Research Paper

Role of human gut microbiota metabolism in the anti-inflammatoryeffect of traditionally used ellagitannin-rich plant materials

Jakub P. Piwowarski a,n, Sebastian Granica a, Marta Zwierzyńska a, Joanna Stefańska b,Patrick Schopohl c, Matthias F. Melzig c, Anna K. Kiss a

a Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw, Faculty of Pharmacy,Banacha 1, 02-097 Warsaw, Polandb Department of Pharmaceutical Microbiology, Medical University of Warsaw, Oczki 3, 02-007 Warsaw, Polandc Institute of Pharmacy, Freie Universitaet Berlin, Königin-Luise-Straße 2 and 4, 14195 Berlin, Germany

a r t i c l e i n f o

Article history:Received 23 April 2014Received in revised form11 June 2014Accepted 14 June 2014

Keywords:EllagitanninsUrolithinsInflammationGut microbiota

Chemical compounds studied in this article:Urolithin A (PubChem CID: 5488186)Urolithin B (PubChem CID: 5380406)Urolithin C (Reaxys registry number:5050777)SB-203580 (PubChem CID: 176155)

a b s t r a c t

Ethnopharmacological relevance: Ellagitannin-rich plant materials are widely used in traditional medicine aseffective, internally used anti-inflammatory agents. Due to the not well-established bioavailability ofellagitannins, the mechanisms of observed therapeutic effects following oral administration still remainunclear. The aim of the study was to evaluate if selected ellagitannin-rich plant materials could be the sourceof bioavailable gut microbiota metabolites, i.e. urolithins, together with determination of the anti-inflammatory activity of the metabolites produced on the THP-1 cell line derived macrophages model.Materials and Methods: The formation of urolithins was determined by ex vivo incubation of human fecalsamples with aqueous extracts from selected plant materials. The anti-inflammatory activity study ofmetabolites was determined on PMA differentiated, IFN-γ and LPS stimulated, human THP-1 cell line-derivedmacrophages.Results: The formation of urolithin A, B and C by human gut microbiota was established for aqueous extractsfrom Filipendula ulmaria (L.) Maxim. herb (Ph. Eur.), Geranium pratense L. herb, Geranium robertianum L. herb,Geum urbanum L. root and rhizome, Lythrum salicaria L. herb (Ph. Eur.), Potentilla anserina L. herb, Potentillaerecta (L.) Raeusch rhizome (Ph. Eur.), Quercus robur L. bark (Ph. Eur.), Rubus idaeus L. leaf, Rubus fruticosus L.and pure ellagitannin vescalagin. Significant inhibition of TNF-α productionwas determined for all urolithins,while for the most potent urolithin A inhibition was observed at nanomolar concentrations (at 0.625 μM29.276.4% of inhibition). Urolithin C was the only compound inhibiting IL-6 production (at 0.625 μM13.972.2% of inhibition).Conclusions: The data obtained clearly indicate that in the case of peroral use of the examined ellagitannin-rich plant materials the bioactivity of gut microbiota metabolites, i.e. urolithins, has to be taken underconsideration.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Ellagitannin-rich plant materials are widely used in traditionalmedicine as effective anti-inflammatory agents. In the case ofexternal use, the results of in vitro experiments using extracts orisolated ellagitannins are easily applied to the observed in vivoeffects. Yet ambiguities arise when internal use is considered.Ellagitannin-rich plant materials are often recommended by folkmedicine in internal treatment of different inflammation-associateddiseases (Table 1). In Europe the Filipendula ulmaria flower and the

whole herb are used internally as a tea in rheumatism, influenzalinfections and fever as well as in respiratory tract and urogenitaldisorders (Sarić-Kundalić et al., 2011; Vogl et al., 2013). In the Lahul-Spiti region of Western Himalaya the whole plant of Geraniumpratense is used in a powdered form in the treatment of liver andgastric disorders (Singh and Lal, 2008). Herbal preparations ofGeranium robertianum are applied in the treatment of gallbladderand urogenital tract inflammation-associated diseases, while teaprepared from the Geranium robertianum herb is recommend inthe treatment of sinus diseases (Menković et al., 2011). Its use innephritic complaints and jaundice in Europe is mentioned in King'sAmerican Eclectic Dispensatory (King, 1856). The Geum urbanum rootis used in the form of a decoction in the treatment of dysentery,gastro-enteritis and uterine disorders (Tita et al., 2009) as well as in

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Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.06.0320378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

n Corresponding author. Tel./fax: þ48 22 572 09 85.E-mail address: jakub.piwowarski@wum.edu.pl (J.P. Piwowarski).

Please cite this article as: Piwowarski, J.P., et al., Role of human gut microbiota metabolism in the anti-inflammatory effect oftraditionally used ellagitannin-rich plant materials. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.032i

Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

the form of a tea in rheumatism and gout, infections and fever (Voglet al., 2013). Deschauer (1945) recommended a Lythrum salicaria herbdecoction in the treatment of fever and liver disorders, while it wasalso recommended for different bowel disorders such as colorectitis(King, 1856; Felter and Lloyd, 1905). Infusion from the aerial parts ofPotentilla anserina is internally used in the treatment of inflammationand urogenital tract disorders (Tita et al., 2009), and Potentilla erectarhizome infusions and decoctions were often used in Europe andNorth America in the treatment of enterocolitis and dysentery (Felterand Lloyd, 1905; Tita et al., 2009). Quercus robur bark has very widetraditional internal uses. It is considered a remedy in the case ofanemia, increased blood circulation, insomnia, nervousness, backpain, gastrointestinal bleeding, pulmonary bleeding, renal and urin-ary bladder stones, spasms, blood purification, ague and liverailments (Sarić-Kundalić et al., 2011). Moreover, boiled bark is usedin the treatment of diabetes (Neves et al., 2009). Leaves from Rubusfruticosus are used in the form of an infusion in the treatment ofbronchitis and urinary disorders (Tita et al., 2009), while the wholeplant is used as an important diuretic and analgesic remedy (Popovicet al., 2012). Rubus idaeus aerial parts are used as diuretic and anti-inflammatory agent (Popovic et al., 2012), while a leaf infusionworksas a remedy in enterocolitis, cough, fever, bronchitis and prostatedisorders (Tita et al., 2009; Soukand and Kalle, 2013).

Unfortunately, due to the not well-established bioavailability ofellagitannins, their direct contribution to the therapeutic effectsappears at least controversial.

