Metabolic transformations of dietary polyphenols: comparison between in vitro colonic and hepatic models and in vivo urinary metabolites

    Metabolic transformations of dietary polyphenols: comparison between in vitro colonic and hepatic models and in vivo urinary metabolites Claudia Vetrani, Angela A. Rivellese, Giovanni Annuzzi, Martin Adiels, Jan Bor´en, Ismo Mattila, Matej Oreˇsiˇc, Anna-Marja Aura PII: DOI: Reference:

S0955-2863(16)30027-4 doi: 10.1016/j.jnutbio.2016.03.007 JNB 7568

To appear in:

The Journal of Nutritional Biochemistry

Received date: Revised date: Accepted date:

28 October 2015 29 February 2016 7 March 2016

Please cite this article as: Vetrani Claudia, Rivellese Angela A., Annuzzi Giovanni, Adiels Martin, Bor´en Jan, Mattila Ismo, Oreˇsiˇc Matej, Aura Anna-Marja, Metabolic transformations of dietary polyphenols: comparison between in vitro colonic and hepatic models and in vivo urinary metabolites, The Journal of Nutritional Biochemistry (2016), doi: 10.1016/j.jnutbio.2016.03.007

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ACCEPTED MANUSCRIPT Metabolic transformations of dietary polyphenols: comparison between in vitro colonic and hepatic models and in vivo urinary metabolites

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Claudia Vetrania*, Angela A. Rivellesea, Giovanni Annuzzia, Martin Adielsb,c, Jan Borénb, Ismo

Department of Clinical Medicine and Surgery, “Federico II” University, Naples, Italy.

b

Sahlgrenska Center for Cardiovascular and Metabolic Research/Wallenberg Laboratory,

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a

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Mattilad, Matej Orešičd, Anna-Marja Aurae

Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden. Department of Mathematical Sciences, University of Gothenburg, Gothenburg, Sweden.

d

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c

Steno Diabetes Center, Gentofte, Denmark.

e

VTT Technical Research Centre of Finland, Espoo, Finland.

*corresponding author: Claudia Vetrani, Department of Clinical Medicine and Surgery, “Federico

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II” University, 5, S. Pansini 80131, Naples, Italy. Tel. +39 81 7462306, Fax +39 81 7462321, E-

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mail: [email protected]

Financial Support: This work was supported by ETHERPATHS project (European Commission

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Grant FP7-KBBE-222639) and Ministero dell’Istruzione, dell’Università della Ricerca, Rome, Italy (PRIN n. 2010JC WWKM).

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Short title: Metabolic transformations of dietary polyphenols

Chemical compounds studied in this article: 3,4-dimethoxybenzoic acid (PubChem CID: 7121); 3,4-dihydroxybenzoic acid (PubChem CID: 72); 2-(3´,4´-dihydroxyphenyl)acetic acid (PubChem CID: 547);

3-(3´,4´-dihydroxyphenyl)propionic

acid

(PubChem

CID:

348154);

3,5-

dihydroxybenzoic acid (PubChem CID: 7424); 3-hydroxybenzoic acid (PubChem CID: 7420); 4hydroxybenzoic acid (PubChem CID: 135); 4-methylcatechol (PubChem CID: 9958); Benzoic acid (PubChem CID: 243); Gallic acid (PubChem CID: 370), Ferulic acid (PubChem CID: 445858); Sinapic acid (PubChem CID: 637775); Caffeic acid (PubChem CID: 689043); 4-coumaric acid (PubChem CID: 637542); Enterodiol (PubChem CID: 115089); Vanillic acid (PubChem CID: 8468); Hippuric acid (PubChem CID: 464).

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ACCEPTED MANUSCRIPT Abstract

Studies on metabolism of polyphenols have revealed extensive transformations in the carbon

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backbone by colonic microbiota; however, the influence of microbial and hepatic transformations

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on human urinary metabolites has not been explored. Therefore, the aims of this study were: 1) to compare the in vitro microbial phenolic metabolite profile of foods and beverages with that excreted in urine of subjects consuming the same foodstuffs, and 2) to explore the role of liver on post-

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colonic metabolism of polyphenols by using in vitro hepatic models.

24h-urinary phenolic metabolite profile was evaluated in 72 subjects participating in a 8-week

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clinical trial during which they were randomly assigned to diets differing for polyphenol content. Polyphenol-rich foods and beverages used in the clinical trial were subjected to human faecal microbiota in the in vitro colon model. Metabolites from green tea, one of the main components of the polyphenol-rich diet, were incubated with primary hepatocytes to highlight hepatic conversion of polyphenols. The analyses were performed using targeted gas chromatography with mass

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spectrometer (GCxGC-TOFMS:colon model; GC-MS: urine and hepatocytes). A significant correlation was found between urinary and colonic metabolites with C1-C3-side-chain

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(p=0.040). However, considerably higher amounts of hippuric acid, 3-hydroxybenzoic acid and ferulic acid were detected in urine than in the colon model. The hepatic conversion showed

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additional amounts of these metabolites complementing the gap between in vitro colon model and the in vivo urinary excretion.

Therefore, combining in vitro colon and hepatic models may better elucidate the metabolism of

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polyphenols from dietary exposure to urinary metabolites.

Key words: polyphenols, metabolism, phenolic carbon backbone, in vitro colon model, primary hepatocytes.

