Chemical Characterization, Free Radical Scavenging, and Cellular Antioxidant and Anti-Inflammatory Properties of a Stilbenoid-Rich Root Extract of Vitis vinifera

Dietary stilbenoids are receiving increasing attention due to their potential health benefits. However, most studies concerning the bioactivity of stilbenoids were conducted with pure compounds, for example, resveratrol. The aim of this study was to characterize a complex root extract of Vitis vinifera in terms of its free radical scavenging and cellular antioxidant and anti-inflammatory properties. HPLC-ESI-MS/MS analyses of the root extract of Vitis vinifera identified seven stilbenoids including two monomeric (resveratrol and piceatannol), two dimeric (trans-ɛ-viniferin and ampelopsin A), one trimeric (miyabenol C), and two tetrameric (r-2-viniferin = vitisin A and r-viniferin = vitisin B) compounds which may mediate its biological activity. Electron spin resonance and spin trapping experiments indicate that the root extract scavenged 2,2-diphenyl-1-picrylhydrazyl, hydroxyl, galvinoxyl, and superoxide free radicals. On a cellular level it was observed that the root extract of Vitis vinifera protects against hydrogen peroxide-induced DNA damage and induces Nrf2 and its target genes heme oxygenase-1 and γ-glutamylcysteine synthetase. Furthermore, the root extract could induce the antiatherogenic hepatic enzyme paraoxonase 1 and downregulate proinflammatory gene expression (interleukin 1β, inducible nitric oxide synthase) in macrophages. Collectively our data suggest that the root extract of Vitis vinifera exhibits free radical scavenging as well as cellular antioxidant and anti-inflammatory properties.


Introduction
Stilbenoids are secondary plant metabolites which are mainly present in Vitis vinifera L. species, the latter belonging to the plant family Vitaceae [1]. Vitis vinifera derived stilbenoids exist as monomers, that is, trans-resveratrol or piceatannol, and oligomers [1] and mostly occur in the plant kingdom as trans-isomers (E) [1]. The stilbene aglycone consists of two aromatic rings linked by an ethylene bridge [1]. Beside Vitis vinifera, dietary sources of resveratrol (trans-3,5,4trihydroxystilbene) and its oligomers are tea, peanuts, and pistachios [1][2][3][4]. Table 1 summarizes the occurrence of different stilbenoids in cell suspension culture, berries, stems, leaves, roots, and wine of Vitis vinifera according to Pawlus et al. [5]. Stilbenoids exhibit antimicrobial properties and as phytoalexins they play an important role in plants defending pathogens [5].
Up to now, the majority of studies concerning stilbenoids were conducted with resveratrol as a purified standard compound. However, studies in which a complex stilbene extract of Vitis vinifera was applied are scarce. The use of an extract may lead to synergistic effects of the various stilbenoids as far as their bioactivity is concerned. Stilbenoids are known to exhibit potential health benefits, that are, antioxidant [3,6], anti-inflammatory [7,8], anticancerogenic [9], antiatherogenic [10], antiviral [11], and neuroprotective properties [12]. 2 Oxidative Medicine and Cellular Longevity Table 1: Stilbenoids in cell suspension culture, berries, stems, leaves, roots, and wine of Vitis vinifera according to Pawlus et al. [5].

Plant part Stilbenoids
Cell suspension culture Monomer:
In the current study, we investigated potential free radical scavenging and cellular antioxidant and anti-inflammatory activities of the root of Vitis vinifera, which may be highly enriched with various stilbenoids. A standardized ethanol extract of the root of Vitis vinifera purified with ethyl acetate/n-hexane was applied for all studies. The qualitative and quantitative stilbenoid composition was analyzed by HPLC-ESI-MS/MS and HPLC-PDA.
Plant bioactives may prevent the oxidation of lipids, proteins, and DNA either directly by free radical scavenging or indirectly by induction of endogenous antioxidant defense mechanisms. Free radical scavenging activity was monitored by ESR spectroscopy and as spin trapping and the prevention of DNA damage was determined by the Comet assay.
The redox sensitive transcription factor nuclear factor erythroid 2-related factor-2 (Nrf2) partly regulates the expression of genes encoding antioxidant enzymes. Nrf2 is bound in the cytoplasm to its inhibitor Keap1 (Kelch-like ECH-associated protein 1). When Nrf2 is activated by electrophiles, it is released from its cytosolic protein Keap1 and binds to the antioxidant response element of the DNA in the nucleus thereby regulating the transcription of target genes including -glutamylcysteine synthetase ( GCS) and heme oxygenase-1 (HO-1) [13]. Nrf2 transactivation and its target genes HO-1 and GCS were determined by a reporter gene assay, real-time PCR, and Western blotting, respectively. Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL) associated enzyme which is primarily synthesized in the liver [14]. PON1 prevents low-density lipoproteins (LDL) from oxidation and thereby mediats antiatherogenic effects [14]. PON1 transactivation was measured by a reporter gene assay. Biomarkers of inflammation including interleukin 1 (IL-1 ) and inducible nitric oxide synthase (iNOS) were determined in cultured cells by real-time PCR.