Studies performed on ellagitannin-containing food productssuch as oak-aged red wine, pomegranate juice, walnuts, almonds,hazelnuts, strawberries and raspberries, revealed that their intakeis associated with gut microbiota metabolites, i.e. urolithins,appearance in blood and urine (Cerda et al., 2005b). Urolithins

are dibenzopyran-6-one derivatives, which possess good bioavail-ability and can be found in plasma at low micromolar concentra-tions (Seeram et al., 2006). Urolithins can be formed fromellagitannins possessing the hexahydroxydiphenoyl group, whichrelease ellagic acid in the jejunum. Gut flora metabolism leads todecarboxylation of one of the lactone rings of ellagic acid and thesequential removal of hydroxyl groups from different positions,leading to lipophilicity increase (Espin et al., 2007). Many inter-esting biological activities have been determined for urolithins sofar. They were shown to possess anti-inflammatory associatedin vitro activities, such as inhibition of MMP-9 release and expres-sion by stimulated monocytes (Dell’agli et al., 2010), inhibition offibroblasts migration and monocyte adhesion to fibroblasts andendothelial cells (Gimenez-Bastida et al., 2012a; Gimenez-Bastidaet al., 2012b). NF-κB-dependent anti-inflammatory activity wasdetermined on human colonic fibroblasts model (Gonzalez-Sarriaset al., 2010b). The observed anti-inflammatory effects can occurdue to influence of urolithins on histone acetylation status, whichwas determined by Kiss et al. (2012). Strong antioxidant propertieson different in vitro models were also established (Bialonska et al.,2009; Verzelloni et al., 2011).

Despite ellagitannin-rich plant materials being used internallyin traditional therapy of different diseases with inflammatorybackground, studies concerning their microbial metabolism havenot been performed yet.

Macrophages are present in virtually all tissues. They differ-entiate from circulating peripheral mononuclear cells, whichmigrate into tissue in the steady state or in response to inflamma-tion. Macrophages are responsible for maintaining tissue home-ostasis, responding to microorganisms and mediating immuneregulation (Mosser and Edwards, 2008). The pro-inflammatory

Table 1Ethnopharmacological significance of internally used ellagitannin-rich plant materials.

Family Species Part Plant material origin Voucherspecimen

Totaltannincontent(%)

Traditional internal use Reference

Rosaceae Filipendulaulmaria (L.)Maxim (Ph.Eur.)

Herb Natural habitat inMasuria region

HE0803 21.071.5 Rheumatism, infections, fever,respiratory tract and urogenital disorders

(Sarić-Kundalić et al., 2011; Voglet al., 2013)

Geraniaceae Geraniumpratense L.

Herb Natural habitat inMasuria region

HE0807 15.171.1 Anti-inflammatory, jaundice, gastricdisorders

(Kupeli et al., 2007; Singh andLal, 2008)

Geraniaceae Geraniumrobertianum L.

Herb Natural habitat inMasuria region

HE0804 20.771.3 Intermittent fever, inflammatoryconditions of gallbladder and urogenitaltract, sinus diseases

(King, 1856; Menković et al.,2011)

Rosaceae Geum urbanumL.

Rootandrhizome

Experimental farmof WarsawUniversity of LifeSciences

HE0805 20.270.9 Rheumatism and gout, infections, fever,dysentery, gastro-enteritis, uterinedisorders

(Tita et al., 2009; Vogl et al.,2013)

Lythraceae Lythrumsalicaria L. (Ph.Eur.)

Herb Natural habitat inMasuria region

HE0806 27.470.8 Dysentery, colorectitis and otherinfections of the bowels, fever, liverdisorders

(Deschauer, 1945; Felter andLloyd, 1905; King, 1856)

Rosaceae Potentillaanserina L.

Herb Natural habitat inMasuria region

HE0810 10.070.9 Anti–inflammatory, renal and uterinedisorders

(Tita et al., 2009; Tomczyk andLatte, 2009)

Rosaceae Potentilla erecta(L.) Raeusch(Ph. Eur.)

Rhizome Natural habitat inPodlasie region

HE0811 37.770.9 Dysentery, enterocolitis, respiratory tractdisorders

(Felter and Lloyd, 1905; Sarić-Kundalić et al., 2011; Tita et al.,2009; Tomczyk and Latte, 2009)

Fagaceae Quercus roburL. (Ph. Eur.)

Bark Local market HE0813 35.471.7 Anti-diabetic, anti-dysenteric, bloodsystem and pulmonary disorders, renaland urinary bladder stones, analgesic

(Neves et al., 2009; Sarić-Kundalić et al., 2011)

Rosaceae Rubusfruticosus L.

Leaf Natural habitat inPodlasie region

HE0809 18.470.8 Bronchitis, urinary disorders, analgesic (Popovic et al., 2012; Tita et al.,2009)

Rosaceae Rubus idaeus L. Leaf Natural habitat inPodlasie region

HE0808 12.670.3 Enterocolitis, bronchitis, prostatedisorders, analgesic, cold, cough, fever

(Popovic et al., 2012; Soukandand Kalle, 2013; Tita et al., 2009)

Hippocastanaceae AesculushippocastanumL.

Bark Local market HE0801 24.571.5

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Please cite this article as: Piwowarski, J.P., et al., Role of human gut microbiota metabolism in the anti-inflammatory effect oftraditionally used ellagitannin-rich plant materials. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.032i

cytokines that are produced by macrophages, such as TNF-α orIL-6, are important components of host defense. However, exces-sive inflammatory response of macrophages can cause extensivedamage to the host. Over-activated macrophages are the keymediators of the immunopathologies that occur during autoim-mune diseases such as rheumatoid arthritis (Szekanecz and Koch,2007) and inflammatory bowel disease (Zhang and Mosser, 2008).Activated macrophages produce TNF-α ,a pleiotropic cytokine,whose role in inflammation and metabolism is complex. Togetherwith other pro-inflammatory cytokines, chemokines and variousimmune cells, TNF-α is an important contributor to the develop-ment of acute and chronic inflammatory conditions (Popa et al.,2007). Interleukin-6 (IL-6), secreted by activated macrophages,promotes TH17 cells accumulation by preventing their apoptosis.These persistent inflammatory T cells produce cytokines andchemokines, which propagate macrophage activation resulting ina positive feedback loop (Zhang and Mosser, 2008). TNF-α and IL-6can interfere with insulin signaling in adipocytes, leading to type2 diabetes (Bastard et al., 2006).

