli finally resulting in a number of specific adaptive and defensive responses. Despite a consensus about this “hormetic” (para-hormetic) activity,3 comprehensive knowledge about the absorption, metabolism, and specific biological activities of the different classes of polyphenols is still scarce. Tannins are a specific family of polyphenols having a relatively large molecular structure. Tannins are considered to be “secondary” compounds of plant physiology, not functioning in “primary” metabolism such as biosynthesis or energy conversions, although they serve biological purposes such as protecting plants from herbivores and microbial infections. Tannins are divided into two groups on the basis of their structural characteristics: hydrolyzable and condensed tannins. In the hydrolyzable tannins, a carbohydrate is partially or totally esterified with phenolic groups such as gallic acid (gallotannins, GTs) or ellagic acid (ellagitannins, ETs). These compounds are present in significant amounts in many berries, nuts, pomegranates, muscadine grapes, and edible dark-colored fruits.4,5 © XXXX American Chemical Society

The French oak water extract Robuvit is a registered proprietary water extract obtained from fresh Quercus robur wood, standardized to provide a specific profile of tannins and in particular a mixture of ≥20% roburins (A−E) including grandinin. The main ETs in Q. robur are two diastereoisomers, vescalagin and castalagin, which were originally isolated and described by Mayer et al.6 Sugar derivatives of vescalagin with aldopentoses xylose and lyxose are defined as roburin E and grandinin, respectively (Figure 1).7 Roburins A−D are dimers, with roburin D consisting of vescalagin and castalagin and roburin A of two vescalagin subunits. Roburins B and C resemble the structure of roburin A with additional xylose (roburin C) or lyxose (roburin B) bound to one vescalagin unit (Figure 2). Further to the roburins, Robuvit contains monomeric vescalagin and castalagin, as well as ellagic acid (EA) and gallic acid (GA). Oak wood is currently the only known source of roburins, and according to this specificity, the major source of roburins in human diets results from the consumption of wine and spirits (cognac and whiskey) traditionally matured, aged, and stored in oak barrels. In fact, oak wood constituents contribute to the improvement of the aroma, taste, and color and to the building up of the sensation of “mouth-fullness”. As a result of this technological practice, humans have been exposed to roburins for centuries. In wines and spirits, roburins (including grandinin and the monomers Received: August 7, 2013 Revised: December 16, 2013 Accepted: December 19, 2013

A

dx.doi.org/10.1021/jf403493a | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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.

on gene expression in cultured endothelial, neuronal, and keratinocyte cells to obtain a solid background for further studies addressing the understanding of possible beneficial effects of this family of molecules on human health.

castalagin and vescalagin) have been found to range between 1.2 mg/L (bourbon whiskey) and 9.4 mg/L (red wines).8 A number of indications suggest that, similarly to other polyphenols, ETs and their metabolites may exert beneficial effects on human health.9 Despite the traditional consumption of roburins with wine and spirits, very little is currently known about their bioavailability in humans. In general, ET from berries, pomegranate, walnuts, pecans, and pistachios are known to require the involvement of the colonic microflora to be metabolized into dibenzopyranone species (urolithins).10 The consumption of oak-aged red wine was found to result in only trace amounts of urolithin-B aglycone and glucuronide in urine.11 Furthermore, several kinds of grapes have been found to contain EA, and therefore excreted urolithins may not necessarily be attributed to oak constituents extracted following wine aging in barrels. The aim of our study is to investigate the absorption and metabolism of Robuvit extract in healthy volunteers and to evaluate the effect of Robuvit consumption

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MATERIALS AND METHODS

Chemicals. MeOH, HOCOOH, gallic acid, ellagic acid, Trolox, fluorescein, perchloric acid, and 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) were purchased from Sigma-Aldrich (Milano, Italy); randomly methylated β-cyclodextrin (RMCD) was purchased from Cyclolab (Budapest, Hungary). In Vivo Study. Experimental Design. Three healthy, nonsmoker, volunteers participated in this study, which was approved by the CRANUT ethical committee. The investigation conforms to the principles outlined in the Declaration of Helsinki and its amendments. None of subjects was taking any medication or vitamin supplementation for the 2 weeks before the beginning of the experiment. The day before the experiment subjects abstained from drinking coffee, tea, wine, and beer, and they ate no chocolate, fruits, and vegetables. The first day of the in vivo experimental protocol, blood was withdrawn in fasting B