Materials and Methods
2.1. Chemicals. Methanol, HPLC grade was purchased from VWR (Leuven, Belgium) and methanol, LC-MS grade was purchased from Fisher Chemical (Loughborough, UK). Acetic acid, HPLC grade was obtained from AppliChem (Darmstadt, Germany). Doubly deionized water using a Nanopure resin (Nanopure, Barnstead) was used for highperformance liquid chromatography (HPLC) analyses.
Vitisin grapevine root extract (Vitis vinifera cultivated from vines of the area Bordeaux) was kindly provided by Wolfgang Loersch (Breko, Bremen, Germany).

Free Radical Scavenging Activity Measured by Electron
Spin Resonance Spectroscopy (ESR). ESR and spin trapping measurements were conducted according to Esatbeyoglu et al. [16] using a JEOL JES-FR30EX free radical monitor (JEOL Ltd., Akishima, Japan). The amplitude was set at 200 for DPPH radicals, 250 for galvinoxyl, 400 for hydroxyl radicals, and 500 for superoxide radicals.

Hydroxyl Radical Scavenging Experiments.
To the reaction mixture of 25 L 200 mM DMPO, 20 L 50 mM hydrogen peroxide, 35 L distilled water, and 10 L 0.5 mM ferric chloride, 10 L of the root extract of Vitis vinifera (1, 1.25, 1.67, 2.5, 5, and 10 mg/mL) was added. The reaction mixture was stirred, vortexed, and put on ice for 10 sec.

Cell
Lines. The detailed cell culture conditions regarding the human liver hepatoma cell line Huh7, stably transfected PON1-Huh7 cells, and human colonic adenocarcinoma cell line HT-29 are described by Esatbeyoglu et al. [16].
Murine RAW264.7 macrophages (obtained from the Institute of Applied Cell Culture, Munich, Germany) were cultured in Dulbecco's modified Eagle's medium high glucose (4.5 g/L) containing sodium pyruvate and L-glutamine supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin and grown in a humidified atmosphere of 5% CO 2 at 37 ∘ C.

Oxidative Medicine and Cellular Longevity
For all cell culture studies, 100 mg/mL stock solutions of the root extract of Vitis vinifera in ethanol and resveratrol in DMSO were prepared and stored at −80 ∘ C until further use. LPS from Salmonella enterica serotype Enteritidis (Sigma) was dissolved in DPBS to a stock solution of 1 mg/mL and stored at −20 ∘ C until further use.

Oxidative DNA Damage (Comet Assay).
HT-29 cells were treated with 50 g/mL root extract of Vitis vinifera and 50 M resveratrol as positive control for 14 h at 37 ∘ C. Subsequently, cells were treated with 25 M H 2 O 2 in DPBS for 15 min to induce DNA damage. Oxidative DNA damage in HT-29 cells was measured using the Comet assay as described earlier [16].

Nrf2 Transactivation (Dual-Luciferase Reporter Gene
Assay). Transient transfection and luciferase reporter gene assay for measuring Nrf2 transactivation were conducted as described elsewhere [16].
Transiently transfected Huh7 cells were incubated with the root extract of Vitis vinifera (1, 5, 10, 25, and 50 g/mL) and 25 M resveratrol was used as a positive control.

Determination of Nrf2 Target Genes Heme Oxygenase-1 (HO-1) and -Glutamylcysteine Synthetase ( GCS) (RNA Isolation and Real-Time PCR).
Human Huh7 liver cells were seeded in a 6-well plate at a density of 0.9 × 10 6 cells/well for 24 h. Subsequently, cells were treated with 1, 10, 25, and 50 g/mL root extract of Vitis vinifera for 6 h. Cells were washed with DPBS and RNA was isolated using peqGOLD TriFast via phenol-chloroform extraction according to manufacturer's description.
SensiMix one-step kit (Quantace, Berlin, Germany) was used for real-time PCR. Human GAPDH was used as housekeeping gene.

