Tea is a significant dietary source of ellagitannins and ellagic acid

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Bioactive Constituents, Metabolites, and Functions

Tea is a significant dietary source of ellagitannins and ellagic acid Francisco A. Tomas-Barberan, and Xiao Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05010 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 12, 2018

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Journal of Agricultural and Food Chemistry

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Tea is a significant dietary source of ellagitannins and ellagic acid

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Xiao Yang,†, ‡ and Francisco A. Tomás-Barberán †,*

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†

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Soil Science and Biology of the Segura, the Spanish National Research Council (CEBAS-

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CSIC), Murcia, 30100, Spain

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‡ School

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China

Research Group on Quality, Safety, and Bioactivity of Plant Foods, Center for Applied

of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240,

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* Correspondence to Dr. Francisco A. Tomás-Barberán

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Address: CEBAS-CSIC, P.O. Box 164, Espinardo, Murcia 30100, Spain. Tel.:+34-

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968396200 (ext. 6334); Fax: +34-968396213; E-mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The ellagitannin composition and the total content of ellagitannins in different types of tea

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were studied by high-performance liquid chromatography/ion-trap mass spectrometry.

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Strictinin and other seven isomers, tellimagrandin I, and ellagic acid were identified from

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tea infusions. The ellagitannins content in tea infusions was determined after acid

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hydrolysis and ranged from 0.15 to 4.46 mg ellagic acid equivalent /g tea in the infusions.

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The intake of ellagic acid after drinking a cup of tea brewed with 4g tea could range

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between 0.59 and 17.89 mg. These results indicate that tea can be a significant contributor

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to the dietary intake of ellagitannins. Urolithins, the gut microbiota metabolites produced

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in vivo from ellagic acid and ellagitannins, were detected in human urine after dietary tea

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beverage intake. Urolithin metabotypes A, B, and 0 were identified in volunteers after tea

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intake. These results suggest that the daily intake of ellagitannins from tea can have a role

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in tea health effects.

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Keywords: ellagitannins, ellagic acid, tea, urolithins, strictinin, tellimagrandin I


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INTRODUCTION

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The ellagitannins (ETs), complex derivatives of ellagic acid (EA), is the largest group

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of hydrolyzable tannins and have been reported to play positive roles in the prevention of

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some chronic diseases.1, 2 ETs and its hydrolyzed metabolite, EA, are beneficial for human

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health, 2, 3 but the absorption and bioactivity of ETs in vivo is generally poor. 2, 4 Although

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ETs have not been detected in human plasma, 2 the ET corilagin, 5 an isomer of strictinin,

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and the pomegranate ET punicalagin6 have been detected in rat plasma. In the human

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gastrointestinal tract, ETs release EA by an unknown mechanism, and both ETs and EA

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are converted in the colon by gut microbiota to dibenzopyranone metabolites, named

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urolithins (Figure 1).7 Unlike ETs, urolithins are better absorbed in the gastrointestinal

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tract and are considered as biomarkers of ETs intake.8, 9 Recent evidence suggested that

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urolithins have antiproliferative effects on prostate, and colon cancer cell lines,10, 11 anti-

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inflammatory effects,12 extend the lifespan of Caenorhabditis elegans, and improve the

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muscle function in rodents.11 All these health benefits are considered to be associated with

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the consumption of food containing ETs.4

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Tea, manufactured with the leaves of Camellia sinensis, is the most consumed beverage

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worldwide.14 5.95 Million tons of tea were produced in 2016.15 Due to the substantial tea

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annual consumption and drinking population, even a small effect in humans could result in



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considerable implications for the health status of individuals.16 Strictinin consists of a

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hexahydroxydiphenoyl moiety (HHDP) esterified to a glucose and a galloyl residue, which

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occurrence has been reported in green, black and white tea (Figure 1).17-19 However, the

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dietary burden of ETs after the intake of different types of tea has not been evaluated yet.

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Therefore, this study aimed to analyze the presence of ETs in different tea products and

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to determine the EA content in different tea beverages. We also evaluated the production

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of the different urolithin metabolites in the urine of volunteers after dietary tea intake, and

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if this could be used to stratify volunteers among tea drinkers according to their urolithin

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metabotypes.

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MATERIALS AND METHODS

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Chemicals and reagents. Ultra-pure water was prepared using a Milli-Q system (Millipore

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Laboratory, Bedford, MA, USA). LC-MS grade of acetonitrile was purchased from Fisher

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Scientific (Pittsburgh, PA, USA); Dimethyl sulfoxide (DMSO) and methanol (HPLC grade)

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were bought from Scharlau (Scharlab, S. L., Barcelona, Spain) and J.T Baker (Avantor

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Performance Materials, Center Valley, PA, USA), respectively. Ellagic acid (≥95%, HPLC)

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was from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); Urolithin standards

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including urolithin A 3-glucuronide, urolithin A 8-glucuronide, isourolithin A 3-


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glucuronide, urolithin B glucuronide, urolithin M6, urolithin M7, isourolithin A, urolithin

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A, and urolithin B were chemically synthesized and purified (Villafarma, Parque

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Tecnológico de Fuente Alamo, Murcia, Spain). Urolithin C and urolithin D were purchased

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from Dalton Pharma Services (Toronto, Canada), and urolithin A-sulphate and urolithin B-

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sulphate were prepared according as reported.20 The purity of all the urolithin standards

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was > 95%.

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Tea collection and samples preparation. In this study, 29 kinds of tea products including

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two yellow, three black, six dark, three white, four green, two oolong, and nine

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reprocessing teas were purchased from online shops or local markets. Details are shown in

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Table 1.

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Tea samples (0.5g) were brewed with 25mL of boiling water and kept at room

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temperature until cooling down (approximately 2h), then centrifuged at 4200 x g for 15

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min (Sorvall™ ST 16, Thermo Fisher Scientific, Waltham, MA, USA). The supernatant

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(tea infusion) was collected and freeze-dried; the leaves after brewing (tea residue) were

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stored at -80 oC for further analysis.