Abbreviations: 3,4-diMeBA: 3,4-dimethoxybenzoic acid; 3,4-diOHBA: 3,4-dihydroxybenzoic acid;

3,4-diOHPAc:

dihydroxyphenyl)propionic hydroxybenzoic

acid;

2-(3´,4´-dihydroxyphenyl)acetic acid;

3,5-diOHBA:

3-OHPAc:

acid;

3,4-diOHPPr:

3,5-dihydroxybenzoic

2-(3´-hydroxyphenyl)acetic

acid;

acid;

3-(3´,4´-

3-OHBA:

3-OHPPr:

3-

3-(3´-

hydroxyphenyl)propionic acid; 3-PPr: 3-phenylpropionic acid; 4-OHBA: 4-hydroxybenzoic acid; 4mecatechol: 4-methylcatechol; 4-OHPPr: 3-(4´-hydroxyphenyl)propionic acid.

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ACCEPTED MANUSCRIPT 1. Introduction

Polyphenols is a group of phytochemicals which have been studied extensively. Polyphenol intake

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is associated with reduced risk of cardiovascular disease and diabetes as shown by epidemiological

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findings [1-4]. In addition, some clinical trials have shown the effect of polyphenol exposure on cardiometabolic risk factors, mainly lipid and glucose metabolism and oxidative stress [5-9]. Hence, the benefits of polyphenol intake are becoming more and more apparent.

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Moreover, the metabolism of polyphenols has been explored. After the intake, a small amount of the ingested polyphenols are absorbed from intestine and can be converted to glucuronidated and

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sulphated conjugates by the enzymes of intestinal tissue, and enter the bloodstream. In addition, in the liver they can be further metabolized to glucuronidated, sulphated and glycinated derivatives [10,11]. Furthermore, studies on metabolism of polyphenols has revealed extensive microbial transformations in the carbon backbone by colonic microbiota [12,13]. After colonic metabolism microbial metabolites are absorbed and have a long residence time (up to 24-48 hours) in the

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bloodstream before being excreted in urine [14,15]. Moreover, human intervention studies have investigated the metabolism of single foodstuffs or compound group (i.e. flavan-3-ols,

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anthocyanins, phenolic acids) [15-20]. The study presented here is a continuation of our previous publication [21] in which a polyphenol-rich diet was consumed by subjects having features of

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metabolic syndrome. Urinary excretion was analysed for hydroxylated phenylpropionic and –acetic acids, some benzoic acid derivatives, and mammalian lignans; the phenolic profile showed a significant difference between the polyphenol-diet consuming group and the control group that was

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achieved by strict control of polyphenol-rich foodstuffs [21]. In addition, some transformations of carbon backbone of dietary polyphenols were observed. However, it was not clear what were the roles of colonic and post-colonic hepatic metabolism in the carbon backbone transformations of excreted phenolic metabolites in urine. Hence, in this study the same polyphenol-rich foodstuffs used in the clinical trial were subjected to the conversions in the in vitro colon model to find the correlation between microbial and urinary metabolites. Furthermore, green tea microbial metabolite extract was exposed to the post-colonic hepatic conversions, because microbial phenolic acid metabolites of green tea at 6 hour-time point were diverse and the extract could represent a microbial metabolite profile of the polyphenol-rich diet better than any other single food or beverage used in the study. The hypothesis of this study is that transformations of carbon skeleton of dietary polyphenols reflect both colonic and subsequent hepatic metabolism and this can be shown in the urinary excretion profile.

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ACCEPTED MANUSCRIPT 2. Materials and methods

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2.1. Clinical trial

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A description of the clinical trial has been published earlier [21]. Briefly, 86 overweight/obese subjects with a high cardiovascular and metabolic risk profile were enrolled in the study. They had a waist circumference above the standard cut-off and at least one of the features of the metabolic

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syndrome according to the NCEP/ATP III criteria [22]. They followed a high-polyphenol diet (HPdiet; 2868 mg/day) or a low-polyphenol control diet (LP-diet, 363mg/day) for 8 weeks. The two

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diets were isoenergetic and had the same composition for nutrients. The main dietary sources of polyphenols in the HP-diet were: artichokes, fennel, onion, spinach, arugula salad (rucola), orange, dark chocolate bar, decaffeinated coffee and green tea. The intake of alcoholic beverages was not allowed. All foods and beverages were provided to the subjects during the intervention. At baseline and after the intervention all subjects collected 24h-urine for the evaluation of the excretion of

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phenolic metabolites. The volunteers received careful instructions and motivation to complete 24hurine collection. After a 12h-overnight fast, they collected all excreted urine to a 3000-ml-plastic

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urine containers (SARSTEDT s.r.l. Verona, Italy) until the following morning. The study was conducted according to the guidelines of the Declaration of Helsinki, and all

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procedures involving human subjects were approved by “Federico II” University Ethics Committee. Written informed consent was obtained from all subjects. The study was registered at

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www.clinicaltrials.gov (identifier NCT01154478).