THP-1 is a human monocytic leukemia cell line. After treatmentwith phorbol esters, THP-1 cells differentiate into macrophage-like cells, which mimic native monocyte-derived macrophages.(Auwerx, 1991; Daigneault et al., 2010).

The aim of the study was to evaluate if ellagitannin-rich medi-cinal plant materials are potent to be the source of bioavailablemicrobial metabolites, i.e. urolithins, together with determinationof these metabolites' influence on TNF-α and IL-6 production byTHP-1 cells, triggered by stimulation of toll-like receptor 4 (TLR-4).

2. Materials and methods

2.1. Compounds

Urolithins A, B and C were synthetized according to Bialonskaet al. (2009). Their identity was confirmed by NMR and MSspectra. Vescalagin was isolated from Lythrum salicaria L. byPiwowarski and Kiss (2013).

2.2. Plant sources and extraction

Plant materials were collected from natural habitats, purchasedfrom the local market or obtained from Warsaw University of LifeSciences. Pharmacopoeial plant materials were identified based ontheir monographs, the identity of others was confirmed anatomi-cally and morphologically in the Department of Pharmacognosy andMolecular Basis of Phytotherapy, Medical University of Warsaw,where all voucher specimens were deposited. Details of plantmaterials’ origins are shown in Table 1. Five grams of each plantmaterial was extracted thrice for 30 min with 50 ml of distilledwater in 40 1C using an ultrasonic bath, filtered and lyophilized.

2.3. Phytochemical screening

The UHPLC–DAD–MS/MS phytochemical analysis was per-formed using the UHPLC-3000 RS system (Dionex, Germany) withDAD detection and an AmaZon SL mass spectrometer with ESIinterface (Bruker Daltonik GmbH, Germany) on a reversed-phaseKinetex C8 analytical column (100 mm; 2.1 mm; 1.7 μm), Phenom-enex (Torrance, CA, USA). The column temperature was 25 1C.Mobile phase A was water:formic acid (99.9:0.1, v/v) and mobilephase B was acetonitrile:formic acid (99.9:0.1, v/v). A multistepgradient solvent system was used: 0–5 min 0% B, 5–35 min 0–35%B. The flow rate was 0.3 ml/min. The eluate was introduceddirectly to the ESI interface of the mass detector. The nebulizerpressure of the ESI interface of mass detector was 50 psi; dry gas

flow was 10 L/min; dry temperature 300 1C; and capillary voltage4.5 kV. 5 μL of extract solution (5 μg/ml in mobile phase A)was injected into the UHPLC column. The mass scan ranged from100 to 2200m/z. UV spectra were recorded in the range of200–400 nm.

2.4. Formation of ellagitannin metabolites

Human fecal samples were donated by healthy volunteers aged25 to 30 (without history of gastrointestinal disease). Donors hadnot used antibiotics in the six months before sample collection.The study complied with the Helsinki Declaration. The intake ofellagitannin-containing products was strictly forbidden for 1 weekbefore sample collection. Samples were processed within 30 minfrom defecation. The growth medium—brain heart infusion (BHI)(DIFCO, Detroit, MI, USA)—was prepared according to the manu-facturer's instructions. To acquire anaerobic conditions, BHI wasboiled and immediately cooled down before experiment. Fecalslurries (FS) were prepared by suspending 1 g of human feces in10 ml of BHI (37 1C). 40 mg of each extract or 10 mg of vescalaginwas dissolved in 1 ml of distilled water and sterilized by filtrationthrough Ophtalsart hydrophilic syringe filters (0.2 mm) (SartoriusStedim Biotech GmbH, Germany). 1 ml of FS and 0.5 ml of extractsolution were added to 8.5 ml of BHI (37 1C). 0.5 ml of distilledwater was added to the control sample. The batch cultures wereincubated in a sealed container under anaerobic conditions usingGENbox anaer sachets (bioMerieux, France) at 37 1C. Samples werecollected after 24 h. As a control, incubation of extracts withoutFS and FS without extract was performed. 10 ml of the batchculture was extracted thrice with 10 ml of diethyl ether. Theorganic phases were pooled, evaporated to dryness under reducedpressure and re-dissolved in 1 ml of methanol. HPLC-DAD-MS/MSanalysis was performed using the apparatus described inSection 2.3. Separation was performed using the Zorbax SB-C18

column (150 mm; 2.1 mm; 1.9 μm) (Agilent, USA). The mobilephase consisted of water/acetonitrile/formic acid (95:5:0.1; v/v/v)(A) and acetonitrile/formic acid (99:0.1; v/v) (B). The gradient was:0–10 min 1–10% B, 10–20 min 20–30% B, 20–30 min 30–50% B and30–35 min 50–100% B. Column temperature was maintained at251C, the flow rate was 0.2 ml/min. The LC eluate was introducedinto the ESI interface without splitting and compounds wereanalyzed in the negative ion mode with the following settings:nebulizer pressure of 40 psi; drying gas flow rate of 9 L/min;nitrogen gas temperature of 3001C; capillary voltage of 4.5 kV. Themass scan ranged from 100 to 2200 m/z. UV spectra were recordedin the range of 200–400 nm. The presence of urolithins wasconfirmed by comparison of retention times, UV spectra and m/zspectra with corresponding standards.

2.5. THP-1 cell culture

THP-1 human monocytic cell line was purchased from DSMZ(Braunschweig, Germany). Cells were cultivated at 37 1C underhumidified 5% CO2 in the RPMI 1640 (Biochrom, Cambridge, UK)medium containing 10% FBS (Biochrom) and 2 mM glutamine(Biochrom). For experiments, cells were seeded in 24-well platesat a density of 250,000 cells per well and differentiated for 24 h inthe presence of phorbol myristate acetate (PMA, 100 ng/ml)(Sigma-Aldrich GmbH, Steinheim, Germany). After 24 h cells wereincubated with urolithins (dissolved in DMSO) at the concentra-tion of 1, 5 and 20 μM for 1 h prior to 24 h stimulation with LPSfrom Escherichia coli (100 ng/ml) (Invivogen, San Diego, CA, USA)and IFN-γ (10 ng/ml) (Cell Signaling Technology, Beverly, MA,USA). After stimulation supernatants were collected and storedat �70 1C. The levels of TNF-α and IL-6 were determined usingcommercially available ELISA tests (Human TNF-α total module set

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ELISA, eBioscience; IL6 Human ELISA Kit, R&D systems) accordingto manufacturer's instructions. MAPK inhibitor SB-203580 (Cay-man Chemical, Ann Arbor, MI, USA) was used as a positive controlaccording to Saklani et al. (2012).