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Quantification of EA in tea after acid hydrolysis. When ETs suffer acid hydrolysis, they

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release one or more HHDP groups, which then lactonize to EA spontaneously. Therefore,

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the ETs in this study were quantified as ellagic acid equivalents (EAE) after acid

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hydrolysis.21 The protocol was as previously reported with slight modifications.22 Briefly,

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the samples of freeze-dried tea infusion and the tea residue after infusion were placed in

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50 mL centrifuge tubes, and 3.34 mL ultrapure-water and 1.66 mL 37% HCl (Scharlau,

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Barcelona) were added to get a final concentration of 4 M HCl in the solution. Then the

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samples were vortexed and incubated in an oven at 90 oC for 4h. After cooling down, the

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pH of the samples was adjusted to 3.5 with 5M NaOH in water and centrifuged at 4200 x

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g for 15 min (Sorvall™ ST 16, Thermo Fisher Scientific). The supernatant was collected,

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adjusted to a final volume of 10 mL, and filtered through a 0.45 μm PVDF filter before

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injection. The remaining part was extracted with 10 mL of a methanol: DMSO solution

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(1:1, v/v), filtered through a 0.45 μm PVDF filter and analyzed in an Agilent 1100 series

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HPLC system (Agilent Technologies, Waldbronn, Germany). 20 μL samples were injected

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onto a Pursuit XRs C18 column (Agilent Technologies) with a flow rate of 0.8 mL min-1.

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The mobile phases were water with 0.1% formic acid (A) and acetonitrile (B). The gradient

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conditions were: 0-6 min, 3-9% B in A; 6-15min, 9-16% B in A; 15-45 min, 16-50% B in

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A; 45-47 min, 50-90% B in A; 47-52 min, 90% B in A; finally, the solvents returned to the


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initial conditions in 2 min and were maintained for 5 min before the next injection. EA

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content in each sample was calculated by the peak area of the EA standard curve and

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expressed as mg EAE per g tea.

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Identification of ETs in tea beverages. We selected ten types from the 29 available types

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of tea to further profile the ETs in the tea infusions. These teas included mengding huang

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ya (yellow tea), qimen black tea (black tea), pu-erh raw tea (tea cake, dark tea), pu-erh tea

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(tea cake, dark tea), bai mu dan tea (white tea), taiping hou kui tea (green tea), long jing

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tea (green tea), tie guan yin tea (oolong tea), organic UJI matcha (reprocessing tea), and

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Earl Grey (reprocessing tea). The tea beverages were prepared as described above. The

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supernatant was collected and filtered through a reversed-phase C18 cartridge (Macherey-

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Nagel, Düren, Germany) and eluted with methanol (2.5 mL). Then the sample was filtered

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through a 0.22 μm PVDF filter before analysis in a high-performance liquid

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chromatography/ion-trap mass spectrometry platform (HPLC-IT, Bruker Daltonics,

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Bremen, Germany).

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The analysis of MSn fragments of tea samples was performed in HPLC-IT with a

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Pursuit XRs C18 column (4 × 250 mm, 5 μm, Agilent Technologies). Chromatographic



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separation and MS conditions were set according to a previous study.22 The MSn fragments

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data were analyzed by Bruker Daltonics DataAnalysis software (version 4.3).

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Analysis of urolithin metabolites in human urine after tea intake. Ten healthy

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volunteers including six males and four females with ages ranging from 25 to 60 years old

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and belonging to the three known urolithin metabotypes [A (n=3), B (n=4) and 0 (n=3)],9

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were recruited and informed of the study to be carried out. They were asked to avoid

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consumption of ET-containing food (a list was provided, Supplemental Table S1) during

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the three days previous to the tea intervention. The tea infusion was prepared with 5g of

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tea brewed with 250 mL boiling water (tea: water = 1: 50, w/v) in a teapot for 1 min, then

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shared with ten volunteers averagely. During the intervention, eight types of tea infusion

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were consumed by volunteers both in the morning and the afternoon; volunteers consumed

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a total volume of 400 mL tea beverage with approximate 3 mg EAE per day for two days.

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Urine samples were then collected, vortexed and centrifuged at 18000 x g for 10 min (1-

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16K microcentrifuge, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany),

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followed by dilution 1:2 with ultrapure-water before filtration through a 0.22 μm PVDF

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filter.


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The chromatographic method for urine sample analysis was previously developed.23

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In brief, 3 μL of diluted urine samples were analyzed in a UPLC-QTOF system (6550

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iFunnel Q-TOF, Agilent Technologies) with a reversed C18 column (Poroshell 120, 3 ×

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100 mm, 2.7 μm, Agilent Technologies). Water: formic acid (99.9: 0.1, v/v) and acetonitrile:

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formic acid (99.9: 0.1, v/v) were used as mobile phases A and B, respectively. The flow

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rate was 0.4 mL min-1 and the gradient conditions were 0 min, 5% B in A; 0-3 min, 5-15%

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B in A; 3-11 min, 15-30% B in A; 11-15 min, 30-50% B in A; 15-21 min, 50-90% B in A;

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21-22 min, 90% B in A; 22-23 min, 90-5% B in A, and the initial conditions were

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maintained for 5 min. The MS condition was: capillary voltage, 3500V; gas temperature,

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280 oC; drying gas, 11 L min-1; nebulizer pressure 45psi; MS spectra were acquired in the

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negative mode with a full scan range m/z 100-1100. The mixture of urolithin standards was

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injected before the samples. The raw data were processed (including baseline, denoising,

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smoothing, deconvolution, and alignment) by Agilent Mass Hunter Profinder (version

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B.06.00, Agilent Technologies). The identification of urolithin derivatives was based on

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the retention time and MS information of the available standards.

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Statistical analysis. Quantified data were expressed as mean ± SD of three biological

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replicates per sample. One-way analysis of variance was performed in IBM SPSS



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Statistics 22 (IBM, Armonk, NY, USA) based on LSD analysis; the significance level

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was set at 0.05; figures were drawn by OriginPro 2016 (OriginLab, Northampton, MA,

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USA).

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RESULTS

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Quantification of EA in tea after acid hydrolysis. The content of ETs in the infusion of

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the selected 29 kinds of tea was assessed by the acid hydrolysis method as shown in Figure

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2. The ETs content exhibited wide differences in individual types of tea beverage samples

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from 0.15 ± 0.02 to 4.46 ± 0.44 mg EAE /g of tea used to prepare the beverage with a 30-

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fold difference, and the average arithmetic value was 1.59 mg EAE /g in the 29 tea beverage

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samples. Two types of yellow tea, mengding huang ya tea (4.26 ± 0.17 mg EAE /g) and

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junshan yin zhen tea (4.19 ± 0.75 mg EAE /g) and one dark tea, pu-erh raw tea (quadrel,

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4.46±0.44 mg EAE /g) had the highest amount of ETs, while tie guan yin tea had the lowest

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content with a value of 0.15 ± 0.02 mg EAE /g among the 29 kinds of tea.

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For the tea residue part (the leaves remaining after preparation of the tea beverage), the

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ETs content varied from 0.55 ± 0.05 to 3.84 ± 0.32 mg EAE /g with a 7-fold difference

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(Figure 3). A significantly higher content of ETs was found in two dark tea samples

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including cake and quadrel pu-erh teas with respective values of 3.84 ± 0.32 and 3.63 ±


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0.43 mg EAE /g, but PG tips black tea had the lowest content of ETs (0.55 ± 0.05 mg EAE

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/g).

