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1 In vivo biotransformation of (poly)phenols and anthocyanins of red- 1 2 2 fleshed apple and identification of intake biomarkers 3 4 5 3 6 7 4 8 9 1+ 1,2+ 1 1 10 5 Silvia Yuste , Iziar A. Ludwig , Laura Rubió , Maria-Paz Romero , Anna 11 2,3 2 2,4 1,5 1* 12 6 Pedret , Rosa-Maria Valls , Rosa Solà , Maria-José Motilva , Alba Macià 13 14 7 15 16 1 17 8 Food Technology Department, XaRTA-TPV, Agrotecnio Center, Escola 18 19 9 Tècnica Superior d’Enginyeria Agrària, University of Lleida. Avda/ Alcalde 20 21 22 10 Rovira Roure 191, 25198-Lleida, Catalonia, Spain 23 24 11 2Universitat Rovira i Virgili, Facultat de Medicina i Ciències de la Salut, 25 26 27 12 Functional Nutrition, Oxidation, and Cardiovascular Diseases Group (NFOC- 28 29 13 Salut), C/Sant Llorenç 21, 43201-Reus, Spain. 30 31 14 3Eurecat, Centre Tecnològic de Catalunya, Unitat de Nutrició i Salut, Reus, 32 33 34 15 Spain. 35 36 16 4Hospital Universitari Sant Joan de Reus, Reus, Spain. 37 38 5 39 17 Current address: Instituto de Ciencias de la Vid y del Vino-ICVV (CSIC- 40 41 18 Universidad de La Rioja-Gobierno de La Rioja), Finca “La Grajera”, Carretera 42 43 44 19 de Burgos km 6, 26007-Logroño, Spain 45 46 20 47 48 49 21 50 + 51 22 SY and IAL contributed equally to the study. 52 53 23 54 55 56 24 *Corresponding author: E-mail: [email protected] 57 58 25 Phone: +34 973 702825 59 60 61 62 63 64 1 65 26 Abstract 1 2 27 The aim of this study was to investigate comprehensively the metabolic 3 4 5 28 pathways and human bioavailability of anthocyanins and other phenolic 6 7 29 compounds in apple matrix, and to elucidate potential intake biomarkers. After 8 9 10 30 the acute intake of a red-fleshed apple snack, plasma and urine were collected 11 12 31 and analyzed by UPLC-MS/MS. A total of 37 phase-II and microbial phenolic 13 14 32 metabolites were detected in plasma and urine. Among these, phloretin 15 16 17 33 glucuronide, cyanidin-3-O-galactoside (plasma and urine) and peonidin-3-O- 18 19 34 galactoside (urine) were the only metabolites detected in all the volunteers and 20 21 22 35 not detected at basal conditions. The maximum urine excretion was detected at 23 24 36 2-4 h, and the main increase in plasma of phloretin glucuronide and cyanidin-3- 25 26 27 37 O-galactoside was observed at 2h post-intake (61.0  6.82 and 10.3  1.50 nM, 28 29 38 respectively). These metabolites could be selected as the best intake 30 31 32 39 biomarkers of red-fleshed apple that might be useful in human intervention 33 34 40 studies when studying the bioactivity of red-fleshed apple. 35 36 37 41 38 39 42 40 41 43 42 43 44 44 45 46 45 47 48 49 46 50 51 47 52 53 54 48 55 56 49 Keywords: anthocyanins, metabolic pathways, phenolic compounds, red- 57 58 50 fleshed apple, UPLC-MS/MS. 59 60 61 51 62 63 64 2 65 52 1. INTRODUCTION 1 2 53 Apples are one of the most commonly consumed fruits and their diverse and 3 4 5 54 high (poly)phenol content is considered one of the most important determinants 6 7 55 of their health-promoting properties (Hyson, 2011; Bondonno et al., 2018). 8 9 10 56 In the last few years, there has been a rapidly increasing interest in potential 11 12 57 crops for coloring food naturally without transgenic or cysgenic programs. In 13 14 58 order to obtain better-quality apples with added healthy properties, new 15 16 17 59 genotypes of apple with red-flesh have been obtained by innovative breeding 18 19 60 strategies through cross-breeding programs with wild red-fleshed apple 20 21 22 61 varieties (with poor taste) and commercial good-flavored white-fleshed apples 23 24 62 (Deacon, www.suttonelms.org.uk). The resulting red-fleshed apples contain a 25 26 27 63 high amount of anthocyanin compounds in their flesh and have a good-tasting. 