Cytotoxicity of urolithins was determined using a standard MTTtest. Cells were seeded in 96-well plates at a density of 125,000cells per well and treated according to the method describedabove. After 24 h of incubation of differentiated cells with com-pounds at the concentration of 1, 20 and 50 μM with or withoutstimulation with IFN-γ and LPS, cells were washed twice withfresh culture medium and afterwards 150 μL of the mediumcontaining MTT (Carl Roth GmbH, Karlsruhe, Germany) at theconcentration of 0.5 mg/ml was added. After 1 h of incubation in37 1C, the medium was removed, cells were washed with freshmedium and formazan crystals were dissolved in 100 μL of DMSO.Absorbance was measured in 580 nm.

To determine the influence of urolithins on cell proliferation,the undifferentiated THP-1 cells were seeded in 24-well plates ata density of 250,000 cells per well and incubated with urolithins atthe concentration of 1, 20 and 50 μM for 24 h. Afterwards incuba-tion cells were centrifuged (1000 g), washed twice with PBS anddissolved in 400 μL of deionized water. After cell lysis achieved bythree freeze-thaw cycles, 100 μL of each cell lysate was dispensedon black 96-well plate and 100 μL of buffer (82 mM Na2HPO4,18 mM NaH2PO4, 4 M NaCl, 4 mM EDTA) was added. DNAwas stained by addition of 10 μL of Hoechst 33258 (10 μg/ml)(Sigma-Aldrich GmbH, Steinheim, Germany). Fluorescence wasmeasured at 360 nm excitation wavelength and 465 nm emissionwavelength.

For all assays DMSO at the final concentration of 0.2% was usedas the untreated control.

2.6. Statistical analysis

The results were presented as mean values7SEM of theindicated number of experiments. Statistical significance of differ-ences between means was determined by one-way ANOVA. Forcomparison of results with the control group, Dunnett's post hoctest was used. To compare the differences between the inhibitoryactivities of compounds, Tukey's post hoc test was performed.Results with p-valueo0.05 were considered statistically signifi-cant. All analyses were performed using Statistica 10 software.

3. Results and discussion

To characterize the ellagitannin composition of the ellagitannin-rich plant materials examined, the UHPLC–DAD–MS/MS method wasused. Peaks representing ellagitannins were initially denoted basedon the UV spectra with characteristic maximum around 220 nm.Compounds possessing the hexahydroxydiphenoyl (HHDP) group,which is potent to be transformed by gut microbiota to urolithins(Cerda et al., 2005a), were assigned based on MS2 spectra showingcharacteristic fragments at m/z 301 [HHDP�H]� , 303 [HHDPþH]þ

and/or neutral loss of 302 amu (Table S1). Vescalagin, castalagin,casuarinin, stachyurin, gemin A, pedunculagin and ellagic acid wereunambiguously identified using previously isolated compounds.Other ellagitannins were tentatively assigned based on the literaturedata regarding phytochemical characterization of certain species orgenera (Fig. 1) .

Filipendula ulmaria was shown to contain two ellagitannins atm/z 787 [MþH]þ , which were assigned as isomers of bis-galloyl-HHDP-glucose (according to Moilanen et al. (2013), one of themcan be identified as telimagrandin I) together with two dominat-ing peaks: one at m/z 1106 [M�H]� and second at m/z 937[M�2H]2� , which according to Nitta et al. (2013) can be identified

as rugosin A and rugosin D, respectively. One unknown ellagitan-nin was assigned at m/z 769 [M�H]� . Chromatogram of Geraniumpratense indicated dominating ellagitannin at m/z 951 [M�H]� ,which according to Ushiki et al. (1997) can be identified asgeraniin. This compound was also detected in Geranium robertia-num, but the peak intensity was significantly lower. Geum urba-num contained as dominating compounds gemin A (m/z 935[M�2H]2�), pedunculagin (m/z 783 [M�H]�), stachyurin (m/z935 [M�H]�; RT¼16.3) and casuarinin (m/z 935 [M�H]�;RT¼17.0), which were previously isolated from this plant material(Piwowarski et al., 2014). Lythrum salicaria as determined in ourprevious study contained C-glucosidic ellagitannins: vescalagin(m/z 933 [M�H]�; RT¼3.4), castalagin (m/z 933 [M�H]�;RT¼5.7), salicarinin A (m/z 933 [M�2H]2�; RT¼9.5), salicarininB (m/z 933 [M�2H]2�; RT¼12.9) and salicarinin C (m/z 933[M�2H]2�; RT¼14.3) as dominating compounds (Granica et al.,2014; Piwowarski and Kiss, 2013). Dimeric ellagitannin at m/z 935[M�2H]2� was determined in Potentilla anserina, which accordingto Fecka (2009) can be identified as agrimoniin. In Potentilla erectathe dominating compound was ellagitannin at m/z 1567 [M�H]� ,which according to Geiger and Rimpler (1990) can be assigned aslaevigatin B or F. In Qercus robur two C-glucosidic ellagitannins:vescalagin (m/z 933 [M�H]�; RT¼3.4) and castalagin (m/z 933[M�H]�; RT¼5.7) were identified. Two ellagitannins with flavan-3-ol moiety at m/z 1205 [M�H]� (RT¼15.8 and RT¼18.3) canbe identified as acutissimin A and B together with one at m/z1055 [M�H]� , which can be identified as stenophynin A. Thesecompounds were not previously determined in Quercus robur, butwere isolated from the bark of Quercus acutissima and Quercusstenophylla, respectively (Nishimura et al., 1986; Ishimaru et al.,1987). The above assignments are supported by the presence offragment ions at m/z 915 and 765, which most probably resultfrom the neutral loss of flavan-3-ol moiety (290 amu). In the caseof Rubus fruticosus and Rubus idaeus, the majority of denotedellagitannins had not been previously isolated from the Rubusgenus. Only one ellagitannin at m/z 1103 [M�H]� in Rubus idaeuscan be initially identified as sanguiin H-2 according to Tanaka et al.(1993). Apart from Filipendula ulmaria, all extracts containedsignificant amounts of ellagic acid (EA), visible on chromatogramsat 22.0 min. Extract from Aesculus hippocastanum did not containellagitannins or ellagic acid. The HPLC-DAD-MS/MS analysisunambiguously excluded the presence of signals at m/z character-istic to urolithins in any of the examined extracts. In Aesculushippocastanum bark no ellagitannins were detected and dominat-ing peaks at m/z 339 [M�H]� and m/z 369 [M�H]� wereidentified as aesculin and fraxin, respectively; other compoundswere identified mainly as flavan-3-ol derivatives.