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For the combined value of the tea beverage and the tea residue fractions, pu-erh raw tea

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(quadrel), mengding huang ya tea, and junshan yin zhen tea had significantly higher ETs

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content with values of 7.20 ± 0.81, 6.78 ± 0.26, and 6.70 ± 1.06 mg EAE /g, respectively

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(Figure 4). The ET content of the seven subclasses of teas ranged in the following order:

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yellow tea (6.74 mg EAE /g), white tea (5.09 mg EAE /g), dark tea (4.86 mg EAE /g),

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green tea (3.66 mg EAE /g), black tea (2.88 mg EAE /g), reprocessing tea (1.62 mg EAE

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/g), and oolong tea (1.51 mg EAE /g). The tea beverage fraction contributed to approximate

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40% of total ET content in all brewed tea samples. However, the ET contents of the tea

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residue samples were generally higher than those found in the tea infusion. The ETs content

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of the tea residue in nine of the samples was higher than 70% of the total content of tea.

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For example, 80.7% of the ETs were detected in the tea residue of tie guan yin tea (Oolong

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tea) while the value in the tea infusion was just 19.3%.

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Characterization of ETs in tea infusions. According to the results of EA content in

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different tea beverages, the composition of ETs in ten tea samples belonging to the different

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tea types was analyzed by HPLC-IT-ESI-MS. Structurally, ETs comprise a polyol



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(primarily glucose) esterified with one or two HHDP moieties and often also with

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additional galloyl moieties; the HHDP consists of two neighboring galloyl residues linked

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by C-C coupling.1 Eight [M-H]- ions at m/z 633 with retention times of 10.6, 12.1, 12.6,

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14.3, 16.8, 20.5, 22.0, and 22.8 min (Table 2 and Figure 5) were detected and all yielded

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an MS2 product ion at m/z 301, indicating the neutral loss of a glucogallin residue [M-H-

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332]- from the parent ions. Moreover, several MS3 ions at m/z 257, 229 and 189 were

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detected form MS2 ion of m/z 301, which suggested the presence of the HHDP moiety.

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Some deprotonated ions at m/z 633 also had product ions at m/z 463 (peaks at 12.1, 12.6,

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20.5, 22.0, and 22.8 min) corresponding to the cleavage and loss of a gallic acid residue

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[M-H-170]-. Therefore, these compounds were tentatively identified as galloyl-HHDP-

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glucose isomers (Figure 6). Among the isomeric forms, the peak at 20.5 min, presenting

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the most abundant intensity compared to other galloyl-HHDP-glucose isomers, was

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tentatively assigned to strictinin (1-galloyl-4,6-HHDP-glucose), which had already been

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reported in tea.17, 19 The isomers could include other combinations of glucose, gallic acid,

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and HHDP acid as is the case of corilagin (1-galloyl-3,6-HHDP-glucose) and gemin-D (2-

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galloyl-3,6-HHDP-glucose). According to the fragmentation pattern above, the compound

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at 29.6 min with a molecular ion of m/z 785 was tentatively identified as tellimagrandin I,

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(2,3-digalloyl-4,6-HHDP-glucose) resulting from the MS2 fragment at m/z 633 and its MS3


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fragments at m/z 463, 301 and 275 (Figure 7). By comparing with an authentic standard,

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we also confirmed the occurrence of free EA in tea beverages. We further injected the

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samples into UPLC-QTOF, indicating the m/z of strictinin at 633.0736 m/z (mass error 0.3

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mDa) and tellimagrandin I m/z at 785.0836 (mass error -0.7 mDa).

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Metabolic profiling of urolithins in human urine after tea intake. To evaluate if the

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dietary consumption of tea could be used to stratify individuals by their urolithin

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metabotypes, these metabolites were analyzed in urine. The identification of urolithin

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metabolites was performed based on chromatographic comparisons with authentic

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standards (m/z and retention time). Nine known urolithin derivatives were identified in the

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urine samples of the different volunteers after the intake of dietary doses of tea, as shown

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in Table 3. For all the identified compounds, stable retention time (drift time for same

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compounds between samples ≤ 0.02 min) and low mass error (≤ 0.8 mDa) were observed.

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Urolithin A, urolithin B, isourolithin A, and their glucuronide or sulfate conjugates were

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present in the urine samples from the different subjects with the characteristic profiles of

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metabotypes A, B and 0 for every volunteer.

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DISCUSSION

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Strictinin is the major ET in tea beverages. Although tea was known to be a potential

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source of ETs, the composition and content of ETs had never been studied in detail so far.

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In this study, we performed an LC-MSn approach to gain a deeper insight of the ETs present

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in different tea samples, and ten metabolites belonging to the ET group were annotated,

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which included strictinin and seven isomers with the same mass and fragments,

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tellimagrandin I, and EA. Among the ETs family in tea, EA was firstly described in green

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tea leaves in 1941,24 and strictinin was purified and identified from green tea leaves in

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1984,17 but tellimagrandin I had never been reported to be present in tea.

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Strictinin is present in tea as a relatively minor phenolic compound compared with

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the total amount of flavan-3-ols (catechins).19 This ET had been detected in the leaves of

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C. sinensis, and in some tea products, such as black, green, oolong, white and pu-erh tea.18,

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25, 26

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ET in tea. Its presence was relatively higher in the infusion of yellow tea (mengding huang

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ya tea) and pu-erh raw tea in comparison with other teas. In the tea plant, the content of

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strictinin differs at different leaf maturity stages and order.27 Strictinin content decreases

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sharply as the tea plant matured, and the younger parts (such as the bud and the first leaf)

In this study, strictinin was detected in all the samples and was identified as the main


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bear more strictinin than the older leaves or stems. The yellow tea had a relatively higher

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content of strictinin and this is mainly a consequence of the criteria used for the selection

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of tea leaves for processing, as in this case only the earliest spring tea buds could be used

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as materials to produce mengding huang ya tea.28 The reason for pu-erh raw tea to contain

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a higher amount of strictinin than other teas is that the raw materials were produced from

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the leaves of the large-leaf tea species C. sinensis var. assamica rather than from the

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cultivated shrub species C. sinensis var. sinensis,29 and the content of strictinin in cultivated

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tea trees is higher than that found in the cultivated tea shrubs.26

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Dietary tea intake can be used for determination of urolithin metabotypes. ETs and

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EA intake correlates with the presence of urolithin metabolites in plasma and urine, and