Inhibition of LPS-Mediated Interleukin-1 (IL-1 ) and Inducible Nitric Oxide Synthase (iNOS) (RNA Isolation and
Real-Time PCR). RAW264.7 macrophages were seeded in 12-well plates at a density of 0.2 × 10 6 cells/well for 24 h. Afterwards, cells were treated with 20 g/mL of the root extract of Vitis vinifera for 24 h. LPS (10 ng/mL) was added to the cells for 4 h. RNA was isolated by peqGOLD TriFast according to manufacturer's protocol. Remaining DNA was lysed using DNAse according to manufacturer's instructions (New England Biolabs, Ipswich, USA).
SensiMix one-step kit (Quantace, Berlin, Germany) was used for real-time PCR. Gene expression was normalized to the housekeeping gene GAPDH.

HO-1 Protein Levels (Western Blot Analysis).
Whole cell extracts, total protein determination, and Western blot analysis were performed according to Wagner et al. [18] and Esatbeyoglu et al. [16].

Statistical Analyses.
Data obtained from cell culture experiments were expressed as means + standard error of the mean (SEM) or standard deviation (SD) of three independent experiments and compared to untreated cells (control) or LPS-stimulated control cells. HPLC analyses of stilbenes were expressed as means + standard deviation of five injections. Statistical analysis was performed by PASW Statistics Software Version 18 (IBM, Chicago, Illinois, USA). Data were tested for normality of distribution (Shapiro-Wilk test). Significant differences between groups were analyzed by Student's -test. In case of not normally distributed data the non-parametric Mann-Whitney test was applied. Significance was accepted at < 0.05.

Characterization and Quantification of the Vitis vinifera
Root Extract. The grapevine root extract derived from Vitis vinifera was analyzed by HPLC-ESI-MS/MS and quantified by HPLC-PDA. HPLC-ESI-MS/MS analyses were performed using electrospray ionization operated in negative ion mode. Seven stilbenoids including two monomeric (resveratrol and piceatannol), two dimeric (trans--viniferin and ampelopsin A), one trimeric (miyabenol C), and two tetrameric (r-2-viniferin = vitisin A and r-viniferin = vitisin B) were detected in the root extract of Vitis vinifera. Chemical structures of all detected compounds are given in Figure 1. A representative HPLC chromatogram at = 280 nm is shown in Figure 2. The main compounds in the root extract of Vitis vinifera were the dimer trans--viniferin (125.1 g/kg), a dehydrodimer of resveratrol, and the tetramer  r-2-viniferin (87.1 g/kg), composed of two resveratrol dimers (+)--viniferin and ampelopsin B, followed by the monomer resveratrol (46.3 g/kg) ( Table 2). The monomer piceatannol, the dimer ampelopsin A, the trimer miyabenol C, and the tetramer r-viniferin were present in considerably lower amounts (∼4-16 g/kg) ( Table 2).

Radical Scavenging Activity of the Root Extract of Vitis vinifera.
The free radical scavenging activity of the root extract of Vitis vinifera was determined by ESR and spin trapping analysis. The root extract of Vitis vinifera exhibited relatively potent free radical scavenging activity in terms of DPPH, hydroxyl, and galvinoxyl radicals and scavenged these free radicals in a dose-dependent manner (Figures 3(a)-3(c)). Superoxide radicals were scavenged at higher concentrations of the root extract (Figure 3(d)).

Induction of Antioxidant Defense Mechanisms through
Nrf2 Transactivation. Treatment of Huh7 cells with the root extract of Vitis vinifera at concentrations of 25 g/mL and 50 g/mL significantly ( < 0.001) upregulated Nrf2 transactivation. This effect was comparable with the Nrf2 inducing activity of 25 M resveratrol ( Figure 5). Moreover, mRNA and protein levels of the Nrf2 target genes HO-1 and GCS were analyzed by real-time PCR and Western blotting in human liver Huh7 cells. The root extract of Vitis vinifera (50 g/mL) significantly induced HO-1 both on the mRNA ( < 0.001) (Figure 6(a)) and protein levels ( Figure 6(b)). Accordingly, a significant induction of GCS was observed at 50 g/mL root extract of Vitis vinifera ( < 0.05; Figure 7).

PON1 Transactivation.
Under the conditions investigated, luciferase reporter gene activity of stably transfected PON1-Huh7 cells was significantly ( < 0.001) induced by the root extract of Vitis vinifera in a dose-dependent manner (Figure 8).