The HPLC-DAD-MS/MS analysis of human gut microbiotaex vivo cultures revealed the formation of urolithins from theexamined ellagitannin-rich plant materials. Peaks representingurolithins were identified based on retention times as well as UVand MS spectra of synthesized chemical standards (Fig. 2 andFig. 3). After 24 h of incubation, the dominating peak—represent-ing urolithin A—appeared together with less intensive urolithin Cpeak and small urolithin B peak. Urolithin production was alsoestablished for pure ellagitannin, i.e. vescalagin (Fig. 4), which wasused as a standard compound present in Lythrum salicaria L. andQuercus robur L. Incubation of extract from Aesculus hippocastanumL. bark (containing only condensed tannins), which was usedas a negative control, did not result in urolithins formation. Theamount of urolithins produced by gut microbiota significantlydiffered between extracts and did not correlate with total tannincontent or with the estimated ellagic acid amount. However, theproportions between urolithin A, B and C peak intensitiesremained stable and in the case of all extracts the general patternof urolithins formation was similar. The formation of urolithins,

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which was not initially detected in extracts, indicates that theellagitannins present in Filipendula ulmaria (L.) Maxim. herb (Ph.Eur.), Geranium pratense L. herb, Geranium robertianum L. herb,Geum urbanum L. root and rhizome, Lythrum salicaria L. herb (Ph.Eur.), Potentilla anserina L. herb, Potentilla erecta (L.) Raeuschrhizome (Ph. Eur.), Quercus robur L. bark (Ph. Eur.), Rubus idaeus L.leaf and Rubus fruticosus L. leaf are potent to be metabolized by gutmicrobiota to urolithins. This is the first time when ellagitannin-richmedicinal plant materials were shown to be a source of bioavailablegut microbiota metabolites, i.e. urolithins. Despite the vast impor-tance of selected ellagitannin-rich plant materials in therapy ofdiseases with inflammatory background no in vivo or ex vivo studies

have been conducted so far revealing their potency to become thesource of urolithins. Urolithins formation shown previously forsingle compounds such as ellagic acid, geraniin and punicalagin(Cerda et al., 2005a; Gonzalez-Barrio et al., 2011; Ito, 2011; Garcia-Villalba et al., 2013) (from which the former two were determinedin the examined extracts) can suggest such possibility, but directevidence indicating complex extracts as a source of urolithins hasnot been established yet.

To evaluate the influence of urolithins on pro-inflammatoryfunctions of macrophages the PMA differentiated THP-1 cellsmodel was used. The production of cytokines was triggered bypriming cells with IFN-γ and stimulation of the TLR-4 receptor by

Fig. 1. UHPLC–DAD–MS/MS chromatograms acquired at 245 nm with indicated m/z values of ellagitannins possessing HHDP group together with ellagic acid (EA).

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LPS. All urolithins have shown strong, dose dependent inhibitionof TNF-α production while for the most active urolithin A inhibi-tion was observed in nanomolar range and the effect at concen-tration of 20 μM was statistically stronger than MAPK inhibitorSB-203580 (po0.05) (Fig. 5). In the case of IL-6 production, only

urolithin C has shown significant activity (at the concentration of10, 2.5 and 0.625 μM 25.574.0, 17.972.1, 13.972.2% of inhibition,respectively) at the level comparable to positive controlSB-203580 (20.371.8, 14.873.0, 12.674.2% of inhibition, respec-tively) (all values were statistically significant, i.e. po0.001 versus

Fig. 2. UHPLC–DAD–MS/MS chromatograms acquired at 280 nm presenting rolithins A, B, and C (UA, UB, UC respectively) formation after 24 h incubation of aqueous extractsfrom selected plant materials and vescalagin with human gut microbiota ex vivo culture.

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stimulated control). The urolithins at concentrations of up to50 μM did not affect the viability of THP-1 derived macrophageseither in IFN-γ and LPS treated or in un-stimulated cells (Fig. S1).The proliferation of un-differentiated THP-1 cells was also notinfluenced (Fig. S2).

Because macrophages play an important role in the develop-ment of different diseases with inflammatory background, thespecific influence of urolithins on TNF- α production expressed atconcentrations achievable in gut and bloodstream can help to at

least partially explain the different directions of therapeutic usesof chosen ellagitannin-rich plant materials. The observed effectsare consistent with previous reports on the anti-inflammatoryactivities of urolithins. Determined with the colonic fibroblastsmodel, modulation of NF-κB and MAPK pathway can be involvedin the inhibition of cytokine release (Gonzalez-Sarrias et al.,2010b) as well as in the influence on HAT activity, establishedwith the THP-1 cell line model (Kiss et al., 2012). Urolithins werepreviously shown to inhibit haemozoin-induced MMP-9 secretionand expression in THP-1 cells. (Dell’agli et al., 2010). The influenceof urolithins’ mixture on colonic fibroblasts was determined byGimenez-Bastida et al. (2012b). The mixture as well as singleurolithin A and B were shown to inhibit colon fibroblast migrationand monocyte adhesion to fibroblasts, which was followed bysignificant decrease of inflammatory mediators such as PGE2, PAI-1 and IL-8. Studies conducted on human aortic endothelial cellsalso shown urolithin A and B to be active against TNF-α triggeredcell migration and monocyte adhesion. The effect was associatedwith down-regulation of the CCL2 and PAI-1 levels (Gimenez-Bastida et al., 2012a).

In contrast to parental compounds—ellagitannins, urolithinspossess well-established bioavailability, which was widely exam-ined in studies conducted on different food products. Afterpomegranate juice consumption, urolithins in free and conjugatedforms were detected in plasma and urine. Six hours after con-sumption the plasma levels of UA and UB were shown to reachCmax of 0.14 and 0.01 μM/L, respectively (Seeram et al., 2006).Urolithins seem potent to cumulate in organism during regularellagitannin intake, as previous studies have shown that totalcirculating urolithins are able to reach the concentration of

Fig. 3. Chemical structures, MS and UV spectra of urolithin A, B and C.