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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. Volunteers were then supplemented with one capsule of Robuvit (containing 100 mg of Q. robur extract) three times a day for 5 days. During the 5 days of the supplementation, the subjects were encouraged to reduce as much as possible the consumption of polyphenol-rich foods. The last day of the supplementation, subjects were administered 300 mg of Robuvit in fasting condition, about 1 h before blood withdrawal, and serum was collected and isolated again as described above. Q. robur Capsule Analyses. Robuvit capsule was extracted with 50 mL of MeOH/H2O 80:20 (v/v) in a shaking bath overnight in reduced light and temperature conditions. Subsequently, the extract was centrifuged for 10 min at 2000g and 4 °C. The supernatant was filtered through a 0.20 mm PVDF filter (Whatman, USA) and diluted 1:1 with methanol before the analyses. Plasma Sample Preparation. Plasma samples were acidified immediately to pH 3 with 1 N HCl immediately after blood withdrawal and stored at −80 °C. Once thawed, samples were mixed with acetonitrile 1:4 (v/v) to precipitate plasma proteins and then centrifuged for 10 min at 25000 rpm and 4 °C. The supernatant was recovered, dried under nitrogen (N2), and reconstituted with 0.5 mL of H2O acidified with 0.1% HCOOH. Extraction of metabolites was performed using solid-phase extraction (SPE) OASIS HLB 1 cm3 (30 mg) extraction cartridges (Waters, Milford, MA, USA). The SPE procedure consisted of the following steps: activation of SPE cartridges

with 1 mL of MeOH acidified with 0.1% HCOOH, conditioning with 1 mL of H2O acidified with 0.1% HCOOH, loading of the sample, washing with H2O acidified with 0.1% HCOOH (2 mL) and with water/methanol (95:5) (2 mL), and finally elution of metabolites with MeOH acidified with 0.1% HCOOH (2 mL). The eluted fraction was evaporated under nitrogen, and the residue was reconstituted with mobile phase to 0.3 mL and filtered through a 0.2 mm PVDF filter (Whatman) into a vial insert for LC-MS/MS analyses. We determined only the principal compounds that were extracted in MeOH/H2O. However, several ETs are insoluble and should be measured after hydrolysis (by detection of the produced EA). Moreover, our MS has a maximum limit of m/z 1500; therefore, ETs that have higher molecular weights (such as roburin dimers) cannot be measured. ESI-MS, ESI-MS/MS, LC-ESI-MS, and LC-ESI- MS/MS Analyses. Quantitative online HPLC-ESI-MS/MS analysis of the capsule extract and plasma sample were performed using a Perkin-Elmer series 200 HPLC system interfaced to an Applied Biosystems (Foster City, CA, USA) API 3200 instrument equipped with a Turbo ionspray source. A gradient elution was performed by using a mobile phase A represented by water acidified with formic acid (0.1%) and a mobile phase B represented by acetonitrile acidified with formic acid (0.1%). The gradient started from 0% of eluent B, remained at 0% of B for 10 min, then rose from 0 to 60% B in 40 min and subsequently to 100% B in 25 min. The flow (0.300 mL min−1) generated by chromatographic separation was directly injected into the electrospray ion source. The C