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urolithins can be considered biomarkers of ETs and EA intake.4 Depending on the different

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urolithin metabolites excreted by individuals, they can be consistently grouped into three

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metabotypes, including metabotype A (producer of urolithin A derivatives), metabotype B

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(producer of urolithin A, urolithin B, and isourolithin A and their derivatives), and

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metabotype 0 (non-producer).9 In this study, we aimed to evaluate if the dietary intake of

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tea could be used for the determination of the volunteer metabotype, in spite of the low

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amount of EA ingested. In this study, volunteers belonging to the three known urolithin



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metabotypes were selected and invited to participate. The three volunteers of metabotype

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A only excreted urolithin A and its glucuronide or sulfate derivatives. The four volunteers

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of metabotype B produced both urolithin B and isourolithin A in addition of urolithin A as

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could be expected. Furthermore, no urolithin derivatives were detected in the urine samples

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of the three volunteers of metabotype 0, who were urolithin non-producers. The

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identification of the urolithin metabotypes after regular tea drinking was consistent with

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previous results observed after dietary interventions with ET-rich foods, such as

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pomegranate, walnuts, or strawberry. Here we show here that this stratification is also

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possible after the intake of a dietary dose of tea, and this is interesting, as these metabotypes

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have been associated with different health status, particularly regarding metabolic

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syndrome, cardiovascular disease biomarkers and obesity,30, 31 as well as with differences

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in gut microbiota composition.

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Tea is a dietary source of ETs more relevant than previously believed. The ETs have

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been reported to be present in a wide range of food products including strawberry,

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raspberry, and pomegranate which contain 77-85, 51-330, and 35-75 mg/100g of EA fresh

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weight, respectively.32, 33 In this study, 29 kinds of commercial tea products were studied,

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including all the seven known subclasses of tea (yellow, black, green, white, oolong, dark,


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and reprocessing tea). The total ET content in tea ranged from 0.77-7.20 mg EAE /g, and

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more than 40% of the total ETs were extracted in the first infusion. The considerable

281

amount of ETs remaining in the tea residue indicates that additional extractions of the same

282

leaves, as is a common practice in some regions, would additionally increase the ETs intake.

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It is also known that the ET content is more relevant in tea leaves with a low degree of

284

maturity than in the mature ones and that the large-leaf tea tree species contain more ETs

285

than those from the small-leaf tea bush species.27 Different tea products required different

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raw tea leaves picking criteria, and this affects to the ET content of tea products. To reach

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specific flavor properties of tea, different cultivars can also be selected, and this affects the

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ETs content. For instance, the picking criterion for raw tea leaves of bai mu dan tea is one-

289

tip two-leaf of young shoot from cv. ‘fudingdabai’, a tree type of tea plant; while to process

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tie guan yin tea requires the raw tea leaves maintaining a higher maturity (inert bud with

291

terminal facing three-leaf) form shrub cultivar cv. ‘tieguanyin’. Therefore the raw leaves

292

of bai mu dan tea had a higher content of strictinin than that in tie guan yin tea. In addition,

293

to decrease the astringent and bitter flavors, some tea products also require strict shade

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conditions before harvest of the raw tea leaves, such as matcha. Shading enhances the

295

nitrogen metabolism, adjusts the carbon-nitrogen ratio, and the metabolic flux flows to

296

biosynthesize nitrogen compounds (i.e., theanine and caffeine) but decreases the



297

(poly)phenols accumulation.34 This reason explains the relatively low content of ETs in

298

matcha.

299

To date, a few studies have calculated the daily intake of ETs based on the average

300

content of EA in food and the ET-rich food consumption in different populations. Typically,

301

the daily intake of ET is generally less than 5 mg per day in the Western diet,2, 35 and the

302

ET intake is higher in northern European countries, such as Finland, which increased to an

303

average intake of 12 mg per day.36 In this study, we found that a 200 mL cup of tea produced

304

by brewing 4g of tea leaves contains at most 17.82 mg ETs in the tea infusion. According

305

to the annual tea consumption per capita in 2016,37 Turkey is the most extensive tea

306

consumer with 3.16 kg per capita, which means an average intake of 13.8 mg ETs per day,

307

followed by Ireland (2.19 kg per capita) and the UK (1.94 kg per capita), where the ETs

308

consumption by drinking tea could average 9.5 and 8.5 mg/day respectively. Therefore, the

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daily consumption of dietary ETs can be much higher than previously reported, particularly

310

in those countries drinking tea regularly.

311 312 313 314


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AUTHOR INFORMATION

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Corresponding Authors

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* Correspondence

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Espinardo, Murcia 30100, Spain. Tel.:+34-968396200 (ext. 6334); Fax: +34-968396213;

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E-mail address: [email protected]

320

Funding

to Francisco A. Tomas-Barberan, Address: CEBAS-CSIC, P.O. Box 164,

321

This research was partially supported by CSIC 201870E014 and Fundación Séneca de

322

la Región de Murcia, Ayudas a Grupos de Excelencia 19900/GERM/15. XY was supported

323

by the State Scholarship Fund of China Scholarship Council (No. 201706230173).

324

Author Contributions

325

XY and FATB designed and performed all the experimental measurements, analyzed the

326

data, and drafted the manuscript.

327

Notes

328

The authors declare that they have no conflict of interest.

329 330

ACKNOWLEDGMENTS

331

The authors are grateful to Mr. Carlos J. Garcia, Dr. Diego A. Moreno, Dr. Rocio Garcia-

332

Villalba and Mr. Alberto M. Blazquez for assisting in the experimental part, and to Mr.



333

Hongkai Zhu from the University of Copenhagen for providing some advice in the

334

preparation of the manuscript.

335 336 337

REFERENCES 1.

Landete, J. M., Ellagitannins, ellagic acid and their derived metabolites: a review

338

about source, metabolism, functions and health. Food Res. Int. 2011, 44, 1150-1160.

339

2.

340

health, and research perspectives for innovative functional foods. Crit. Rev. Food Sci. Nutr.

341

2014, 54, 1584-98.

342

3.

343

Ellagitannins, ellagic acid and vascular health. Mol. Aspects Med. 2010, 31, 513-539.

344

4.

345

M. A.; Selma, M. V.; García-Conesa, M. T.; Espín, J. C., Urolithins, the rescue of “old”

346

metabolites to understand a “new” concept: Metabotypes as a nexus among phenolic

347

metabolism, microbiota dysbiosis, and host health status. Mol. Nutr. Food Res. 2017, 61,

348

1500901.