2.2. In vitro colon model

2.2.1. Materials Onion (cultivated in Finland), fennel (cultivated in The Netherlands), artichoke (cultivated in France), spinach (cultivated in Italy), oranges (cultivated in South Africa), arugula salad (cultivated in Italy), green tea (China Gun Powder, Nordqvist, Helsinki, Finland) and dark cocoa powder (Van Houten®, Lebecke Wieze, Belgium) were purchased from a local store in Finland, whereas decaffeinated coffee (Lavazza DeK, Torino, Italy) was purchased in Italy. The internal standard during the comprehensive profiling of small polar metabolites from the colon model was 2-hydroxycinnamic acid (Aldrich Inc. St. Louis, USA). The following compounds were used as standards: benzoic acid, 3-hydroxybenzoic acid (3-OHBA), 3-(4´-hydroxyphenyl)propionic acid (4-OHPPr), 4-methylcatechol (4-mecatechol) and 3-(3´,4´-dihydroxyphenyl)propionic acid 4

ACCEPTED MANUSCRIPT (3,4-diOHPPr) were purchased from Aldrich (Steinheim, Germany); 4-hydroxybenzoic acid (4OHBA), 2-(3´-hydroxyphenyl)acetic acid (3-OHPAc), 2-(3´,4´-dihydroxyphenyl) acetic acid (3,4diOHPAc), 4-hydroxycinnamic acid and ferulic acid (3-methoxy-4-hydroxy-cinnamic acid) from

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Sigma (St. Louis, USA); 3-phenylpropionic acid (3-PPr), vanillic acid (3-methoxy-4-

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hydroxybenzoic acid) and 3,4-dihydroxybenzoic acid (3,4-diOHBA) from Fluka (Buchs, Switzerland); 3-(3´-hydroxyphenyl)propionic acid (3-OHPPr) from Alfa Aesar (Karlsruhe, Germany) and gallic acid from Extrasynthése (Genay, France).

N-methyl-N-trimethylsilyl-

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trifluoracetamide (MSTFA) from Pierce (Rockford, USA) and methoxyamine hydrochloride (2%)

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in pyridine (MOX; Pierce, Rockford, USA) were used in the derivatization of the metabolites.

2.2.2. Preparation of foods for the colon model.

In order to remove free sugars, orange, onion and fennel were pre-digested in enzymatic digestion in vitro model and freeze-dried, as described below (2.2.4 Enzymatic in vitro digestion). Other foods or beverages were not pre-digested in the upper intestinal model to preserve soluble

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indigestible material. Spinach and arugula salad were directly freeze-dried, because they do not contain starch, sugar or protein in excess to disturb microbial metabolism and to avoid loss of

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polyphenols in the upper intestinal model. Artichoke was cooked for 45 min in acidified (15 ml of 10 % acetic acid in 3 L of water) and freeze-dried. Green tea infusion was prepared by incubating

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50 g of green tea in 500 ml of boiling water for 5 min and filtering the tea leaves, which were discarded. Green tea infusion was cooled and freeze-dried. Decaffeinated espresso was prepared using an SIC® certified espresso maker (Morenita Express, Ornavasso, Italy) according to the

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instructions of use. The coffee from several 3-cup-portions were combined and cooled rapidly on ice in the anaerobic chamber to avoid oxidation. Large aliquots of coffee were also frozen and freeze-dried prior to the hydration and dosage for use in the colon model. Commercial dark cocoa powder (Van Houten®, Lebbeke-Wiese, Belgium) was used as it is.

2.2.3. Characterization of the foods and beverages The dry weight (d.w.) of the foods were determined by weighing at least 3 samples after freezedrying (when applicable) and the remnant moisture was determined by Karl-Fischer titration. The samples were weighed for the colon model experiment on the basis of the absolute d.w.

2.2.4. Enzymatic in vitro digestion Enzymatic in vitro digestion of samples (10.5 g d.w.) was performed as described by Aura et al. [23] with following exceptions: the method was scaled up (7-fold) and the digestion and filtration 5

ACCEPTED MANUSCRIPT were performed at 37 ºC under anaerobic conditions to prevent oxidation of the phenolic components. Strictly anaerobic conditions were accomplished by operating in an anaerobic chamber (Don Whitley, Shipley, West Yorkshire, UK) using a gas mixture: nitrogen 80%, hydrogen 10%,

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carbondioxide 10%, and by using reduced 20 mM sodium phosphate buffer (pH 6.9) by heating the

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buffer to 80° C and cooling under nitrogen stream and placing the buffer into the anaerobic chamber for two days before using for the upper intestinal model. The atmosphere of the chamber

rapidly and freeze-dried in vacuum.

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2.2.5. Incubation in the in vitro colon model

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was verified by detective strips sensitive for oxygen. Digested and filtrated samples were frozen

Colon model experiments were performed under strictly anaerobic conditions according to Aura et al. [24,25]. In the colon model anaerobic conditions were maintained in a same manner as in the upper intestinal model described in the section 2.2.4, but the buffer was 0.11 M carbonate-0.02 M pheosphate buffer pH 5.5 containing cysteine-HCl (0.5 g/L) as a reducing agent.

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Faecal suspensions (10 %, w/v) for each experiment were prepared from freshly passed faeces from at least four healthy volunteers who had made a written informed consent. Faecal samples were

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numbered to hide the identity of the donors. The suspensions were filtered through a 1 mm sieve and diluted to 10 % (w/v). Each solid food was dosed 10 mg d.w./1 mL of faecal suspension,

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whereas freeze-dried green tea and coffee, and dark cocoa powder were dosed 2.5 mg d.w./1 mL of faecal suspension. Triplicate samples were incubated in a water bath at 37 ºC for 0, 2, 4, 6, 8 and 24 hours and stirred magnetically (250 rpm). Aliquots were drawn from the bottles and microbiota was

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removed by centrifugation (Heraeus, Biofuge Primo R, Thermo Scientific Inc. Waltham, MA. USA; 7000g, 4°C, 10 min) and 0.2 µm PTFE-filters (Millipore Corp., Bedford, MA, USA) before extraction of phenolic metabolites from 1 mL of microbe-free faecal water. The samples were extracted twice by ethylacetate and silylated as described in Aura et al. [23].