Inhibition of Proinflammatory Biomarkers like IL-1 and iNOS due to Vitis vinifera Root
Extract. Furthermore, the root extract of Vitis vinifera (20 g/mL) significantly decreased the NF-B target genes IL-1 (Figure 9(a)) and iNOS (Figure 9(b)) on the mRNA level in LPS-stimulated murine RAW264.7 macrophages.

Discussion
Stilbenoids are currently receiving increasing attention due to their potential health benefits [1,6,7,[10][11][12]20]. In this study, we combined ESR and spin trapping measurements with cellular assays in order to determine the free radical scavenging and antioxidant and anti-inflammatory properties of a root extract of Vitis vinifera.
Our analyses indicate that the root extract of Vitis vinifera contained substantial amounts of dimeric and oligomeric stilbenoids including the dimer trans--viniferin and the tetramer r-2-viniferin which may have contributed to its free radical scavenging properties. The free radical scavenging activity of stilbenoids seems to be partly related to proton abstraction as previously reported [21,22].
Since the root extract contained a portfolio of various stilbenoids, these compounds may interact synergistically thereby exhibiting free radical scavenging and antioxidant activity [23].
On a cellular level, free radicals are inactivated by endogenous antioxidant and stress response mechanisms. We found that root extract of Vitis vinifera exhibited HO-1 and GCS inducing activity which is most likely due to Nrf2 activation. Oxidized LDL plays a central role in atherogenesis [24]. Stilbenoids, such as resveratrol, have been shown to prevent Oxidative Medicine and Cellular Longevity copper mediated LDL oxidation in vitro through free radical scavenging activity [10]. Alternatively, our data indicate that a stilbenoid-rich extract may prevent LDL oxidation via cell signaling due to PON1 induction. Thus, stilbenoids may exhibit antiatherogenic properties due to both free radical scavenging and induction of antioxidant defense mechanisms. Interestingly, HO-1, GCS, and PON1 decrease with age [25]. Thus, it is tempting to speculate that our root extract may counteract an aging phenotype which warrants further investigations in appropriate in vivo models such as laboratory rodents. Furthermore, other age-related molecular targets including sirtuins [25,26] and FOXO [26] as well as autophagy related pathways [27] should be taken into account since they have been reported to be modulated by resveratrol in cultured cells and various model organisms.
Recent studies suggest cross talk between Nrf2 and proinflammatory gene expression. Nrf2 counteracts inflammatory processes by downregulating NF-B [28,29]. In the present study, the root extract of Vitis vinifera significantly decreased the expression of the NF-B target genes IL-1 and iNOS in  * indicates significant differences compared to untreated control cells; < 0.05, Student's -test.
murine macrophages suggesting anti-inflammatory activity. These anti-inflammatory properties of the Vitis vinifera root extract and other related plant extracts may be beneficial in pathologies characterized by an overproduction of nitric oxide and inflammatory cytokines [30]. Additionally, other biological properties of the root extract including its effect on platelet aggregation [31], smooth muscle cell proliferation [32], cellular adhesion [33], and vasodilation [34] should be taken into consideration.

Summary and Conclusion
In this study, seven stilbenoids including resveratrol, piceatannol, trans--viniferin, ampelopsin A, miyabenol C, r-2-viniferin = vitisin A, and r-viniferin = vitisin B were identified in the root extract of Vitis vinifera by HPLC-PDA. The root extract of Vitis vinifera scavenged DPPH, hydroxyl,  galvinoxyl, and superoxide free radicals. Accordingly, a protection against hydrogen peroxide-induced DNA damage was observed in cultured cells. Furthermore, Nrf2 and its target genes HO-1 and -GCS as well as PON1 were induced by Vitis vinifera root extract. Moreover, the root extract downregulated proinflammatory gene expression including IL-1 and iNOS in cultured macrophages. To sum up, our results suggest free radical scavenging and cellular antioxidant and anti-inflammatory activities of the Vitis vinifera root extract in vitro. However, little is known about the bioavailability, metabolism, and bioactivity of root-derived stilbenoids in vivo. Therefore, future studies should address the question to which extent stilbenoids from the roots of Vitis vinifera are bioavailable and may exhibit potential health benefits in humans. In addition, both the food industry and the consumer exhibit an increasing demand for natural antioxidants [35]. Thus, further studies are needed to elucidate to which extent the stilbenoid-rich Vitis vinifera root extracts could prevent oxidation processes, such as lipid peroxidation, in the food matrix. Additionally, it needs to be established whether the present Vitis vinifera root extract may be used as a nutraceutical in functional foods.