Fig. 4. Chemical structure of vescalagin.

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18.6 μM when pomegranate juice (4.3 g of ellagitannins) wasadministered daily for 5 days (Cerda et al., 2004). Studies con-ducted on volunteers consuming strawberries, raspberries, wal-nuts, and oak-aged wines revealed that the main metaboliteappearing in urine was urolithin B glucuronide (with maximumexcretion values of 12 mg (6.3% excretion), 32 mg (7.6% excretion),155 mg (81% excretion) and 0.75 mg (7.4% excretion), respec-tively), while in some samples significant amounts of unconju-gated urolithin B were detected. The excretion of metabolites wasnot proportional to the amount of ellagitannins consumed, sug-gesting the strong impact of gut flora composition (Cerda et al.,2005b). In other studies concerning the urinary excretion ofurolithins after consumption of strawberries, the determined areaunder the urinary excretion rate curve (AURCAll) for urolithinA and its glucuronide was 0.04 and 0.52 mg�h/ml, respectively,while for urolithin B and its glucuronide it was 0.03 and0.06 mg�h/ml, respectively (Truchado et al., 2012). After con-sumption of 300 g of raspberries, the total levels of urine excretionof urolithin glucuronides ranged between 0 and 44 μM (Gonzalez-Barrio et al., 2010). Urolithin A glucuronide (at the concentrationrange of 0.05–0.2 μM) together with urolithin C glucuronide wasdetected in plasma after consumption of walnuts or pomegranatejuice (Gonzalez-Sarrias et al., 2010a).

The formation of systematically absorbed urolithins fromellagitannin-rich plant material could be the link between theobserved biological activities of extracts in vivo and the questionablebioavailability of ellagitannins. A preclinical study conducted on thePotentilla erecta root extract revealed its strong anti-inflammatoryeffect in patients with ulcerative colitis. The authors clearly con-cluded that although the effects were significant, the active com-pounds remained unidentified as no ellagitannins were detected inthe patients’ sera (Huber et al., 2007). Orally administered polarextracts from the Geranium pratense and Lythrum salicaria herb haveshown anti-nociceptive and anti-inflammatory activities in mice(Kupeli et al., 2007; Tunalier et al., 2007). The Rubus fruticosus leafextract, when administered orally, was shown to possess hypogly-caemic activity in the case of alloxan-induced diabetes in rabbits. Themost significant effect was observed for pure infusion 4 h afteradministration (Alonso et al., 1980). Hypoglycaemic effects were also

determined on animal models for the Lythrum salicaria herb (Lamelaet al., 1986) and the Geranium robertianum leaves decoction (Ferreiraet al., 2010).

Due to ex vivo determination of bioavailable gut microbiotametabolites formation, the bioactivity studies conducted for uro-lithins need to be quoted while describing in vivo effects of selectedellagitannin-rich medicinal plant materials. When external applica-tions are discussed, the effects of intact ellagitannins should beexamined but in the case of their peroral use the consideration ofthe urolithins’ bioactivity becomes necessary. Further in vivo clinicalstudies combined with thorough pharmacokinetic analysis areneeded to fully determine the participation of urolithins in ther-apeutic effects of ellagitannin-rich plant materials.

Acknowledgments

The project was carried out with the use of CePT infrastructurefinanced by the European Regional Development Fund withinthe Operational Programme ‘Innovative economy’ for 2007–2013.The project was financially supported by Polish National ScienceCenter PhD scholarship ETIUDA decision number DEC-2013/08/T/NZ7/00011.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.jep.2014.06.032.

References

Alonso, R., Cadavid, I., Calleja, J.M., 1980. A preliminary study of hypoglycemicactivity of Rubus fruticosus. Planta Medica Suppl. 40, 102–106.

Auwerx, J., 1991. The human leukemia cell line, THP-1: a multifacetted model forthe study of monocyte-macrophage differentiation. Experientia 47, 22–31.

Bastard, J.P., Maachi, M., Lagathu, C., Kim, M.J., Caron, M., Vidal, H., Capeau, J., Feve, B.,2006. Recent advances in the relationship between obesity, inflammation, andinsulin resistance. European Cytokine Network 17, 4–12.

Bialonska, D., Kasimsetty, S.G., Khan, S.I., Ferreira, D., 2009. Urolithins, intestinalmicrobial metabolites of pomegranate ellagitannins, exhibit potent antioxidant

Fig. 5. Effect of urolithins A, B and C and SB-203580 on TNF-α production by PMA-differentiated, IFN- γ primed and LPS stimulated THP-1 cells. Data were expressed asmean7SEM of three separate experiments assayed in triplicate. npo0.05, nnpo0.001 versus stimulated control. a- statistically significant (po0.001) versus non-stimulatedcontrol; ST- stimulated control; NST- non-stimulated control.

J.P. Piwowarski et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎8

Please cite this article as: Piwowarski, J.P., et al., Role of human gut microbiota metabolism in the anti-inflammatory effect oftraditionally used ellagitannin-rich plant materials. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.032i

activity in a cell-based assay. Journal of Agricultural and Food Chemistry 57,10181–10186.

Cerda, B., Espin, J.C., Parra, S., Martinez, P., Tomas-Barberan, F.A., 2004. The potentin vitro antioxidant ellagitannins from pomegranate juice are metabolised intobioavailable but poor antioxidant hydroxy-6H-dibenzopyran-6-one derivativesby the colonic microflora of healthy humans. European Journal of Nutrition 43,205–220.

Cerda, B., Periago, P., Espin, J.C., Tomas-Barberan, F.A., 2005a. Identification ofurolithin A as a metabolite produced by human colon microflora from ellagicacid and related compounds. Journal of Agricultural and Food Chemistry 53,5571–5576.

Cerda, B., Tomas-Barberan, F.A., Espin, J.C., 2005b. Metabolism of antioxidant andchemopreventive ellagitannins from strawberries, raspberries, walnuts, andoak-aged wine in humans: identification of biomarkers and individual varia-bility. Journal of Agricultural and Food Chemistry 53, 227–235.

Daigneault, M., Preston, J.A., Marriott, H.M., Whyte, M.K., Dockrell, D.H., 2010. Theidentification of markers of macrophage differentiation in PMA-stimulatedTHP-1 cells and monocyte-derived macrophages. PloS One 5, e8668.