2.3. Human primary hepatocyte experiments

2.3.1. Hepatic incubation Human primary hepatocytes (Biopredic International, Rennes, France) were incubated according to the protocol and media supplied by Biopredic (Rennes, France). Cells were incubated for at least 24 hours in fresh maintaining media, after which they were subjected to cell medium or exposure to diluted (1:10) extracts: green tea extract after colonic conversion (converted green tea), faecal microbial extract (no green tea), or green tea extract before colonic conversion (no microbiota) for 8 6

ACCEPTED MANUSCRIPT or 24 hours. Faecal microbial extract (no green tea) and green tea extract before colonic conversion (no microbiota) were used as controls. To exclude spontaneous chemical conversions in the medium, the medium without hepatocytes with

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the same extracts and controls were incubated for 8 and 24 hours. Cells were washed with PBS (2

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ml) twice and 400 μl PBS and protease inhibitors (leupeptin, trasylol, PMSF, pepstatin, Calpain,

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EDTA) were added. 50 μl were used for cell counting and 350 μl were frozen for extraction.

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2.3.2. Cell extraction

A mixture of 25 ml Tris-HCl (1M), 15 ml NaCl (5M), 5 ml EDTA (0.5 M), 2.5 ml PMSF and 5 ml Triton X-100 were diluted to 100 ml (pH 7.4) and 87.5 μl was added to the frozen cells. After three intervals of ultrasound treatment (15s), 1 ml of methanol and 15 μl of 2-hydroxycinnamic acid (1:8) were added and the mixture was vortexed for 1 minute and let to rest at room temperature. The

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lysed hepatocytes and media samples (500 µl) were extracted twice with 1 ml methanol. The combined extracts were evaporated to dryness, and 500 µl of 0.15 M acetic acid buffer (pH 4.1)

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with the addition of ascorbic acid (25 mg) were added. Samples were hydrolysed using βglucuronidase enzyme from Helix pomatia (10 mg; SIGMA G0751-500KU, St. Louis, USA) at

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37°C for 16 h and an internal standard (123 ppm 2-hydroxycinnamic acid, Aldrich, St Louis, USA). All samples were extracted twice using 3 ml of ethyl acetate and evaporated to dryness and closed

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under nitrogen flow.

2.4. Analysis of the samples and calculation

Microbial metabolites of foods and beverages from the colon model were analysed using the twodimensional gas chromatography coupled with time-of-flight mass detection (GCxGC-TOFMS; Leco Pegasus 4D) instrument as described by Aura et al.[26]. Time course of the food specific metabolite concentrations (μM) for each food or beverage are shown in Supplement 1.

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metabolites from human urine and primary hepatocytes were analysed using the targeted analysis of microbial metabolites by gas chromatography with mass detection (GC-MS) using SCAN mode as described by Aura and co-workers [26] with authentic standards and 2-hydroxycinnamic acid as the internal standard. Metabolite concentrations in hepatocyte media were expressed in nmol/L whereas in urine in nmol/mg creatinine. For further details on the analyses of urinary metabolites see Vetrani et al. [21] . 7

ACCEPTED MANUSCRIPT The estimated daily exposure to polyphenol metabolites from polyphenol-rich food and beverages was calculated as follows: metabolite profile of 6-hour-time point in the in vitro colon model was chosen and the concentration of each metabolite in faecal control was subtracted. 6-hour time point

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was selected because polyphenols are fully converted to the diverse microbial metabolites and

concentration of each metabolite was expressed as nmol/day.

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would represent the likely daily absorption profile of the metabolites from the colon. The

In addition, the differences between green tea infusions and cocoa samples used in the clinical trial

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and in the in vitro colon model were taken into account in the calculations. Green tea infusion used in the colon model (50 g green tea/500 ml hot water) was stronger than the infusion used in the

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clinical trial (12 g green tea/400 ml hot water). Hence, metabolite concentrations from green tea in the colon model were divided by coefficient 3.333. (Figure 1a) Dark cocoa powder used in the colon model differed from the dark chocolate bar used in the clinical trial and fat and sugar contents needed to be subtracted, respectively, according to the list of the ingredients: 100 g of dark chocolate bar corresponded to 16.7 g of fatless and sugarless cocoa and so the coefficient for dark

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chocolate bar is 0.167. Furthermore, 100 g of dark cocoa powder corresponds to 78.6 g fatless and sugarless cocoa and so the coefficient is 0.786. The concentrations of dark cocoa microbial

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metabolites were divided by 0.786 (dark cocoa powder) and multiplied by 0.167 (dark chocolate bar) (Figure 1b).

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Finally, the daily dose (g/portion) and the frequency (portion/week) of the foods and beverages used in the trial [21] were taken into account and the total concentration (nmol/day) of each metabolite

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was calculated as the sum of concentrations from all the foodstuffs.

2.5. Statistical analysis

The statistics of the metabolites concentrations (μM) in the colon model in triplicates were analysed using MatLab Version R2008b and Two-Way ANOVA with repeated measures using a Bonferroni adjustment. The metabolite was considered a foodstuff-related one, when the response differed significantly (p<0.05) from the faecal control (no added substrate). For primary hepatocytes experiments, post-hoc tests were applied to distinguish significant (p<0.05) differences between extracts of green tea after colonic conversion (converted green tea) and controls: green tea before colonic conversion (no microbiota) or faecal control (no green tea), and between the corresponding responses of hepatocytes and of the media without hepatocytes. Bivariate associations between estimated and urinary metabolites were analysed by Pearson’s correlation coefficients and a p value <0.05 was considered significant. These analyses were 8

ACCEPTED MANUSCRIPT performed according to standard methods using the Statistical Package for Social Sciences software version 21.0 (SPSS/PC; SPSS, Chicago, IL, USA).