Garcia-Munoz, C.; Vaillant, F., Metabolic fate of ellagitannins: implications for

Larrosa, M.; García-Conesa, M. T.; Espín, J. C.; Tomás-Barberán, F. A.,

Tomás-Barberán, F. A.; González-Sarrías, A.; García-Villalba, R.; Núñez-Sánchez,


Page 20 of 40

Page 21 of 40


349

5.

Chen, Q.Q.; Guo, J.; Fan, H.; Wang, C.; Xu, F.; Zhang, W. Determination of

350

corilagin in rat plasma using a liquid chromatography-electrospray ionization tandem mass

351

spectrometric method. Biomed. Chromatogr. 2015, 29, 1553-1558.

352

6.

353

of the bioavailability and metabolism in the rat of punicalagin, and antioxidant polyphenol

354

of pomegranate juice, Eur. J. Nutrition., 2003, 42: 18-28.

355

7.

356

Espín, J. C., Urolithins, ellagic acid-derived metabolites produced by human colonic

357

microflora, exhibit estrogenic and antiestrogenic activities. J. Agric. Food Chem. 2006, 54,

358

1611-1620.

359

8.

360

Metabolites: Bioactives or Biomarkers? J. Agric. Food Chem. 2018, 66, 3593-3594.

361

9.

362

Espín, J. C., Ellagic acid metabolism by human gut microbiota: consistent observation of

363

three urolithin phenotypes in intervention trials, independent of food source, age, and

364

health status. J. Agric. Food Chem. 2014, 62, 6535-6538.

365

10.

366

metabolites, ellagic acid and urolithin a, synergistically inhibit androgen-independent

Cerdá, B.; Llorach, R.; Cerón, J.J.; Espín, J.C.; Tomás-Barberán, F.A. Evaluation

Larrosa, M.; González-Sarrías, A.; García-Conesa, M. T.; Tomás-Barberán, F. A.;

Tomas-Barberan, F. A.; Selma, M. V.; Espín, J. C., Polyphenols’ Gut Microbiota

Tomás-Barberán, F. A.; García-Villalba, R.; González-Sarrías, A.; Selma, M. V.;

Vicinanza, R.; Zhang, Y.; Henning, S. M.; Heber, D., Pomegranate juice



367

prostate cancer cell growth via distinct effects on cell cycle control and apoptosis.

368

Evidence-Based Complement. Altern. Med. 2013, DOI: 10.1155/2013/270418.

369

11.

370

Colon cancer chemopreventive activities of pomegranate ellagitannins and urolithins. J.

371

Agric. Food Chem. 2010, 58, 2180-2187.

372

12.

373

Ortuño, M.; Toti, S.; Tomás-Barberán, F.; Dolara, P.; Espín, J. C., Anti-inflammatory

374

properties of a pomegranate extract and its metabolite urolithin-A in a colitis rat model and

375

the effect of colon inflammation on phenolic metabolism. J. Nutr. Biochem. 2010, 21, 717-

376

725.

377

13.

378

Félix, A. A.; Williams, E. G.; Jha, P.; Lo Sasso, G.; Huzard, D.; Aebischer, P.; Sandi, C.;

379

Rinsch, C.; Auwerx, J., Urolithin A induces mitophagy and prolongs lifespan in C. elegans

380

and increases muscle function in rodents. Nature Med. 2016, 22 879-888.

381

14.

382

Tsubono, Y.; Tsuji, I., Green tea consumption and mortality due to cardiovascular disease,

383

cancer, and all causes in Japan: the Ohsaki study. Jama 2006, 296, 1255-65.

Kasimsetty, S. G.; Bialonska, D.; Reddy, M. K.; Ma, G.; Khan, S. I.; Ferreira, D.,

Larrosa, M.; González-Sarrías, A.; Yáñez-Gascón, M. J.; Selma, M. V.; Azorín-

Ryu, D.; Mouchiroud, L.; Andreux, P. A.; Katsyuba, E.; Moullan, N.; Nicolet-dit-

Kuriyama, S.; Shimazu, T.; Ohmori, K.; Kikuchi, N.; Nakaya, N.; Nishino, Y.;


Page 22 of 40

Page 23 of 40


384

15.

United Nations Food and Agriculture Organization, Tea production in world of

385

2016. URL (http://www.fao.org/faostat/en/#data/QC/visualize) (22 August 2018).

386

16.

387

2004, 292, 1490-1492.

388

17.

389

from green tea. Phytochemistry 1984, 23, 1753-1755.

390

18.

391

R. J.; de Vos, R. C. H.; Vervoort, J., Structural annotation and elucidation of conjugated

392

phenolic compounds in black, green, and white tea extracts. J. Agric. Food Chem. 2012,

393

60, 8841-8850.

394

19.

395

polyphenolic composition in leaves of Camellia irrawadiensis. Chem. Pharm. Bull. (Tokyo)

396

2009, 57, 1284-1288.

397

20.

398

Barberán, F.; Álvarez, A. I.; Espín, J. C., The Gut Microbiota Ellagic acid-derived

399

metabolite urolithin a and its sulfate conjugate are substrates for the drug efflux transporter

400

breast cancer resistance protein (ABCG2/BCRP). J. Agric. Food Chem. 2013, 61, 4352-

401

4359.

Rimm, E. B.; Stampfer, M. J., Diet, lifestyle, and longevity-the next steps? Jama

Nonaka, G.; Sakai, R.; Nishioka, I., Hydrolysable tannins and proanthocyanidins

van der Hooft, J. J. J.; Akermi, M.; Ünlü, F. Y.; Mihaleva, V.; Roldan, V. G.; Bino,

Yagi, K.; Goto, K.; Nanjo, F., Identification of a major polyphenol and

González-Sarrías, A.; Miguel, V.; Merino, G.; Lucas, R.; Morales, J. C.; Tomás-



402

21.

Daniel, E. M.; Krupnick, A. S.; Heur, Y.; Blinzler, J. A.; Nims, R. W.; Stoner, G.

403

D., Extraction, stability, and quantitation of ellagic acid in various fruits and nuts. J. Food

404

Compos. Anal. 1989, 2, 338-349.

405

22.

406

G.; Voorspoels, S.; Koivumäki, T.; Kroon, P. A.; Pelvan, E., Saha, S., Tomas-Barberan, F.

407

Validated method for the characterization and quantification of extractable and

408

nonextractable ellagitannins after acid hydrolysis in pomegranate fruits, juices, and extracts.

409

J. Agric. Food Chem. 2015, 63, 6555-6566.

410

23.

411

spectroscopic characterization of urolithins for their determination in biological samples

412

after the intake of foods containing ellagitannins and ellagic acid. J. Chromatogr. A 2016,

413

1428, 162-175.