28 29 64 Apart from anthocyanins, red-fleshed apples are also a rich source of other 30 31 65 (poly)phenols that are also detected in common apple varieties such as 32 33 34 66 phenolic acids, dihydrochalcones, flavan-3-ols, and flavonols (Bars-Cortina et 35 36 67 al. 2017). Due to the enhanced content of anthocyanins reported in these red- 37 38 39 68 fleshed apples, different studies have shown that the total phenolic content and 40 41 69 antioxidant capacity were significantly higher in red-fleshed apples compared to 42 43 44 70 traditional white-fleshed apples, which indicates that these apples could have 45 46 71 presumably added healthy properties (Rupasinghe et al., 2010; Bars-Cortina et 47 48 49 72 al. 2017). 50 51 73 Regarding the bioavailability of apple phenolic compounds, only a few 52 53 74 studies have investigated the metabolism of these compounds in common 54 55 56 75 varieties of apple and most of them were focused on the bioavailability after 57 58 76 apple juice (Kahle et al.; 2011; Trošt et al. 2018) or apple cider consumption 59 60 61 62 63 64 3 65 77 (DuPont et al., 2002; Marks et al., 2009), with only one study reporting the 1 2 78 phenolic metabolites after consumption of apple fruit (Saenger et al., 2017). 3 4 5 79 Concerning the bioavailability of anthocyanins, there are plenty of studies 6 7 80 reporting their human bioavailability and metabolism, however, they have been 8 9 10 81 only studied in other food matrices such as blueberries, elderberries, 11 12 82 blackcurrants, strawberries and red grapes or red wine (Wu et al., 2002; Bitsch 13 14 83 et al., 2004; Stalmach et al., 2012; Kuntz et al., 2015; Zhong et al., 2017). So, to 15 16 17 84 our knowledge, no study has been reported in the literature regarding the 18 19 85 bioavailability of common apple phenolic compounds together with 20 21 22 86 anthocyanidins in the same food matrix, which represents a specific 23 24 87 characteristic of red-fleshed apple varieties. 25 26 27 88 In the case of anthocyanins, various types of food samples have been used 28 29 89 to determine the effects of food matrix on their bioavailability. For instance, 30 31 90 anthocyanins in strawberries, blood oranges and red wine have been reported 32 33 34 91 to be highly bioavailable with their urinary levels varying between 1-5% of the 35 36 92 ingested dose (Wallace et al., 2016). The differences reported in anthocyanin 37 38 39 93 bioavailability from different food sources, to a large extent, is due to the 40 41 94 presence of several structurally diverse anthocyanins in these foods, and the 42 43 44 95 interactions between food matrix and these specific anthocyanins. Therefore, 45 46 96 human postprandial studies are very useful and can contribute to knowledge 47 48 49 97 about the food matrix affecting polyphenol bioavailability (Motilva et al. 2015). 50 51 98 Moreover, the measurement of dietary exposure and reliable intake 52 53 99 biomarkers before investigating the potential health benefits of a new food 54 55 56 100 product is of crucial importance for the discovery of unbiased associations 57 58 59 60 61 62 63 64 4 65 101 between the intake of bioactive compounds and the observed effects (Dragsted 1 2 102 et al. 2018). 