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3. Results

3.1. Clinical trial

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Seventy-eight subjects completed the trial but only 72 participants provided 24h-urine samples and were considered for the analyses: 37 subjects for the LP-diet and 35 for the HP-diet [21].

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Complementary to the previous publication[21], the urinary metabolite profile detected after the HP-diet and the absolute changes from baseline values are reported in table 1. A significantly higher concentrations were found for colonic metabolites with C2-C3-side-chain (hydroxylated phenylacetic, -propionic acids), but not for C1-side chain (benzoic acid). Hippuric acid and 3-hydroxybenzoic acid represented C1-side chain and ferulic acid

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3.2. In vitro colon model

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group than in the LP group.

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hydroxycinnamic acids showing significantly higher concentrations in urinary excretion in the HP

The time course of microbial metabolite formation from each food or beverage are shown in Supplement 1.

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The calculated concentration of phenolic microbial metabolites is shown in Table 2. The abundant metabolites (> 5000 nmol/24h) in the calculated concentrations and their source foods were 3-(3’hydroxyphenyl)propionic acid (3-OHPPr: artichoke, orange and coffee), 3-phenylpropionic acid (3PPr: artichoke, orange and green tea), 3-(3’,4’-dihydroxyphenyl)propionic acid (3,4-diOHPPr: coffee,

arugula

salad

and

fennel),

2-(3’,4’-dihydroxyphenyl)acetic

acid

(3,4-diOHPAc:

arugulasalad, spinach and onion), 3-(4’-hydroxyphenyl)propionic acid (4-OHPPr: artichoke and orange), benzoic acid (BA: orange, onion and fennel), and finally 2-(3-hydroxyphenyl)acetic acid (3-OHPAc: Cocoa powder). Metabolites showing low calculated average concentrations (< 5000nmol/day) were 3-hydroxybenzoic acid (3-OHBA: artichoke, fennel, arugula salad and spinach), sinapic acid (arugulasalad), 4-coumaric acid (spinach), 3,4-dihydroxybenzoic acid (3,4diOHBA: spinach, cocoa powder and fennel), 4-hydroxybenzoic acid (4-OHBA: artichoke, fennel and cocoa powder), 4-methyl catechol (coffee, artichoke and cocoa powder), ferulic acid (cocoa and spinach), gallic acid (fennel and onion), vanillic acid (arugulasalad) and finally enterodiol (orange, 9

ACCEPTED MANUSCRIPT onion and green tea). Ferulic acid and 3-OHBA showed low concentrations in the 6-h-metabolite profile taking into account the dose and frequency of their precursor foodstuffs: cocoa and spinach, and artichoke, fennel, arugulasalad and spinach, respectively.

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A moderate but significant correlation between average calculation of 6-h-colonic metabolites from

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in vitro colon model and 24-h-urinary excretion in vivo was observed (r=0.280, p =0.040; Figure 2). Since most of the 3-PPR and 4-OHBA originated from the faecal background (Supplement 1), these

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metabolites were excluded from the correlation analysis as a non-relevant artefact.

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3.3. Human primary hepatocytes

When hepatocytes were incubated with green tea extract after colonic conversion (converted green tea), significant concentrations of 3-OHBA, 3,4-diOHPPr , ferulic acid and hippuric acid were formed as post-colonic hepatic metabolites (Figure 3). The same metabolites exhibited low

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concentrations or were completely absent from the control incubations: green tea extract before colonic conversion (no microbiota) or faecal control (no green tea), or in the absence of hepatocytes

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(media with each extract).

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4. Discussion

This study showed that the microbial polyphenol metabolism of the occurring in the carbon

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backbone of the phenolic compounds is partly due to microbial conversion and partly by postcolonic hepatic conversions. A significant correlation was found between the metabolite profiles in 24h-urine from the clinical trial [21] and the estimated average of each metabolite formed in the in vitro colon model (Table 2). Microbial metabolites formed from green tea in 6 hours in the in vitro colon model were chosen as representatives of microbial metabolites from all food and beverages to the hepatic incubation study. This was considered the best choice because green tea is soluble in medium and its microbial metabolites were diverse and typical for a polyphenol-rich diet. The colonic model profile contained benzoic acid but no hippuric acid. In contrast, the urinary excretion showed significantly higher hippuric acid concentrations, but no difference in benzoic acid concentrations between high- and low-polyphenol groups [21]. Hippuric acid is a hepatic glycine conjugate of benzoic acid [27] and its high levels in urine is consistent with a previous study showing increased excretion of hippuric acid in urine after green tea intake [28]. Hippuric acid can be a metabolite in hepatocytes from colonic microbial metabolites; benzoic acid as such or 10

ACCEPTED MANUSCRIPT from phenylpropionic acid (3-PPr, in abundance in the colon model) or phenylacetic acid (not analysed), the side chain of which can be shortened by β-oxidation [29,30]. The resulting benzoic acid is finally glycinated to hippuric acid, as has been reported earlier [27,28,31]. Thus, the gap

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between urinary excretion in vivo and colonic conversions in vitro concerning benzoic acid and

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hippuric acid could be explained by the hepatic metabolites.