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Ex Vivo Study. Serum isolated before and after the supplementation of Robuvit from the three healthy subjects was used to enrich the medium of different cell cultures, that is, a permanent human endothelial cell, obtained by cell fusion of HUVEC, primary human endothelial cells, and immortalized human alveolar epithelial cells (EA.hy 926), human keratinocytes (HaCaT), and human neuroblastoma cells (SH-SY5Y). Cells were cultured in the absence of bovine serum in culture media supplemented with 10% (v/v) of the isolated human serum for 3 h. RNA Extraction and Gene Array. At the end of the incubation, RNA was isolated using an RNAeasy Plus Mini kit (Qiagen). The concentration, purity, and integrity were analyzed using the Agilent 2100 bioanalyzer, before gene expression analysis was performed. Two micrograms of each isolated RNA samples was sent to the FIRC Institute of Molecular Oncology Foundation (IFOM), Milano, Italy, to be processed and hybridized on Gene 1.0 ST array chips according to the Affymetrix protocol. These arrays allow the assessment of the expression level of 28869 genes represented by approximately 26 probes spread across the full length of the gene, providing a more complete and accurate picture of gene expression than the classical 3′ -based expression array designs. Cell intensity files for each Gene 1.0 ST chip processed were generated utilizing Command Console software. Microarray statistical analysis was performed utilizing the OneChannelGUI R package.16 Raw signal intensities were normalized utilizing the gene-chip robust multiarray average (GCRMA) method employing the empirical Bayes approach for background correction followed by quantile normalization. Differentially expressed genes were identified using the “limma package”, applying linear models and moderated t statistics that implement empirical Bayes regularization of standard errors. Differences in expression values were expressed by means of log2 fold change (FC). A minimum difference threshold of FC ± 0.5 with a p value ≤0.05 was selected as gene differentially expressed in cells incubated with serum isolated after the supplementation with respect to cells incubated with serum isolated before the supplementation. To highlight small variations on a quantitative scale, we decided to decrease the threshold of FC. The lists of significantly differentially expressed genes in association with the administration of Robuvit-enriched serum were analyzed by using DAVID17 and FIDEA18 Web servers to identify statistically overrepresented biological processes annotated in gene ontology (GO). Enriched GO biological processes (level 5) and Kegg pathways were identified according to p value threshold 1 and 12 genes upregulated with a FC between 0.5 and 1. Eight genes were downregulated with a FC between −0.5 and −1 (Table 6). In SHSY5Y cells (human bone marrow neuroblastoma), two genes (SMNDC1, a component of the spliceosome that removes introns from a transcribed pre-RNA, and FAU, involved in translation) were down-regulated with a FC ≤ 1 and 12 genes were up-regulated with a FC number between 0.5 and 1. Conversely, 23 genes have been found to be downregulated with a FC between −0.5 and −1 (Table 7). Despite these cell-specific differences, the genes modulated by Robuvit supplementation are mainly involved in translation, cell cycle, RNA splicing, and ribosome biogenesis in all cell types, according to the statistical pathway enrichment analysis. On the basis of the evidence of a non-negligible interindividual difference of Q. robur metabolite profile we considered that the effect of Robivit supplementation on gene expression modulation could be flattened out. We therefore assessed the effect of sera isolated from each subject (1,2 or 3) separately on the different cell lines. Table 8 shows the total number of genes modulated within a FC≤ ≥1. However, because most of the genes regulated are pseudogenes, not mapped in any GO term, the pathway analysis is only able to capture a percentage ranging from 30 to 50% of the total number of the modulated genes measured (shown in the second column). Moreover, the table shows the list of the genes, up- and down-regulated by each serum, that were used by the software to enrich the GO pathways and the resulting over-represented pathway in the three cell lines: ribosome, aminoacyl tRNA biosynthesis, and spliceosome. These results show that, independently of the individual profile metabolites resulting by Robuvit supplementation, the over-represented pathways associated with the three sera are similar in the three cell lines considered in the study.

Table 2. Increase of Plasmatic Total Phenols Measured after Supplementation μg GAE equiv/mL plasma

a

lipophilic component

1.8 ± 0.05 1.9 ± 0.01b

a

Data are expressed as μg ellagic acid equivalent (EAE)/tablet.