414

24.

Tsujimura, M., Ellagic acid in green tea. Sci. Pap. IPCR. 1941, 38, 487-489.

415

25.

Dou, J.; Lee, V. S. Y.; Tzen, J. T. C.; Lee, M., Identification and comparison of

416

phenolic compounds in the preparation of oolong tea manufactured by semifermentation

417

and drying processes. J. Agric. Food Chem. 2007, 55, 7462-7468.

García-Villalba, R.; Espín, J. C.; Aaby, K.; Alasalvar, C.; Heinonen, M.; Jacobs,

García-Villalba, R.; Espín, J. C.; Tomás-Barberán, F. A., Chromatographic and


Page 24 of 40

Page 25 of 40


418

26.

Chen, G.; Lin, Y.; Hsu, W.; Hsieh, S.; Tzen, J. T. C., Significant elevation of

419

antiviral activity of strictinin from Pu'er tea after thermal degradation to ellagic acid and

420

gallic acid. J. Food Drug Anal. 2015, 23 116-123.

421

27.

422

Tachibana, H.; Miyase, T.; Sano, M., Changes in epigallocatechin-3-O-(3-O-methyl)

423

gallate and strictinin contents of tea (Camellia sinensis L.) cultivar 'Benifuki' in various

424

degrees of maturity and leaf order. Food Sci. Technol. Res. 2004, 10, 186-190.

425

28.

426

China, 2016; 142-150.

427

29.

428

erh tea: a review. Food Res. Int. 2013, 53, 608-618.

429

30.

430

Örem, A.; Zafrilla, P.; Tomás-Barberán, F.A.; Selma, M.V.; Espin, J.C. Clustering

431

according to urolithin metabotype explains the interindividual variability in the

432

improvement of cardiovascular risk biomarkers in overweight-obese individuals

433

consuming pomegranate: A randomized clinical trial. Mol. Nutr. Food Res., 2017, 61 (5),

434

1600830

Maeda-Yamamoto, M.; Nagai, H.; Asai, K.; Moriwaki, S.; Horie, H.; Kohata, K.;

Tao, X., Eds. Tea Processing, edition third. China Agricultural Press: Beijing,

Lv, H.; Zhang, Y.; Lin, Z.; Liang, Y., Processing and chemical constituents of Pu-

González-Sarrías, A., García-Villalba, R.; Romo-Vaquero, M.; Alasarvar, C.;



435

31.

Selma, M.V.; González-Sarrías, A.; Salas-Salvadó, J.; Andrés-Lacueva, C.;

436

Alasalvar, C.; Orem, A.; Tomas-Barberan, F.A.; Espin, J.C. The gut microbiota

437

metabolism of pomegranate or walnut ellagitannins yields two urolithin-metabotypes that

438

correlate with cardiometabolic risk biomarkers: comparisons between normoweight,

439

overweight-obesity and metabolic syndrome. Clin. Nutr. 2018, 37, 897-905.

440

32.

441

anthocyanins and ellagitannins in selected foods consumed in Finland. J. Agric. Food

442

Chem. 2007, 55, 1612-1619.

443

33.

444

Antioxidant activity of pomegranate juice and its relationship with phenolic composition

445

and processing. J. Agric. Food Chem. 2000, 48, 4581-4589.

446

34.

447

flavonoid biosynthesis in tea (Camellia sinensis (L.) O. Kuntze). Sci. Hortic.-Amsterdam

448

2012, 141, 7-16.

449

35.

450

J. Sci. Food Agric. 2000, 80, 1118-1125.

Koponen, J. M.; Happonen, A. M.; Mattila, P. H.; Törrönen, A. R., Contents of

Gil, M. I.; Tomás-Barberán, F. A.; Hess-Pierce, B.; Holcroft, D. M.; Kader, A. A.,

Wang, Y.; Gao, L.; Shan, Y.; Liu, Y.; Tian, Y.; Xia, T., Influence of shade on

Clifford, M. N.; Scalbert, A., Ellagitannins-nature, occurrence and dietary burden.


Page 26 of 40

Page 27 of 40


451

36.

Ovaskainen, M.; Törrönen, R.; Koponen, J. M.; Sinkko, H.; Hellström, J.; Reinivuo,

452

H.; Mattila, P., Dietary intake and major food sources of polyphenols in Finnish adults. J.

453

Nutr. 2008, 138, 562-566.

454

37.

455

countries (in pounds). URL (https://www.statista.com/statistics/507950/global-per-capita-

456

tea-consumption-by-country/) (August 22, 2018).

Quartz., Annual per capita tea consumption worldwide as of 2016, by leading



Figure captions Figure 1. Gut microbiota catabolic pathway of the tea ellagitannin strictinin to urolithins. Figure 2. The ETs content in 29 kinds of tea infusions. Note: Different small letters indicate significant differences, p < 0.05; LSD analysis (n = 3). Figure 3. The ETs content in 29 kinds of tea residues. Note: Different small letters indicate significant differences, p < 0.05; LSD analysis (n = 3). Figure 4. The total content of ETs in 29 kinds of tea. Figure 5. Extracted ion chromatogram of m/z at 633 in different kinds of tea profile Figure 6. MSn spectrum of strictinin. Figure 7. MSn spectrum of tellimagrandin I.


Page 28 of 40

Page 29 of 40


HO

OH

HO HO HO

Strictinin (tea ellagitannin)

O

O

O O

HO

O

HO

O OH

O OH

OH

Urolithin A

OH

Urolithin C

O

HO

OH O

OH

OH HO

HO

O

OH

O

OH O

HO

O

O

Urolithin B

O

Ellagic acid

HO HO HO

OH

OH

OH

HO

O

O

O

Urolithin M-6

Isourolithin A

Figure 1.


O

O O

an g ya

0

Zh

H

ai di

t zh ea e go n t ea n en Q gs im g fu h a en te n b xia lac a k o zh tea on g te Li Pu u a -e Q bao ia rh t n ea ra l i a w te ng P Pu u-e a (q tea -e rh ua te rh dr ra a ( e qu l) w ad Pu te re -e a (t l) rh e te a ca a (te ke) Ba a i m ca ke u ) d Ag Go an n t e H ua d b g m ea ng a ei sh i m t an u d ea an m Ta ao te ip a fe in n g ho g te a u k Sp Lon ui t e g ec jin a ia lG g un tea Ti p e gu owd er Da an ho yin ng te Ja sm pa a o O ine te rg g a re a en En nic UJ t gl ish I m ea a Br ea tch a Pu kfa re st t ea gr ee Ye n llo te w E ar a la l be G lb re y la ck Pu tea -e rh t PG Wh ea i te tip s b te la a ck te a

yi n

hu

an

in g

sh

gd

Ju n

M en

ETs content in tea infusion (mg EAE per g tea)


5 a

b

2 de

1 b b

3

efg

ijklm

Figure 2.