Dell’agli, M., Galli, G.V., Bulgari, M., Basilico, N., Romeo, S., Bhattacharya, D.,Taramelli, D., Bosisio, E., 2010. Ellagitannins of the fruit rind of pomegranate(Punica granatum) antagonize in vitro the host inflammatory response mechan-isms involved in the onset of malaria. Malaria Journal 9, 208.

Deschauer, T., 1945. Illustrated Phytotherapy. Thos.. Deschauer Publications,Maywood, Ill.

Espin, J.C., Gonzalez-Barrio, R., Cerda, B., Lopez-Bote, C., Rey, A.I., Tomas-Barberan,F., 2007. Iberian pig as a model to clarify obscure points in the bioavailabilityand metabolism of ellagitannins in humans. Journal of Agricultural and FoodChemistry 55, 10476–10485.

Fecka, I., 2009. Development of chromatographic methods for determination ofagrimoniin and related polyphenols in pharmaceutical products. Journal ofAOAC International 92, 410–418.

Felter, H.W., Lloyd, J.U., 1905. King’s American Dispensatory. The Ohio ValleyCompany, Cincinnati.

Ferreira, F.M., Peixoto, F., Nunes, E., Sena, C., Seica, R., Santos, M.S., 2010. “MitoTea:Geranium robertianum L. decoctions decrease blood glucose levels and improveliver mitochondrial oxidative phosphorylation in diabetic Goto-Kakizaki rats.Acta Biochimica Polonica 57, 399–402.

Garcia-Villalba, R., Beltran, D., Espin, J.C., Selma, M.V., Tomas-Barberan, F.A., 2013.Time course production of urolithins from ellagic acid by human gut micro-biota. Journal of Agricultural and Food Chemistry 61, 8797–8806.

Geiger, C., Rimpler, H., 1990. Ellagitannins from Tormentillae rhizoma and Alchemillaeherba. Planta Medica 56, 585–586.

Gimenez-Bastida, J.A., Gonzalez-Sarrias, A., Larrosa, M., Tomas-Barberan, F., Espin, J.C.,Garcia-Conesa, M.T., 2012a. Ellagitannin metabolites, urolithin A glucuronide andits aglycone urolithin A, ameliorate TNF-alpha-induced inflammation andassociated molecular markers in human aortic endothelial cells. MolecularNutrition & Food Research 56, 784–796.

Gimenez-Bastida, J.A., Larrosa, M., Gonzalez-Sarrias, A., Tomas-Barberan, F., Espin, J.C.,Garcia-Conesa, M.T., 2012b. Intestinal ellagitannin metabolites amelioratecytokine-induced inflammation and associated molecular markers in humancolon fibroblasts. Journal of Agricultural and Food Chemistry 60, 8866–8876.

Gonzalez-Barrio, R., Borges, G., Mullen, W., Crozier, A., 2010. Bioavailability ofanthocyanins and ellagitannins following consumption of raspberries byhealthy humans and subjects with an ileostomy. Journal of Agricultural andFood Chemistry 58, 3933–3939.

Gonzalez-Barrio, R., Edwards, C.A., Crozier, A., 2011. Colonic catabolism of ellagi-tannins, ellagic acid, and raspberry anthocyanins: in vivo and in vitro studies.Drug Metabolism and Disposition 39, 1680–1688.

Gonzalez-Sarrias, A., Gimenez-Bastida, J.A., Garcia-Conesa, M.T., Gomez-Sanchez,M.B., Garcia-Talavera, N.V., Gil-Izquierdo, A., Sanchez-Alvarez, C., Fontana-Compiano, L.O., Morga-Egea, J.P., Pastor-Quirante, F.A., Martinez-Diaz, F.,Tomas-Barberan, F.A., Espin, J.C., 2010a. Occurrence of urolithins, gut microbiotaellagic acid metabolites and proliferation markers expression response in thehuman prostate gland upon consumption of walnuts and pomegranate juice.Molecular Nutrition & Food Research 54, 311–322.

Gonzalez-Sarrias, A., Larrosa, M., Tomas-Barberan, F.A., Dolara, P., Espin, J.C., 2010b.NF-kappaB-dependent anti-inflammatory activity of urolithins, gut microbiotaellagic acid-derived metabolites, in human colonic fibroblasts. The BritishJournal of Nutrition 104, 503–512.

Granica, S., Piwowarski, J.P., Kiss, A.K., 2014. Determination of C-glucosidic ellagi-tannins in Lythri herba by ultra-high performance liquid chromatographycoupled with charged aerosol detector: method development and validation.Phytochemical Analysis 25, 201–206, http://dx.doi.org/10.1002/pca.2492.

Huber, R., Ditfurth, A.V., Amann, F., Guthlin, C., Rostock, M., Trittler, R., Kummerer,K., Merfort, I., 2007. Tormentil for active ulcerative colitis: an open-label, dose-escalating study. Journal of Clinical Gastroenterology 41, 834–838.

Ishimaru, K., Nonaka, G.I., Nishioka, I., 1987. Tannins and related compounds. LV.Isolation and characterisation of acutissimins A and B, novel tannins fromQuercus and Castanea species. Chemical and Pharmaceutical Bulletin 35,602–610.

Ito, H., 2011. Metabolites of the ellagitannin geraniin and their antioxidantactivities. Planta Medica 77, 1110–1115.

King, J., 1856. American Eclectic Dispensatory. Moore, Wilstach, Keys, & Co.,Cincinnati.

Kiss, A.K., Granica, S., Stolarczyk, M., Melzig, M.F., 2012. Epigenetic modulation ofmechanisms involved in inflammation: influence of selected polyphenolicsubstances on histone acetylation state. Food Chemistry 131, 1015–1020.

Kupeli, E., Tatli, I.I., Akdemir, Z.S., Yesilada, E., 2007. Estimation of antinociceptiveand anti-inflammatory activity on Geranium pratense subsp. finitimum and itsphenolic compounds. Journal of Ethnopharmacology 114, 234–240.

Lamela, M., Cadavid, I., Calleja, J.M., 1986. Effects of Lythrum salicaria extracts onhyperglycemic rats and mice. Journal of Ethnopharmacology 15, 153–160.

Menković, N., Savikin, K., Tasić, S., Zdunić, G., Stesević, D., Milosavljević, S., Vincek, D.,2011. Ethnobotanical study on traditional uses of wild medicinal plants inProkletije Mountains (Montenegro). Journal of Ethnopharmacology 133, 97–107.