0.63 ± 0.06 1.26 ± 0.18a

before supplementation after supplementation

hydrophilic component

Significantly different, p < 0.005.

four EA-derived metabolites, conjugated with glucuronic acid, namely, urolithin B-glucuronide, urolithin A-glucuronide, urolithin C-diglucuronide, and EA methyl ether glucuronide, were detected. The total concentration of metabolites in plasma was in the order of 0.2 μM. With the exception of EA, none of these metabolites was present in plasma collected before Robuvit supplementation. No sulfate derivatives were detected in our analysis. The interindividual variability observed among the volunteers was quite high. Antioxidant Capacity Analysis. To assess if the administration of Robuvit resulted in an increase of plasmatic antioxidant capacity, we performed a classical “antioxidant capacity test”, according to the ORAC assay. Five days of Robuvit supplementation was associated with a significant increase of the ORAC value related to the hydrophilic plasmatic component, but not to the lipohilic fraction (Table 4). When the assay was directly performed on a Robuvit capsule, an ORAC value equal to 648 nmol Trolox eq/mg Q. robur was obtained. Microarray Results. As mentioned under Materials and Methods, human serum isolated before and after 5 days of Robuvit supplementation was used to enrich the culture medium of cells at a concentration of 10% v/v. Microarray results show that, in this experimental condition, the presence of Robuvit metabolites induces a differential expression of a limited number of genes. In HaCaT (stabilized human keratinocites), only one gene (PRPF8, a component of

Table 3. Analysis of the Metabolites Contained in Plasma Isolated before and after Supplementation from Each Subject before supplementation

after supplementation

subject gallic acid ellagic acid urolithin A glucuronidea urolithin B glucuronidea urolithin C diglucuronidea EA methyl ether glucuronide a

subject

1

2

3

mean

1

2

3

mean

0 13.5 0 0 0 0

0 4.5 0 0 0 0

0 0 0 0 0 0

0 6 0 0 0 0

± ± ± ± ± ±

0 14.5 8.1 96.1 0 5.6

2.9 14.5 11.8 7.1 24.1 0

6.5 9.5 12.2 7.9 46.9 0

3±3 13 ± 11 11 ± 2 37 ± 51 24 ± 23 2±3

0 7 0 0 0 0

Data are expressed as ng of ellagic acid equivalent (EAE)/mL plasma. E



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Table 5. Gene Expression Regulation by the Metabolites of Q. robur in HaCaTa

a

official gene symbol

gene name

FC

p value

representative pathways

USP22 EIF4G2 NPM1 RPS7 C1D PRPF8 TARDBP TP53TG3 LYZL2 CD69 EIF3D CAMTA2 SNX3 FMN1

ubiquitin specific peptidase 22 eukaryotic translation initiation factor 4 γ 2 nucleophosmin 40S ribosomal protein S7 C1D nuclear receptor corepressor pre-mRNA processing factor 8 homologue TAR DNA-binding protein 43 TP53TG3 lysozyme 2 CD69 eukaryotic translation initiation factor 3 subunit D calmodulin binding transcription activator 2 sorting nexin-3 formin 1

0.51 −0.55 −0.77 0.71 −0.60 1.00 −0.77 0.58 0.53 0.51 −0.51 −0.56 −0.76 −0.83

0.007 0.003 0.004 0.002 0.034 0.018 0.027 0.002 0.007 0.032 0.012 0.022 0.041 0.023

cell cycle cell cycle ribosome biogenesis; cell cycle ribosome biogenesis; RNA processing ribosome biogenesis; RNA processing RNA processing RNA processing; cell cycle

FC threshold is ≤ ≥ 0.5; p value ≤0.05.

Table 6. Gene Expression Regulation by the Metabolites of Q. robur in EA.hy.926a

a

official gene symbol

gene name

FC

p value

NPM1 RPL24 SEPT2 SMC4 RPL30 ELMO1 MT-ND2 HNRNPC NONO EIF4E3 ADAM21 B3GNT5 EPCAM IK CYP1A1 MYST2 CD69 C8orf73 C22orf28 AC007688.1 LOC642947 HOMER2

nucleophosmin 60S ribosomal protein L24 septin 2 structural maintenance of chromosomes protein 4 60S ribosomal protein L30 engulfment and cell motility protein 1 NADH-ubiquinone oxidoreductase chain 2 heterogeneous nuclear ribonucleoproteins C1/C2 non-POU domain-containing octamer-binding protein eukaryotic translation initiation factor 4E member 3 ADAM metallopeptidase domain 21 βGal β-1,3-N-acetylglucosaminyltransferase 5 epithelial cell adhesion molecule IK cytokine cytochrome P450, family 1 histone acetyltransferase CD69