3 4 5 103 So, considering the scarce data regarding the human bioavailability and 6 7 104 metabolism of apple phenolic compounds, in the present work we aimed to 8 9 10 105 investigate the bioavailability and the complex metabolic pathways of the red- 11 12 106 fleshed apple as an innovative food source rich in different polyphenols, 13 14 107 including anthocyanins. Among all the identified metabolites, we also aimed to 15 16 17 108 identify and select those plasmatic and urinary metabolites that could be 18 19 109 considered as potential intake biomarkers of red-fleshed apple consumption 20 21 22 110 and might be used to establish the relationship between their intake and health 23 24 111 benefits in future human intervention studies. 25 26 27 112 28 29 113 2. MATERIALS AND METHODS 30 31 114 2.1. Chemicals and reagents 32 33 34 115 Cyanidin-3-O-galactoside, eriodictyol, quercetin-3-O-glucoside, quercetin-3- 35 36 116 O-rhamnoside, dimer B2, phloretin-2’-O-glucoside, p-coumaric acid, and caffeic 37 38 39 117 acid were purchased from Extrasynthese (Genay, France). p-Hydroxybenzoic 40 41 118 acid, 3,4-dihydroxybenzoic acid (aka protocatechuic acid), hippuric acid, 3-(4’- 42 43 44 119 hydroxyphenyl)acetic acid, 3-(3’,4’-dihydroxyphenyl)acetic acid, 3-(3’- 45 46 120 hydroxyphenyl)propionic acid, 3-(3’,4’-dihydroxyphenyl)propionic acid (aka 47 48 49 121 dihydrocaffeic acid), 3-(3’-hydroxy-4’-methoxyphenyl)propionic acid (aka 50 51 122 dihydroferulic acid), epicatechin, and chlorogenic acid were from Sigma-Aldrich 52 53 123 (St. Louis, MO, USA). Vanillic acid and ferulic acid were from Fluka (Buchs, 54 55 56 124 Switzerland). Vanillic acid-4-O-sulphate, catechol-4-O-sulphate, and 4-methyl 57 58 59 60 61 62 63 64 5 65 125 catechol sulphate were synthesized according to Pimpao et al. (2015) and were 1 2 126 kindly supplied by Dr. Claudia N. Santos (Portugal). 3 4 5 127 Methanol (HPLC grade), acetonitrile (HPLC grade), and acetic acid were 6 7 128 purchased from Scharlab Chemie (Sentmenat, Catalonia, Spain). The water 8 9 10 129 used was Milli-Q quality (Millipore Corp, Bedford, MA, USA). 11 12 130 Stock solutions of standard compounds were prepared by dissolving each 13 14 131 compound in methanol at a concentration of 1000 mg/L, and stored in a dark 15 16 17 132 flask at −30 ºC. 18 19 133 20 21 22 134 2.2. Apple snack 23 24 135 The red-fleshed ‘Redlove’ apple variety was provided by NUFRI SAT 25 26 27 136 (Mollerussa, Lleida, Spain), and planted in “La Rasa” experimental plot (La 28 29 137 Rasa, Soria, Spain). To increase the useful life, obtaining good shelf-stability 30 31 138 and, at the same time, minimize changes in the bioactive compounds of red- 32 33 34 139 fleshed apples, the freeze-dried snack format was selected. Before drying, the 35 36 140 apples were washed, and dried. Then, the apple core was removed and the 37 38 39 141 whole apple (with peel) was cut into 1 cm-sized cubes. The apple cubes were 40 41 142 frozen in liquid nitrogen and lyophilization was then performed with a first drying 42 43 44 143 at 0.6 bar with a temperature ramp of −20 to 0 °C for 25 hours, followed by a 45 46 144 second complete vacuum drying with a temperature ramp of 0 to 20 °C for 40 47 48 49 145 hours (Lyophilizer TELSTAR Lyobeta 15, Terrassa, Spain). The freeze-dried 50 51 146 apple cubes were immediately transferred to airtight plastic containers and 52 53 147 refrigerated (2 ºC) until the analysis of their phenolic composition and use in the 54 55 56 148 acute intake study. A photograph of the red-fleshed apple snack is shown in 57 58 149 Graphical Abstract. 59 60 61 62 63 64 6 65 150 The analysis of the phenolic composition of the apple snack was based on 1 2 151 the previous study by Bars-Cortina et al. (2017). Prior to the chromatographic 3 4 5 152 analysis of the apple (poly)phenols, a fine powder of the freeze-dried samples 6 7 153 was obtained with the aid of an analytical mill (A11, IKA, Germany). The 8 9 10 154 ingested portion of the apple snack contained a total of 196 mg of phenolic 11 12 155 compounds. The nutritional composition and the detailed phenolic composition 13 14 156 of the red-fleshed apple snack are presented in the Supporting Material in 15 16 17 157 Table S1 and Table S2, respectively. 