3-OHBA and ferulic acid were also formed in the hepatocytes after incubation with converted green tea and were absent from the media without hepatocytes or controls without microbiota. The

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same metabolites showed significantly higher concentrations in the urine after the polyphenol-rich diet in the clinical trial [21]. In addition to benzoic acid, 3-OHBA may be a β-oxidation product, but

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in this case from of 3-OHPPr. Moreover, ferulic acid (4’-hydroxy-3’-methoxy cinnamic acid) can be formed from 4-OHPPr via methoxylation at position 3’, or via methylation and hydroxylation at the positions 3’ and 4’, respectively, from 3-OHPPr. In addition, oxidation reaction is required to complete the structure of hydroxycinnamic acids (double-bond in the side –chain). Thus the hepatic incubation could also fill in the gap between human urinary excretion and the in vitro colon model

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in respect to 3-OHBA and ferulic acid. Thus, the results obtained in the present study suggest that liver has a role in the post-colon metabolism prior to excretion to urine.

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Several major metabolites of dietary phenolic compounds were common to both colon model and urinary excretion such as 3-OHPAc, 3,4-diOHPPr, 3-OHPPr, and 3,4-diOHPAc, which

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represent the major metabolites of flavonoids and phenolic acids [9] and were the main metabolites contributing to the in vitro-in vivo correlation. However, 3-PPR and 4-OHBA were excluded from the correlation analysis, because their concentration in the faecal background was high and they

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most likely originated from the diets of the donors and may originate from several precursors. 3PPR is considered as non-specific artefact due to its higher presence in the faecal background than in the presence of the precursor foodstuffs, as shown in the experiments 1-4 (Supplement 1). The colon model does not take into account further metabolism of the compounds by tissues, but especially 3-PPr, regardless of its origin, could serve as a precursor of hepatic metabolites benzoic acid and hippuric acid, as mentioned above. The results presented in this study showed the structural transformations occurring in the carbon backbone of the phenolic compounds, excluding glucuronide conjugates, which would be hydrolysed by the colonic microbiota. Therefore, Helix pomatia snail glucuronidase was used in this study in the hydrolysis of urine and hepatic samples. It is possible that sulphate conjugates are not sufficiently cleaved in the preparation of urine or hepatocyte samples and esterase activities may additionally cleave oxygen bridges, or other oxidative reactions may also occur by Helix pomatia enzyme. Esterase side activities have been shown for Helix pomatia enzyme mixture when 11

ACCEPTED MANUSCRIPT chlorogenic acid was not found in urinary or plasma samples hydrolysed with Helix pomatia [3235]. However, it has been shown that intestine has also esterases, which may cause similar hydrolysis of ester or ether bonds [36-37]. In addition, microbiota has a strong capacity to

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deconjugate phenolic compounds [24]. Ferulic acid was found (with methoxyl functional group) in

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the incubation with hepatocytes and in urinary samples, despite the use of the same glucuronidase enzyme at the same pH 4.1. Therefore, a limit of this study is the inadequate hydrolysis of sulphate conjugates, if Helix pomatia enzyme mixture did not contain sufficient amount of sulphatase or the

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operating pH was not optimal, which may have caused underestimation of sulphated metabolites, which also can explain why the correlation is only moderate, even though it is significant.

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Results from the in vitro colon model showed microbial metabolite profiles for phenolic acids as has been shown for the majority of polyphenols or polyphenol-rich foods [12,38-41]. Moreover, as suggested by previous studies [39,41], hydroxylated phenylpropionic and benzoic acids were detected as metabolites of coffee. Finally, small amounts of enterodiol but no enterolactone was detected in the present study as metabolites from plant lignans [11]. Enterolactone formation is

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suppressed in the colon model in vitro, partly because of low pH, as reported previously [42], and partly because the foodstuffs in the presented study were chosen as typical Mediterranean foods rich

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in polyphenols, not as good sources of plant lignans. Enterolactone formation was not in the focus of this study, but was monitored from all the samples. In contrast, the formation of phenolic acid

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metabolites is not affected by the accumulation of the metabolites [42]. Some of the main metabolites of green tea, namely valeric acid and valerolactone derivatives [43,44], were unfortunately not included in the analyses. 5-(3´-hydroxyphenyl) valeric acid and 5-(3´,4´-

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dihydroxyphenyl) valeric acid were detected as microbial metabolites from (+)-catechin and (-)epicatechin stereoisomers using GCxGC-TOFMS metabolomics [43] ; however, the authentic standards of valeric acid and valerolactone derivatives were not available by the time of this work. In addition, gallic acid, surely abundant in green tea is metabolised quickly and it had a minor weight in the average calculations. The comparison of in vivo and in vitro metabolite profiles is challenging due to high individual variability of all colonic metabolites [45], which can partly be caused because of individual variation of microbiota, consisting of over 1000 different species [46,47]. However, the conversion differences are not extensive because the functional genes within colonic microbiota do not differ between subjects as strongly as the microbial composition, due to a ”functional core” of the microbiome [46,47]. This was described in a study of a cohort of individuals where at least 40 % of the microbial genes were shared by half of the individuals within the cohort [46]. Most likely reason of the difference between the urinary excretion and calculated colonic metabolite profile is 12

ACCEPTED MANUSCRIPT that people had the possibility to vary the intake within a week and still be compliant within the frames of the diet, but foods introduced to the colon model were defined and calculated according to averages of the instructed frequency and dose. However, the 6h-incubation time may not give the

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correct metabolite profile for the average. The effect of food matrix in onion and fennel were

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apparent, showing slower release and consequent conversion than isolated compounds or beverages would give. Therefore, the calculations may have showed more relevant metabolite profile for foods than for beverages.