0.82 1.08 0.52 0.50 1.28 0.95 0.75 0.59 0.58 0.57 0.54 0.53 −0.83 −0.51 −0.53 −0.72 −0.73 −0.73 0.73 −0.61 −0.51 0.55

0.042 0.011 0.028 0.007 0.001 0.047 0.037 0.045 0.045 0.005 0.034 0.004 0.013 0.008 0.006 0.011 0.004 0.002 0.021 0.002 0.002 0.007

representative pathways cell cell cell cell

cycle cycle; M phase mitotic cycle cycle; M phase mitotic cycle cycle; M phase mitotic cycle


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DISCUSSION This study represents the first investigation on standardized French oak wood extract, Robuvit, absorption/metabolism and on the effect of Robuvit metabolites on gene expression modulation, by utilizing a combined ex vivo cell culture approach.19 To the best of our knowledge we have reported for the first time absorption of ETs from Q. robur in humans; in fact, to date, metabolites resulting from human consumption of oak wood constituents remained unknown. The HPLC-MS2 analysis of Robuvit capsule shows that it is mainly composed of GA and EA and of several ETs such as castalagin, vescalagin, and roburin E. Our data show that the consumption of about 180 mg of total phenols per day (three capsules a day of Robuvit) resulted in a significant increase of GA and EA in plasma. GA is generally rapidly absorbed and has a good bioavailability,20,21 whereas ETs are hydrolyzed in vivo to EA, which is further metabolized by gut microbiota mostly to

produce the derivative urolithins. Similarly to isoflavone metabolism, where there is a quite clear-cut separation between equol and nonequol producers,22 it has been proposed that humans can be divided into high and low urolithin producers due to their microbiota, supporting the large variability in the metabolism of ETs.23,24 The appearance of three glucuronated urolithin (urolithin B-glucuronide, urolithin A-glucuronide, urolithin C-diglucuronide) moieties in plasma isolated after the supplementation, absent at baseline, suggests that oak wood ETs are bioavailable. Although glucuronidated urolithin A was present in all three subjects, only one subject showed high urolithin B (about 0.1 μg/mL) but not detectable urolithin C levels. ET bioavailability has been already studied addressing the consumption of different foods and beverages.11 EA and urolithin glucoronides were detected in human plasma at 6 h from pomegranate juice (PJ) administration (providing 318 mg of punicalagins and 12 mg of free EA)9,25 or pomegranate F



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Table 7. Gene Expression Regulation by the Metabolites of Q. robur in SHSY-5Ya official gene symbol EIF4G2 RBMY1D HNRNPK SNRNP200 HNRNPM SMNDC1 EIF3D RPL17 RPL4 RPS13 FAU PSMB2 EFCAB4B ACAA1 OR5L1 STARD7 TSPAN33 FLJ25758 BBS9 FOLH1 FOXK2 CR769776.1 LIAS AC068775.1 ZNF846 ANKRD20A4 ZNF117 ARF1 ARF3 CITED1 XCL1 SLC2A14 CBX3 HSD3B1 C19orf50 USP22 SART3 a

gene name eukaryotic translation initiation factor 4 γ 2 RNA binding motif protein heterogeneous nuclear ribonucleoprotein K small nuclear ribonucleoprotein 200 kDa heterogeneous nuclear ribonucleoprotein M survival motor neuron domain containing 1 eukaryotic translation initiation factor 3 subunit D ribosomal protein L17 60S ribosomal protein L4 40S ribosomal protein S13 Finkel−Biskis−Reilly murine sarcoma virus proteasome subunit, β type, 2 calcium release-activated calcium channel regulator 2A acetyl-eoenzyme A acyltransferase 1 olfactory receptor 5L1 StAR-related lipid transfer (START) domain containing 7 tetraspanin 33 Bardet−Biedl syndrome 9 folate hydrolase forkhead box ankyrin repeat domain 20 family lipoic acid synthetase zinc finger protein 846 ankyrin repeat domain 20 family zinc finger protein 117 ADP-ribosylation factor 1 ADP-ribosylation factor 3 Cbp/p300-interacting transactivator chemokine (C motif) solute carrier family 2 (facilitated glucose transporter) chromobox homologue 3 hydroxy-δ-5-steroid dehydrogenase, 3 β- and steroid δ-isomerase 1 ubiquitin specific peptidase 22 squamous cell carcinoma antigen recognized by T cells 3