18 19 158 20 21 22 159 2.3. Human intervention study and biological sample collection 23 24 160 The protocol of the study was approved by the Ethical Committee of the 25 26 27 161 Human Clinical Research Unit at the Arnau Vilanova University Hospital, Lleida, 28 29 162 Spain (Approval Number: 13/2016). Ten healthy participants (five females and 30 31 163 five males, mean age 37.3 ± 8.4 years) with a body mass index (BMI) of 18.5– 32 33 2 34 164 24.9 kg/m were enrolled. Exclusion criteria were pregnancy or lactation, any 35 36 165 chronic medication, any antibiotic treatment during the 4 months prior to the 37 38 39 166 study, cigarette smoking, alcohol intake > 80 g/day and use of dietary 40 41 167 supplements. After two days of a diet low in phenolic compounds, the 42 43 44 168 participants were invited to eat a portion of 80 g of red-fleshed apple snack after 45 46 169 fasting overnight. Human blood samples were obtained by venipuncture before 47 48 49 170 (0 h) and after the apple snack intake at 0.5, 1, 2, 4, 6, and 24 h using 6 mL 50 51 171 Vacutainer™ tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) 52 53 172 containing ethylenediamintetra acetic acid (EDTA) as an anticoagulant. To 54 55 56 173 obtain the plasma samples, the blood tubes were centrifuged at 8784 g for 15 57 58 174 min (Hettich, Tuttlingen, Germany). Aliquots were stored at −80 ºC until the 59 60 61 62 63 64 7 65 175 chromatographic analysis. On the other hand, urine samples were collected 12 1 2 176 h before and at the interval times of 0-2, 2-4, 4-8, and 8-24 h after the apple 3 4 5 177 snack intake. The total volume of each sample was measured before storing the 6 7 178 aliquots at −80 ºC until the chromatographic analysis. 8 9 10 179 11 12 180 2.3.1. Plasma samples 13 14 181 Before the chromatographic analyses, the plasma samples were pre-treated 15 16 17 182 by micro-Elution solid-phase extraction (µSPE) using OASIS HLB (2 mg, 18 19 183 Waters, Milford, MA) micro-cartridges. The methodology used is the one 20 21 22 184 reported in a previous study (Martí et al., 2010), but with some modifications. 23 24 185 Briefly, the micro-cartridges were conditioned sequentially with 250 µL of 25 26 27 186 methanol and 250 µL of 0.2% acetic acid. 350 µL of 4% phosphoric acid was 28 29 187 added to 350 µL of the plasma sample, and then this solution was loaded into 30 31 188 the micro-cartridges. The loaded micro-cartridges were cleaned up with 200 µL 32 33 34 189 of Milli-Q water and 200 µL of 0.2% acetic acid. Then, the retained phenolic 35 36 190 compounds were eluted with 2 x 50 µL of methanol. Each sample was prepared 37 38 39 191 in triplicate. 40 41 192 42 43 44 193 2.3.2. Urine samples 45 46 194 The urine samples were also pre-treated by µSPE. The micro-cartridges and 47 48 49 195 their conditioning and equilibration steps were the same as reported for plasma 50 51 196 samples. In this case, 100 µL of phosphoric acid at 4% was added to 100 µL of 52 53 197 the urine sample, and this solution was loaded into the micro-cartridge. The 54 55 56 198 retained phenolic compounds were then eluted with 2 x 50 µL of methanol. 57 58 199 Each sample was prepared in triplicate. 