ACS Paragon Plus Environment hijkl

m

Page 30 of 40

a

a

4

Yellow tea Black tea Dark tea White tea Green tea Oolong tea Reprocessing tea

b b

c c

def d de

ghi hijk hijk

jklm klm jklm hijk ghij fgh

lm klm


ETs content in tea residue (mg EAE per g tea)

Page 31 of 40

Yellow tea Black tea Dark tea White tea Green tea Oolong tea Reprocessing tea

5 a

a

4

bc

3

b

bc

b cd cd

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de efg

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) ) r y a a a a a a a a a a a a a a a a a a a a a l) l) a te te te i te te te tch t te te te te dre dre ake ake te te Gre te te te i te te de te h te e te k te w n n n n o o g g g g c c a s e k in u t a a fu ack r l m ee E a r jin npo n y hi blac da da fen u k pa ree I m kfa l g ac u - e on uba lian (qu (qu tea tea r l b n h J u u g g g ( a W s ( a o z u o o a n P L i an ng lb ng a a a a en a o yi i gu hon ine ic U Bre ure i m Go ai m ma g h Lo al G te te ig hu tip be te te Q e n n i h P xi h la g han aid Qim Ba w b m Ti an ipi PG er raw -erh n D a Ja s in ec ra ga glis d w s H h r a a d p e s n u h lo h T S u O h n g g l r g u s P r E P J A an g -e en Ye -e u en M Pu Pu H Zh ng

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Figure 3.



PG tips black tea White tea Pu-erh tea Yellow label black tea Earl Grey Pure green tea English Breakfast tea Organic UJI matcha Jasmine green tea Da hong pao tea Tie guan yin tea Special Gunpowder Long jing tea Taiping hou kui tea Huangshan mao feng tea Aged bai mu dan tea Gong mei tea Bai mu dan tea Pu-erh tea (tea cake) Pu-erh raw tea (tea cake) Pu-erh tea (quadrel) Pu-erh raw tea (quadrel) Qian liang tea Liubao tea Zhengshan xiao zhong tea Qimen black tea Haidi gong fu tea Junshan yin zhen tea Mengding huang ya tea

61.7% 38.3% 61.6% 21.0% 46.8% 53.2% 53.9% 46.1%

Tea residue Tea influsion

79.0%

76.5% 40.0% 60.0% 78.1% 22.9% 77.1% 25.5% 74.5% 80.7% 65.2% 34.8% 51.2% 48.8% 36.5% 63.5% 46.6% 53.4% 61.8% 38.2% 56.0% 44.0% 60.6% 39.4% 29.1% 70.9% 51.7% 48.3% 24.0% 76.0% 61.9% 39.1% 60.9% 29.6% 71.4% 47.9% 52.1% 51.8% 48.2% 51.1% 48.9% 62.5% 62.8%

0

1

Page 32 of 40

2

3

4

38.1%

37.5% 37.2%

5

6

ETs content in tea (mg EAE per g tea)

Figure 4.


7

Page 33 of 40


Figure 5.



Figure 6.


Page 34 of 40

Page 35 of 40


Figure 7.



Page 36 of 40

Table 1. Details of tea products used in the study No.

Name

Classification

Type

Trademark

1

Mengding huang ya tea

Yellow Tea

Tea bud type

Wei Du Zhen

Loose tea; 50g/bag

Sichuan, China

2

Junshan yin zhen tea

Yellow Tea

Tea bud type

Jun Shan

5 g/bag; 10 bags in total

Hunan, China

3

Haidi gong fu tea

Black Tea

Congou

ChinaTea

Loose tea, 125 g/ pot

Fujian, China

4

Qimen black tea

Black Tea

Congou

Ba Ma

Loose tea, 250g /bag

Anhui, China

Black Tea

Souchong

Ba Ma

Loose tea, 80 g/ pot

Fujian, China

99 g/ brick

Guangxi, China

5

Zhengshan xiao zhong tea

Individual package

Production Place

6

Liubao tea

Dark Tea

Guangxi dark tea

ChinaTea

7

Qian liang tea

Dark Tea

Hunan dark tea

ChinaTea

8

Pu-erh raw tea (quadrel)

Dark Tea

Yunnan dark tea

TAETEA

60 g/ brick

Yunnan, China

9

Pu-erh tea (quadrel)

Dark Tea

Yunnan dark tea

TAETEA

60 g/ brick

Yunnan, China

Dark Tea

Yunnan dark tea

TAETEA

100 g/ brick

Yunnan, China

Yunnan dark tea

TAETEA

100 g/ brick

Yunnan, China

ChinaTea

Loose tea, 100 g/pot

Fujian, China

ChinaTea

Loose tea; 50 g/ box

Fujian, China

ChinaTea

Loose tea, 75g/pot

Fujian, China

10

Pu-erh raw tea (tea cake)

11

Pu-erh tea (tea cake)

Dark Tea

12

Bai mu dan tea

White Tea

13

Gong mei tea

White Tea

14

Aged bai mu dan tea

White Tea

15

Huangshan mao feng tea

Tea bud and leaf type Tea leaves type Tea bud and leaf type

15 g/ brick, 15 bricks in total

Hunan, China

Green Tea

Roasted green tea

Yi Fu Tang

Loose tea, 250g /pot

Anhui, China

16

Taiping hou kui tea

Green Tea

Roasted green tea

Yi Fu Tang


Anhui, China

17

Long jing tea

Green Tea

Roasted green tea

Ba Ma

Loose tea, 50g/pot

Zhejiang, China

18

Special Gunpowder

Green Tea

Roasted green tea

CAP

Loose tea, 125g /box

China

19

Tie guan yin tea

Oolong Tea

Ba Ma

Loose tea, 100g/bag

Fujian, China

20

Da hong pao tea

Oolong Tea

ChinaTea

Loose tea, 100g/box

Fujian, China

21

Jasmine green tea

Reprocessing Tea

Scented tea

Cuida Te


Europe

22

Organic UJI matcha

Reprocessing Tea

Dust tea

Morihan

Loose tea, 30g/bag

Kyoto, Japan

23

English Breakfast tea

Reprocessing Tea

Tea bag

TWININGS

2 g/bag, 25 bags in total

24

Pure green tea

Reprocessing Tea

Tea bag

TWININGS


South Fujian oolong tea North Fujian oolong tea

Blended and packed in Madrid, Spain Tea from China, Blended and packed in London, the UK


Page 37 of 40


25

Earl Grey

Reprocessing Tea

Tea bag

TWININGS


Blended and packed in Madrid, Spain Tea from Kenya,

26

Yellow label black tea

Reprocessing Tea

Tea bag

Lipton

1.5 g/bag; 20 bags in total

packed in Barcelona, Spain

27

Pu-erh tea

Reprocessing Tea

Tea bag

Carrefour

2 g/bag; 30 bags in total

28

White tea

Reprocessing Tea

Tea bag

Carrefour

1.5 g/bag, 30 bags in total

29

PG tips black tea

Reprocessing Tea

Tea bag

Unilever

2.9 g/bag, 40 bags in total

Packed in Madrid, Spain Packed in Madrid, Spain Packed in the UK

Note: the type and classification of tea refer to Chinese National Standard (Tea Classification, GB/T 307662014).