FC

p value

representative pathways

0.91 0.56 −0.52 −0.69 −0.91 −1.10 0.69 −0.58 0.58 0.56 −1.00 0.85 0.75 0.66 0.62 0.55 0.53 0.50 −0.53 −0.54 −0.56 −0.59 −0.60 −0.61 −0.62 −0.63 −0.65 −0.66 −0.67 −0.69 −0.69 −0.76 −0.80 −0.85 −0.87 −0.92 −1.01

0.000 0.000 0.005 0.029 0.001 0.003 0.014 0.001 0.029 0.013 0.002 0.002 0.002 0.003 0.031 0.005 0.017 0.025 0.039 0.036 0.034 0.008 0.010 0.025 0.015 0.003 0.013 0.047 0.023 0.008 0.013 0.004 0.027 0.039 0.042 0.000 0.027

translation RNA splicing RNA splicing RNA splicing RNA splicing RNA splicing translation translation; ribosome translation; ribosome translation; ribosome translation; ribosome


extract (providing 330 mg of punicalagin and 21 mg of EA).26 Glucuronides were the only urolithin metabolites found in plasma of subjects supplemented for 5 weeks with PJ consumption.27 Urolithin glucuronides and EA derivatives were detected after 3 days of walnut consumption.28 Stoner and co-workers found EA in human plasma at 2 h after the administration of 45 g of freeze-dried black rasberries (containing 13.5 mg of EA).29 However, neither EA nor any of ET metabolites have been found in detectable quantities in human plasma over a 24 h period after 300 g of rasberry consumption.12 According to the authors, this negative result is not a real proof of absence as it could be due to the high detection limit of the method used in the study. In agreement with our data, no sulfate metabolites have been found in humans after ET-rich food supplementation.10 EA structure presents two ο-dihydroxy groups, and it may therefore be transformed by the activity of catechol-Omethyltransferase (COMT) (in the liver) in mono- and/or dimethyl ether of EA. These metabolites can be further metabolized by enzymatic glucuronic acid derivatization. In fact, dimethylellagic acid glucuronide has been found in the

urine9 and plasma28 of subjects supplemented with PJ and in the plasma of rats supplemented with 6% punicalagin.30 In the present study, we detected a low amount of methylellagic glucuronide in plasma isolated after the supplementation in only one of three subjects. Also in agreement with our study, a high interindividual variability of metabolic profile has been already frequently reported.9,24,27,28 Moreover, as expected, the total plasma polyphenol amount increased significantly by on average 100% in all volunteers. The consumption of the aqueous oak wood extract Robuvit led, also, to a statistically significant increase of hydrophilic blood oxygen radical absorbance capacity (ORAC) in healthy volunteers, even if the small change observed may not have a biological relevance. The methodologies utilized to assess plasma polyphenol concentration and plasma antioxidant capacity are known to be non specific. In fact, it has been reported that the Folin assay provides polyphenol estimation consistently 2−4 times greater than that obtained by HPLC methods,31 because it measures the reducing capacity of phenolic components by detecting nonspecific hydroxyl groups. GA and EA contain three and four G


29 52

47

48

50

38

101

95

2 3

1

2

3

1

2

3

up-regulated

ANAPC5, AREG, CANX, CBX3, EIF3F, FNTA, NPM1, RBMYD1D, SLC25A43, SMNDC1, TMED2, ZNF259, ZNF487P, ZNF492 ARF3, C1D, CBX3, EIF3F, GPC2, HSD3B1, KARS, MT-ND3, PTCH2, RABGGTB, RPNS1, SART1