59 60 61 62 63 64 8 65 200 2.4. Ultra-performance liquid chromatography coupled to tandem mass 1 2 201 spectrometry (UPLC-MS/MS) 3 4 TM 5 202 LC analyses were carried out on an AcQuity Ultra-Performance liquid 6 7 203 chromatography and tandem mass spectrometry equipment from Waters 8 9 10 204 (Milford, MA, USA). Two chromatographic methods were used for the analysis 11 12 205 of 1) anthocyanins and their metabolites, and 2) the rest of the phenolic 13 14 206 compounds and their metabolites. In both methods, the flow rate was 0.3 15 16 17 207 mL/min and the injection volume 2.5 μL. The UPLC.MS/MS conditions were the 18 19 208 same used in our previous studies (Martí et al. 2010; Bars-Cortina et al. 2017; 20 21 22 209 Yuste et al. 2018). Tandem MS analyses were carried out on a triple 23 24 210 quadrupole detector (TQD) mass spectrometer (Waters, Milford, MA, USA) 25 26 27 211 equipped with a Z-spray electrospray interface. The selected reaction 28 29 212 monitoring (SRM) transition for quantification and the cone voltage and collision 30 31 213 energy for the analysis of the phenolic metabolites are shown in Table 1. 32 33 34 214 Due to the lack of commercial standards of phenolic metabolites, some of 35 36 215 these compounds were tentatively quantified by using the calibration curve of 37 38 39 216 their precursor or another phenolic compound with a similar structure. Methyl 40 41 217 catechol glucuronide was tentatively quantified by using the calibration curve of 42 43 44 218 4-methyl catechol sulphate. For hydroxybenzoic acid sulphate, the calibration 45 46 219 curve of p-hydroxybenzoic acid was used; hydroxyhippuric acid with hippuric 47 48 49 220 acid; protocatechuic acid sulphate with protocatechuic acid; vanillic acid 50 51 221 glucuronide with vanillic acid; hydroxyphenylacetic acid sulphate and 52 53 222 hydroxyphenylacetic acid glucuronide with 3-(4’-hydroxyphenyl)acetic acid; 54 55 56 223 dihydroxyphenylacetic acid sulphate and dihydroxyphenylacetic acid 57 58 224 glucuronide with 3-(3’,4’-dihydroxyphenyl)acetic acid; hydroxyphenylpropionic 59 60 61 62 63 64 9 65 225 acid sulphate and hydroxyphenylpropionic acid glucuronide with 3-(3’- 1 2 226 hydroxyphenyl)propionic acid; dihydroxyphenylpropionic acid sulphate with 3- 3 4 5 227 (3’,4’-hydroxyphenyl)propionic acid; hydroxymethoxyphenyl propionic acid 6 7 228 sulphate (dihydroferulic acid sulphate) with 3-(3’-hydroxy-4’- 8 9 10 229 methoxyphenyl)propionic acid (dihydroferulic acid); coumaric acid sulphate with 11 12 230 p-coumaric acid; caffeic acid sulphate with caffeic acid; ferulic acid sulphate 13 14 with ferulic acid; hydroxyphenyl- -valerolactone sulphate, dihydroxyphenyl- - 15 231   16 17 232 valerolactone, dihydroxyphenyl--valerolactone glucuronide, dihydroxyphenyl-- 18 19 20 233 valerolactone sulphate glucuronide, epicatechin sulphate, epicatechin 21 22 234 glucuronide, and methyl epicatechin glucuronide with epicatechin; phloretin 23 24 235 sulphate, phloretin glucuronide and phloretin sulphate glucuronide with 25 26 27 236 phloretin-2’-O-glucoside; cyanidin arabinoside and peonidin-3-O-galactoside 28 29 237 with cyanidin-3-O-galactoside. 30 31 32 238 33 34 239 2.5. Statistical analysis 35 36 37 240 The results are presented as mean values ± standard deviation (SD) for 38 39 241 (poly)phenols in red-fleshed apple snacks, and as mean values ± standard error 40 41 42 242 of the mean (SEM) for metabolites in the urine and plasma samples. For the 8 43 44 243 metabolite groups (catechol/pyrogallol derivatives, benzoic acid derivatives, 45 46 244 phenylacetic/phenylpropionic acid derivatives, phenyl--valerolactone 47 48 49 245 derivatives, flavan-3-ol derivatives, phloretin derivatives, and anthocyanin 50 51 246 derivatives), one-way repeated measures analysis of variance (ANOVA) was 52 53 54 247 performed on the urine samples to compare the mean differences at the five 55 56 248 defined time points. Post hoc analysis was conducted using pairwise 57 58 59 249 comparisons with Bonferroni correction. Differences were considered significant 60 61 62 63 64 10 65 250 at p < 0.05. All statistical analyses were performed using the SPSS v 22.0 1 2 251 software package. 