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Different colonic communities share general metabolic activities, which convert food components to specific metabolite profiles [48]. It has also been shown several phenolic precursors in foods are

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converted to a relatively small number of colonic phenolic acids and lactones which represent the predominant microbial metabolites [44]. The in vitro colon model was performed with a pooled human faecal suspension from several donors to make results repeatable and to diminish this interindividual variation in vitro [48]. Such an approach was chosen because metabolites specific for each food or beverage could be distinguished from the faecal control (with no foods or beverages),

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designating the metabolite profile from the polyphenol-rich-diet, when the standard deviations are low for significant metabolites of the diet. If all the foods were incubated with faeces from 5-10

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even 20 individuals, which would have been adequate for statistics, the standard deviations would have been as high as in the urinary excretion, and specific food-related metabolites could not have

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been identified. The perspective would have been in individual responses without the possibility to identify the metabolites for each food. Using several donors for each food, the number of samples and metabolites would also have been too many considering the dimensions of the study. Therefore,

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the pooled faecal suspensions showed the significant metabolite profile for each foodstuff, and the 24-h-urinary excretion showed the natural individual variation in vivo, which served the approach of the study in an optimal manner.

5. Conclusion

Our hypothesis of this study was that urinary excretion profile reflects colonic and subsequent hepatic transformations of carbon skeleton of dietary polyphenols. This hypothesis was verified in our study by complementary concentrations of post-colonic hepatic metabolites in urinary excretion, showing also that colonic conversions reach only moderate, but still significant correlation with urinary metabolite profile. Combination of in vitro colon and hepatic models could be used as a predictive model in finding foodstuff or diet-specific metabolites in human body fluids.

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ACCEPTED MANUSCRIPT Conflict of interest The authors report no personal or financial conflict of interest arising from the present research and

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its publication.

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Acknowledgments

Food for the clinical trial was kindly supplied by Lavazza, Torino, Italy (coffee); Nestlé, Vevey, Switzerland (chocolate); Parmalat S.p.A., Parma, Italy (juice); Zuegg S.p.A., Verona, Italy (jam);

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Pompadour Tè S.r.l., Bolzano, Italy (green tea); Coop. Nuovo Cilento s.c.r.l., San Mauro Cilento, Salerno, Italy (extra-virgin olive oil).

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We gratefully acknowledge the laboratory work done by Maria Heyden and Annika Lundqvist in the hepatocyte study, Annika Majanen, Siv Matomaa, Kari Lepistö, Niina Torttila and Airi Hyrkäs in the execution of the metabolical in vitro colon model and the analyses of the metabolites; Angela Giacco and Marilena Vitale for dietary counselling.

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Authorship

The authors’ responsibilities were as follows: CV and AMA wrote the manuscript with input from

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all authors and were responsible for statistical aspects of the study; CV, AMA, AAR and GA conceived and designed the study, IM performed metabolites analyses with the input of MO; AMA,

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MA and JB carried out in vitro experiments. CV, AAR and GA were involved in the clinical trial. CV, AAR and AMA had primary responsibility for final content. All authors revised the manuscript

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and approved the final version.

Figure 1. Equations used in the correction of green tea (a) and dark chocolate (b) before the calculation for the estimation.

Figure 2. Correlation between urinary metabolites concentrations and phenolic metabolites estimated from the incubation in the in vitro colon model. 3,4-diOHBA: 3,4-dihydroxybenzoic acid; 3,4-diOHPAc: 3,4-dihydroxyphenylacetic acid; 3,4diOHPPr: 3,4-dihydroxyphenylpropionic acid; 3-OHBA: 3-hydroxybenzoic acid; 3-OHPAc: 3hydroxyphenylacetic acid; 3-OHPPr: 3-hydroxyphenylpropionic acid; 4-Cou: 4-coumaric acid; 4MeCat: 4-metylcatechol; 4-OHBA: 4-hydroxybenzoic acid; BA: benzoic acid; GA: gallic acid; FA: ferulic acid; SA: sinapic acid; ED: enterodiol; VA: vanillic acid.

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ACCEPTED MANUSCRIPT Figure 3. Hepatic conversion of microbial metabolites from green tea (light grey bars: green tea extract converted by microbes - converted green tea) in the presence of human primary hepatocytes (Hepatocytes) as compared with control extracts (medium dark grey bars: cell

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medium, black bars: microbial control - no green tea, dark grey bars: non-converted green

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tea - no microbiota), and in absence of hepatocytes (No Hepatocytes).

Of the analysed metabolites, 3,4-DiOHPPr, 3-OHBA, ferulic acid, and hippuric acid were significantly higher in the presence of converted green tea extract and hepatocytes than in all the

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controls (***:p<0.001).

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3,4-DiOHPPr: 3,4-dihydroxyphenylpropionic acid; 3-OHBA: 3-hydroxybenzoic acid.