CDC26, EPCAM, JTB

AREG, ANAPC5, CANX, CBX3, EIF4E3, IK, MT-CYB, NARS, NONO, RPL34, RPL24, RPL30, SEPT2, SERPIND2,SMNDC1, TAF1D, ZNF28

ANAPC5, ARF3, CASZ1, C1D, EIF3F, FNTA, MT-ND2, NPM1, RHOA,RPL11, RPL30, SMNDC1, SART3, SEPT2, SNX32, TARDBP CANX, ELMO1, IL10RA, KARS, NARS, RPL11, RPL17, RPL24, RPL30, RPL34, SART1, SART3

CANX, NARS, PRPF8, RPS7, SART3 IL10RA, LYZL2, NPM1, PRPF8, RPL11, RPL17, SEPT2, ZNF259

APOBE3CA, USP22, ZNF259

FC threshold is ≤ ≥ 1.

36

1

a

total genes modulated

serum

down-regulated

ribosome, aminoacyl-tRNA biosynthesis, steroid hormone biosynthesis ribosome

ribosome

spliceosome spliceosome, ribosome

over-represented pathway

SHSY-5Y ACTR3C, C1D, EIF3D, FAU, GUK1, HNRNPK, EFCAB4B, IK, IL10RA, KARS, aminoacyl-tRNA biosynthesis NARS, NPM1, RPL24, SART1, TARDBP, TCEB2 ARF3, ATP6 V0B, C1D, c22orf28, CCL4, EIF3D, HNRNPK, HNRNPM, ILF2, NARS, NONO, RHOA, RPL4, RPS27A, SEPT2, SNX3, SRP14, USP22, XCL1 C22orf28, CANX, HNRNPC, HNRNPM, IK, INF2, NPM1, PSMB2,PTH2R, spliceosoma RPL11, SART3, SLCO4C1 SMNDC1, TARDBP, TMED2

ARF3, EIF3F, HNRNPK, SART3, TCEB2

CD69, CYP1A1,EPCAM, PDE5A, HSD3B1, IK, ILF2, ILF2, RASA4

CANX, EIF3F, FNTA, HNRNPM, ILF2, NPM1, RPL11, SART3, SEPT2, TCEB2, TARDBP, ZNF737, ANAPC5, EIF3D, FNTA, HRNPC, IK, ZNF259, ZNF737 ARF3, C22orf28,CBX3, EIF3D, HNRNPC, IL18R1, IK, KARS, NONO, SMNCD1, SNX3, SNX32 EA.hy.926 DEPDC5, EPCAM, HNRNPC, RPL34, TCEB2

HaCaT

Table 8. Gene Expression Regulation Associated with Each Serum Isolated from the Healthy Subjects (Sera 1, 2, and 3) in HaCaT, EA.hy.926, and SHSY-5Y Cell Linesa

Journal of Agricultural and Food Chemistry Article

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putative effect of these botanical extracts in human health and disease. Specific interindividual differences in the metabolization of Q. robur extract components may have differently influenced the final outcome in terms of the single gene expression. However, surprisingly, the functional analysis reveals that the three sera, independently of the individual profile metabolites, modulate similar pathways, ribosome, aminoacyl tRNA biosynthesis, and spliceosome, suggesting that the ET metabolites display a uniform behavior, in a cell-type independent way. However, the small differences in FC of most of the genes do not let us assign to the modulated pathways either a positive or a negative health concern. Moreover, it is important to note that the rate of cell proliferation was not affected by serum containing Q. robur metabolites compared to control sera (data not shown). In conclusion, we demonstrate that Robuvit, a proprietary patented extract from Q. robur, is (at least in part) bioavailable to humans and that despite the very low concentration of the metabolite in serum, its consumption is associated with a statistically significant increase of the antioxidant capacity and to specific changes of gene expression profile.