3 4 5 252 6 7 253 3. RESULTS AND DISCUSSION 8 9 10 254 3.1. Red-fleshed apple snack phenolics characterization 11 12 255 The total amount of phenolic compounds in the apple snack portion (80 g) 13 14 256 administered to the volunteers accounted for 196 ± 10.7 mg and the analysis of 15 16 17 257 the phenolic composition showed a wide range of phenolic groups (Table S2 18 19 258 Supporting Material). The more abundant phenolic compounds were phenolic 20 21 22 259 acids (45%), mainly chlorogenic acid; flavan-3-ols (7%), mainly epicatechin and 23 24 260 its dimer; the flavonols (9%), mainly quercetin derivatives, and 25 26 27 261 dihydrochalcones (17%), with phloretin glucoside being the main representative 28 29 262 and a unique compound characteristic for apples. 30 31 263 Different from common apple species, red-fleshed apples, have an added 32 33 34 264 value as they also contain around 22% of anthocyanins. Anthocyanins in red- 35 36 265 fleshed apple are located both in the peel and flesh (Bars-Cortina, et al., 2017), 37 38 39 266 and are mainly represented by cyanidin-3-O-galactoside. This specific 40 41 267 anthocyanin has only been detected in considerable amounts in chokeberry 42 43 44 268 (Aronia melanocarpa) and lingonberry (Vaccinium vitis-idaea) (Zheng et al. 45 46 269 2003), both fruits that usually do not form part of a regular diet. Therefore, 47 48 49 270 cyanidin-3-O-galactoside could be considered a very characteristic compound 50 51 271 from red-fleshed apple and white-flesh red-skin apples. 52 53 272 Regarding the administered dose, anthocyanins have demonstrated 54 55 56 273 beneficial effects at variable administered doses (7.35-640 mg/day) (Wallace, et 57 58 274 al. 2016). Considering this range described in the literature as effective in the 59 60 61 62 63 64 11 65 275 prevention of chronic diseases, in the present study we selected a dose of 1 2 276 anthocyanins around 50 mg/day (Table S2 Supporting Material) administered 3 4 5 277 through a feasible amount of apple snack (80 g/day) that could be consumed 6 7 278 daily by the volunteers without difficulty. 8 9 10 279 11 12 280 3.2. Identification of the biological apple phenol metabolites by UPLC- 13 14 281 MS/MS 15 16 17 282 In order to identify the phenolic metabolites generated after the acute intake 18 19 283 of the red-fleshed apple snack, the detector system tandem MS was used due 20 21 22 284 to its specificity, sensitivity and selectivity. The generated metabolites were 23 24 285 determined and identified by the full scan mode in the MS mode, and in the 25 26 27 286 daughter scan and SRM modes in the tandem MS mode. In addition to the 28 29 287 detector system (MS/MS), authentic standards were also used when they were 30 31 288 available to determine their retention time and identify the phenolic metabolites 32 33 34 289 generated in plasma and urine samples. Their MS spectrum is shown in Figure 35 36 290 S1 in Supporting Material). 