Page 38 of 40

Table 2. HPLC-IT-MS-MS analysis of ETs in tea infusion samples

No.

1

Component name

Galloyl-HHDP-

Observed

RT

(m/z)-

(min)

633.25

10.6

glucose isomer 1

MS2 and MS3 m/z (%)

precursors for MS3 m/z (%)

Detected in tea samples

301.02 (100) a

257.06(100); 228.89(31)

421.08 (10) a

300.96(100)

1, 7

275.01 (8) 2

Galloyl-HHDP-

633.07

12.1

glucose isomer 2

301.04 (100) a

256.90(100); 229.03(76); 184.87(17); 283.87(12)

275.04 (14) a

228.80(100); 256.89(81); 203.00(69)

1, 3, 5, 7

421.10 (5) 463.17 (3) 3

Galloyl-HHDP-

633.17

12.6

glucose isomer 3

327.03 (100) a

137.17(100); 309.16(43); 226.92(26); 282.95(21)

1, 3, 4, 5, 6,

301.03 (19) a

185.00(100)

7, 8, 9, 10

256.93(100); 284.00(16); 228.99(22); 184.94(10)

1, 3, 5, 6, 7

463.18 (15) 4

Galloyl-HHDP-

633.16

14.3

glucose isomer 4

300.96 (100) a 421.13

(10) a

301.00(100); 270.00(5); 307.2(5)

275.09 (6) 5

Galloyl-HHDP-

632.96

16.8

glucose isomer 5

300.96 (100) a

256.91(100); 214.09(51); 283.80(19); 184.80(17)

275.04 (10) a

258.00(100)

1, 7

421.19 (7) 513.06 (7) 6

Galloyl-HHDP-

633.18

20.5

glucose isomer 6

301.01 (100)a

256.99(100); 229.04(52); 185.05(52); 283.94(25); 201.00(11)

1-10

137.08(100); 308.99(44); 240.98(32); 267.07(19); 218.05(8)

1, 2, 3, 4, 6,

327.06(100); 296.92(13); 339.05(9)

7, 8, 9, 10

301.02 (100) a

257.00(100); 272.05(26); 185.00(22); 228.87(18)

1, 7

463.11 (17) a

301.02(100); 274.70(5); 169.20(5)

463.17 (4)

major 7

Galloyl-HHDP-

633.01

22.0

glucose isomer 7

327.09 (100) a 465.16

(8) a

301.00 (8) 497.14 (5) 8

Galloyl-HHDP-

633.11

22.8

glucose isomer 8 9

Tellimagrandin I

785.28

29.6

633.19 (100)

301.05(100); 463.19(6)

1, 2, 3, 7, 10

10

Ellagic acid

300.99

44.2

256.95 (100) a

185.08(100); 200.98(74); 229.10(30); 213.00(22)

1, 2, 3, 4, 6,

229.10 (76) a

172.92(100); 201.00(51)

7, 10

185.07 (48) 284.02 (11) 212.83 (11)

Note: a. The chosen MS2 fragments were broke to gain MS3 fragments; 1. Mengding huang ya tea; 2. Qimen black tea; 3. Pu-erh raw tea (tea cake); 4. Pu-erh tea (tea cake); 5. Bai mu dan tea; 6. Taiping hou kui tea; 7. Long jing tea; 8. Tie guan yin tea; 9. Organic UJI matcha; 10. Earl Grey


Page 39 of 40


Table 3. Characterization of urolithins in urine samples by UPLC-QTOF Retention

Observed

Accurate mass

Mass error

time (min)

m/z

m/z

(mDa)

Urolithin A 3-glucuronide

6.20

403.0663

403.0671

-0.8

C19H16O10

3, 10

Urolithin A 8-glucuronide

6.22

403.0666

403.0671

-0.5

C19H16O10

1, 2, 4, 8, 10

were detected in three

Urolithin A-sulphate

7.87

306.9920

306.9918

0.2

C13H8O7S

1, 2, 4, 6, 8,

volunteers (No. 6, 8 and 10),

Urolithin A

10.99

227.0351

227.0350

0.1

C13H8O4

1, 2, 6, 10

Isourolithin A 3-glucuronide

6.62

403.0670

403.0671

-0.1

C19H16O10

1, 2, 3, 4

Isourolithin A

10.73

227.0351

227.0350

0.1

C13H8O4

4

Urolithin B glucuronide

9.65

387.0721

387.0722

-0.1

C19H16O9

1, 2, 3, 4

Urolithin B-sulphate

10.94

290.9976

290.9969

0.7

C13H8O6S

2

Urolithin B

14.68

211.0406

211.0401

0.5

C13H8O3

1, 2, 4

Urolithin D

6.43

259.0254

259.0248

0.6

C13H8O6

n.d.

No urolithins were detected

Urolithin C

8.38

243.0304

243.0299

0.5

C13H8O5

n.d.

in three volunteers (No. 5, 7

Urolithin M7

9.22

243.0306

243.0299

0.7

C13H8O5

n.d.

and 9), and grouped as

Urolithin M6

7.72

259.0252

259.0248

0.4

C13H8O6

n.d.

Metabotype 0

Name

Molecular

Detected in

formula

Volunteers

Urolithin Metabotypes

Urolithin A derivatives

10

Only urolithin A derivatives

and were classified as Metabotype A

Isourolithin A derivatives

Urolithin B derivatives

Isourolithin A and urolithin B derivatives were detected in four volunteers (No. 1, 2, 3 and 4), and clustered as Metabotype B

Other urolithins



TOC Graphic Tea is a significant dietary source of ellagitannins and ellagic acid

Xiao Yang, and Francisco A. Tomás-Barberán


Page 40 of 40

Tea is a significant dietary source of ellagitannins and ellagic acid

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