Moilanen, J., Sinkkonen, J., Salminen, J.P., 2013. Characterization of bioactive plantellagitannins by chromatographic, spectroscopic and mass spectrometricmethods. Chemoecology 23, 165–179.

Mosser, D.M., Edwards, J.P., 2008. Exploring the full spectrum of macrophageactivation. Nature Reviews Immunology 8, 958–969.

Neves, J.M., Matos, C., Moutinho, C., Queiroz, G., Gomes, L.R., 2009. Ethnopharma-cological notes about ancient uses of medicinal plants in Tras-os-Montes(northern of Portugal). Journal of Ethnopharmacology 124, 270–283.

Nishimura, H., Nonaka, G.I., Nishioka, I., 1986. Tannins and related compounds.XLVI. Isolation and structures of stenophynins A and B, novel tannins fromQuercus stenophylla MAKINO. Chemical and Pharmaceutical Bulletin 34,3223–3227.

Nitta, Y., Kikuzaki, H., Azuma, T., Ye, Y., Sakaue, M., Higuchi, Y., Komori, H., Ueno, H.,2013. Inhibitory activity of Filipendula ulmaria constituents on recombinanthuman histidine decarboxylase. Food Chemistry 138, 1551–1556.

Piwowarski, J.P., Granica, S., Kosiński, M., Kiss, A.K., 2014. Secondary metabolitesfrom roots of Geum urbanum L. Biochemical Systematics and Ecology 53, 46–50.

Piwowarski, J.P., Kiss, A.K., 2013. C-glucosidic ellagitannins from Lythri herba(european pharmacopoeia): chromatographic profile and structure determina-tion. Phytochemical Analysis 24, 336–348.

Popa, C., Netea, M.G., van Riel, P.L., van der Meer, J.W., Stalenhoef, A.F., 2007. Therole of TNF-alpha in chronic inflammatory conditions, intermediary metabo-lism, and cardiovascular risk. Journal of Lipid Research 48, 751–762.

Popovic, Z., Smiljanic, M., Matic, R., Kostic, M., Nikic, P., Bojovic, S., 2012.Phytotherapeutical plants from the Deliblato sands (Serbia): traditional phar-macopoeia and implications for conservation. Indian Journal of TraditionalKnowledge 11, 385–400.

Saklani, A., Hegde, B., Mishra, P., Singh, R., Mendon, M., Chakrabarty, D., Kamath, D.V., Lobo, A., Mishra, P.D., Dagia, N.M., Padigaru, M., Kulkarni-Almeida, A.A.,2012. NF-kappaB dependent anti-inflammatory activity of chlorojanerin iso-lated from Saussurea heteromalla. Phytomedicine 19, 988–997.

Sarić-Kundalić, B., Dobes, C., Klatte-Asselmeyer, V., Saukel, J., 2011. Ethnobotanicalsurvey of traditionally used plants in human therapy of east, north and north-east Bosnia and Herzegovina. Journal of Ethnopharmacology 133, 1051–1076.

Seeram, N.P., Henning, S.M., Zhang, Y., Suchard, M., Li, Z., Heber, D., 2006.Pomegranate juice ellagitannin metabolites are present in human plasma andsome persist in urine for up to 48 h. The Journal of Nutrition 136, 2481–2485.

Singh, K.N., Lal, B., 2008. Ethnomedicines used against four common ailments bythe tribal communities of Lahaul-Spiti in western Himalaya. Journal ofEthnopharmacology 115, 147–159.

Soukand, R., Kalle, R., 2013. Where does the border lie: locally grown plants usedfor making tea for recreation and/or healing, 1970s-1990s estonia. Journal ofEthnopharmacology 150, 162–174.

Szekanecz, Z., Koch, A.E., 2007. Macrophages and their products in rheumatoidarthritis. Current Opinion in Rheumatology 19, 289–295.

Tanaka, T., Tachibana, H., Nonaka, G., Nishioka, I., Hsu, F.L., Kohda, H., Tanaka, O.,1993. Tannins and related compounds. CXXII. New dimeric, trimeric andtetrameric ellagitannins, lambertianins A-D, from Rubus lambertianus Seringe.Chemical and Pharmaceutical Bulletin 41, 1214–1220.

Tita, I., Mogosanu, G.D., Tita, M.G., 2009. Ethnobotanical inventory of medicinalplants from the south-west of romania. Farmacia 57, 141–156.

Tomczyk, M., Latte, K.P., 2009. Potentilla- a review of its phytochemical andpharmacological profile. Journal of Ethnopharmacology 122, 184–204.

Truchado, P., Larrosa, M., Garcia-Conesa, M.T., Cerda, B., Vidal-Guevara, M.L., Tomas-Barberan, F.A., Espin, J.C., 2012. Strawberry processing does not affect theproduction and urinary excretion of urolithins, ellagic acid metabolites, inhumans. Journal of Agricultural and Food Chemistry 60, 5749–5754.

Tunalier, Z., Kosar, M., Kupeli, E., Calis, I., Baser, K.H.C., 2007. Antioxidant, anti-inflammatory, anti-nociceptive activities and composition of Lythrum salicariaL. extracts. Journal of Ethnopharmacology 110, 539–547.

Ushiki, J., Tahara, S., Hayakawa, Y., Tadano, T., 1997. Suppressive effect of Geraniumpratense L. on potato common scab and identification of the effective com-pound. Soil Science and Plant Nutrition 78, 767–768.

Verzelloni, E., Pellacani, C., Tagliazucchi, D., Tagliaferri, S., Calani, L., Costa, L.G.,Brighenti, F., Borges, G., Crozier, A., Conte, A., Del Rio, D., 2011. Antiglycative andneuroprotective activity of colon-derived polyphenol catabolites. MolecularNutrition & Food Research 55, S35–S43.

Vogl, S., Picker, P., Mihaly-Bison, J., Fakhrudin, N., Atanasov, A.G., Heiss, E.H.,Wawrosch, C., Reznicek, G., Dirsch, V.M., Saukel, J., Kopp, B., 2013. Ethnophar-macological in vitro studies on Austria's folk medicine–an unexplored lorein vitro anti-inflammatory activities of 71 Austrian traditional herbal drugs.Journal of Ethnopharmacology 149, 750–771.

Zhang, X., Mosser, D.M., 2008. Macrophage activation by endogenous dangersignals. Journal of Pathology 214, 161–178.

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