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ACCEPTED MANUSCRIPT Table 1. Phenolic metabolites excreted in 24-hour-urine after 8 week-consumption of highpolyphenol diet and their absolute changes from baseline levels [21]. Absolute change after the intervention Mean SD Mean SD 3,4-diOHPPr 0.141 0.16 ** 0.070 0.13 *** 3-OHPPr 0.115 0.12 ** 0.062 0.12 *** 3-PPr 0 0 0 0 4-OHPPr 0.002 0.00 0 0 3,4-diOHPAc 0.100 0.07 ** 0.002 0.09 *** 3-OHPAc 0.330 0.25 ** 0.222 0.22 *** 3,4-diOHBA 0.025 0.01 ** 0.010 0.01 *** 3,5-diOHBA 0.021 0.01 0.002 0.02 3,4-diMeBA 0.013 0.01 0 0.01 3-OHBA 0.014 0.01 ** 0.008 0.01 *** 4-OHBA 0.059 0.04 -0.019 0.06 Benzoic acid 0.063 0.13 ** 0.012 0.08 4-methylCatechol 0.067 0.07 ** 0.004 0.12 *** Gallic acid 0.004 0.00 ** 0.004 0.00 *** Ferulic acid 0.134 0.08 ** 0.034 0.08 *** Sinapic acid 0.100 0.09 ** 0.044 0.10 Caffeic acid 0.063 0.04 ** 0.032 0.04 *** 4-coumaric acid 0.003 0.00 0.001 0.00 Enterodiol 0.001 0.00 * 0.001 0.00 Enterolactone 0.012 0.01 ** 0.005 0.01 *** Vanillic acid 0.112 0.07 0.007 0.10 *** Hippuric acid 0.023 0.02 ** 0.010 0.02 *** † All metabolites are expressed as nmol/mg creatinine, except for hippuric acid (μmol/mg creatinine). 3,4-diOHPPr: 3,4-dihydroxyphenylpropionic acid; 3-OHPPr: 3hydroxyphenylpropionic acid; 3-PPr: 3-phenylpropionic acid; 4OHPPr: 4-hydroxyphenylpropionic acid; 3,4-diOHPAc: 3,4dihydroxyphenylacetic acid; 3-OHPAc: 3-hydroxyphenylacetic acid; 3,4-diOHBA: 3,4-dihydroxybenzoic acid; 3,5-diOHBA: 3,5dihydroxybenzoic acid; 3,4-diMeBA: 3,4-dimethoxybenzoic acid; 3-OHBA: 3-hydroxybenzoic acid; 4-OHBA: 4-hydroxybenzoic acid. *p< 0.05 vs baseline (Wilcoxon test); ** p< 0.05 vs baseline (Wilcoxon test) and low-polyphenol diet (Mann-Whitney test); *** p< 0.05 vs low-polyphenol diet (Mann-Whitney test).

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8-week concentration

Metabolites†

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Table 2. Calculated concentrations of known phenolic microbial metabolites after 6-h-incubation in the in vitro colon model using human faecal

200

200

3

2

7

3,4-diOHPPr

571

2565

8505

3-OHPPr

1884

1511

0

4-OHPPr

403

300

0

0

0

0

3294

444

0

0

9

114

3-OHBA

81.6

216

4-OHBA

26.1

Benzoic acid

4417

4-methylcatechol

18.2

Gallic acid

235

Ferulic acid

0

Sinapic acid

3-PPr 3,4-diOHPAc 3-OHPAc 3,4-diOHBA

4-coumaric acid Enterodiol

190

28

Dark cocoa 25

2

2

7

7

7

7

3142

524

647

15.1

21319

149

37436

392191

6314

32105

701

138649

2167

575522

Orange

Coffee

Sum

16271

1061

1689

15.5

0

483

20223

165176

0

6811

5989

0

0

177976

14060

0

6520

285

438

0

396

25437

0

386

0

258

448

0

5269

6361

D

portion/week

TE

g/portion

0

0

826

46.2

0

3.89

344

1343

190

1331

130

87.2

9.46

9.5

82.8

2137

194

7

887

0

0

4.45

31.1

253

1403

2346

0

1842

0

4519

49.6

280

530

13984

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Human study set-up

Green tea 12

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Fennel

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Arugula Artichoke Spinach salad 90 325 150

Onion

0

0

401

0

20.3

75.9

403

199

1118

303

0

0

0

7.37

13.5

0

17.6

576

0

10.1

0

181

8.71

0.63

0

398

599

0

0

1714

0

0

149

0

0

0

1863

0

0

44.6

0

1481

3.30

6.62

0

247

1783

17.5

0

0

0

0

56.4

5.03

0

0

79

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Metabolites

CR I

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suspension (10 %, w/v) from polyphenol-rich foodstuffs and dose and frequency data from the human trial [21].

21.6 51.9 307 0 0 86.8 0 0 38.7 507 Vanillic acid 3,4-diOHPPr: 3,4-dihydroxyphenylpropionic acid; 3-OHPPr: 3-hydroxyphenylpropionic acid; 4-OHPPr: 4-hydroxyphenylpropionic acid; 3-PPr: 3phenylpropionic acid; 3,4-diOHPAc: 3,4-dihydroxyphenylacetic acid; 3-OHPAc: 3-hydroxyphenylacetic acid; 3,4-diOHBA: 3,4-dihydroxybenzoic acid; 3OHBA: 3-hydroxybenzoic acid; 4-OHBA: 4-hydroxybenzoic acid; 4-mecatechol: 4-methylcatechol.

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Figure 1

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Figure 2

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ACCEPTED MANUSCRIPT HIGHLIGHTS

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 Dietary polyphenol are converted to a relatively small number of colonic microbial metabolites.

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 Microbial phenolic acids with C1-C3 side chain formed an

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estimated profile.

 A significant correlation was detected between estimated and urinary metabolites.

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 Post-colonic conversion included hepatic shortening of the side-

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chain and glycination.

24