hydroxyl groups, respectively, and they strongly contribute to Folin and possess high antioxidant properties. However, in our results the extent of the increase of plasmatic GA and EA, after the supplementation, is not sufficient to explain the observed increase of ORAC and Folin. It is known that urolithins exhibited a significant antioxidant action correlated with the number of hydroxyl groups,32 whereas no antioxidant activity has been reported for the glucuronide derivative forms.24,33 Therefore, we have to hypothesize that a synergic activity of all Q. robur metabolites occurs. Dietary EA and its metabolites, either independently or synergistically, are good candidates to be among the most active molecules responsible for a wide spectrum of molecular effects. In vivo and in vitro studies have shown that EA and urolithins have antiproliferative effects in human bladder cancer cells and antitumoral properties in several lines of cultured human cancer cells,34,35 in hamster buccal carcinoma36 and anti-inflammatory properties in colon fibroblasts,4 and in human primary endothelial cells.37 EA and urolithin A have been demonstrated to down-regulate the expression of COX-2 and iNOS and other inflammatory markers and to modulate the expression of genes involved in cell cycle in colonic mucosa in a colitis rat model.4 Moreover, CaCo2 cells exposed to concentrations of EA and urolithins achievable in the colon from the diet arrested cell growth at the S and G2/M phases. Transcriptional profiling obtained by microarray and functional analysis revealed significant changes in the expression levels genes involved in MAPK signaling such as growth factor receptors (FGFR2, EGFR), oncogenes (K-Ras, c-Myc), and tumor suppressors (DUSP6, Fos) as well as the modulation of genes involved in cell cycle (CCNB1,CCNB1IP1).38 Similarly, GA has been shown to inhibit carcinogenesis in animal models and in vitro cancerous cell lines. The inhibitory effect of GA on cancer cell growth has been reported to be mediated by the modulation of the expression of genes encoding for proteins involved in cell cycle, metastasis, angiogenesis, and apoptosis.39,40 Upstream to gene expression, GA has been reported to inhibit activation of NF-κB and Akt signaling pathways along with the activity of COX and inflammatory cytokines.41,42 The present study provides an original background to the understanding of possible effects of Q. robur extract metabolites on the modulation of gene expression in endothelial, neuronal, and keratinocyte cell lines. Our methodological approach allows the assessment of the effects of Q. robur metabolites at concentrations physiologically achievable in vivo and in a profile prevailing in humans. The transcriptome of each cell line was identified utilizing an Affymetrix customized array that recognizes a total number of 28869 genes. Subsequent, functional analysis revealed that culturing cells in serum obtained from subjects administered Q. robur extract results in a moderate although significant modulation of the expression of genes related to specific GO categories, namely, cell cycle, ribosome biogenesis, and RNA processing pathways in HaCaT (stabilized human keratinocytes) cells; cell cycle, M phase mitotic cycle in Ea.Hy.926 cells (a permanent human endothelial cells); translation, RNA splicing, and ribosome pathways in SHSY5Y cells (human bone marrow neuroblastoma). Obviously, changes of gene expression at the level of mRNA do not necessarily imply that corresponding changes occur at the level of protein. Nonetheless, an understanding of the effect of dietary bioactive molecules at the transcriptional level is important to the building up of a conceptual background to be exploited in future studies addressing the

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AUTHOR INFORMATION

Corresponding Author

*(R. Canali) Phone: +39 06 51494519. Fax: +39 06 51494550. E-mail: raff[email protected]. Author Contributions §

F.N. and G.L. equally contributed.

Funding

This study was supported by Horphag Research, Geneva, Switzerland, and by Italian Ministry of Agriculture, Food and Forestry (MiPAAF) Grants “NUME” (DM 3688/7303/08). Notes

The authors declare the following competing financial interest(s): F.S. worked at Horphag Research Ltd. The other authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Michael Jourdes (Université Bordeaux Segalen, Faculté d’Oenologie, Unité de recherche Oenologie, Villenave d’Ornon, France) for his help with the chemical structure drawing.

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ABBREVIATIONS USED ETs, ellagitannins; EA, ellagic acid; GA, gallic acid REFERENCES

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