37 38 39 291 Although the volunteers spent two days on a diet low in phenolic compounds 40 41 292 prior to the intervention day, some phenolic acids (phenylpropionic, 42 43 44 293 phenylacetic, benzoic, and hydroxycinnamic acids) were detected and 45 46 294 quantified in the analysis of the basal plasma and urine (fasting conditions) 47 48 49 295 collected just before the apple snack intake (see Table S3 and S4 in 50 51 296 Supporting Material). After subtraction of these basal levels of phenolics, a 52 53 297 total of 37 phenolic metabolites were detected in the urine and/or plasma 54 55 56 298 samples in increased amounts after the red-fleshed apple snack intake (Table 57 58 299 1, and Tables S3 and S4 in Supporting Material). These metabolites included 59 60 61 62 63 64 12 65 300 four catechol and pyrogallol derivatives, six benzoic acid derivatives, five 1 2 301 phenylacetic acid derivatives, six phenylpropionic acid derivatives, and four 3 4 5 302 hydroxycinnamic acid derivatives. Four metabolites were hydroxyphenyl-- 6 7 303 valerolactone derivatives, three epicatechin derivatives, two phloretin 8 9 10 304 derivatives and three cyanidin derivatives. 11 12 305 The phenolic metabolites were mainly phase-II sulphated (18), glucuronided 13 14 15 306 (11) and methylated (8) conjugates formed through the action of the enzyme 16 17 307 sulphotransferases (SULFs), uridine-5’-diphosphate glucuronosyltransferases 18 19 308 (UGT), and catechol-O-methyltransferases (COMT), respectively. From all 20 21 22 309 these phase-II metabolites, sulphation was the main transformation. The 23 24 310 generation of simple phenolic acids, such as phenylpropionic, phenylacetic, and 25 26 27 311 benzoic acid derivatives, is probably the result of microbial transformations 28 29 312 occurring in the colon, which include ring fission, reduction, -oxidation (one 30 31 32 313 decarboxylation), -oxidation (two decarboxylations), dehydroxylation and 33 34 314 demethylation. In addition, phase-I metabolism (dehydrogenation or reduction) 35 36 37 315 may also be involved in the formation of these metabolites. These simple 38 39 316 phenolic acids can then undergo phase-II metabolism at the colon level and/or 40 41 42 317 be absorbed and reach the liver, where they would be subject to enzymatic 43 44 318 metabolism before re-entering the systemic blood circulation and finally being 45 46 47 319 excreted in the urine. 48 49 320 50 51 321 3.3. Proposed metabolic pathways of red-fleshed apple phenols 52 53 54 322 Based on the diversity of phenolic metabolites, whose concentration in 55 56 323 plasma or urine increased after the apple snack intake (Table 1), a complex 57 58 59 324 picture of the metabolic pathways of the main apple phenolic compounds, as 60 61 62 63 64 13 65 325 well as their interactions, has been proposed. Figure 1 shows the proposed 1 2 326 metabolic routes to explain the phenolic metabolites generated from chlorogenic 3 4 5 327 acid (in green), vanillic acid hexoside (in blue), cyanidin-3-O-galactoside (in 6 7 328 orange), epicatechin and dimer B2 (in lilac), quercetin derivatives (in pink), and 8 9 10 329 phloretin (xylosyl) glucoside (in brown) determined in the red-fleshed apple 11 12 330 snacks as the main phenolics. The name of the phase-II enzymes is shown in 13 14 331 green, and the colonic catabolism in brown. In the next subsections, the 15 16 17 332 metabolic pathways of the metabolites generated from the apple (poly)phenols 18 19 333 are described. 20 21 22 23 334 24 25 26 335 3.3.1. Anthocyanins 27 28 29 30 336 Cyanidin-3-O-galactoside, and cyanidin arabinoside (but at lower 31 32 337 concentration levels), were the main anthocyanins present in the red-fleshed 33 34 35 338 apple snack (Supporting Material Table S2). These cyanidin glycosides 36 37 339 (galactoside and arabinoside) were also detected in both the plasma and urine 38 39 40 340 samples in their native structure detected in apple snack. Other anthocyanin 41 42 341 metabolites were also identified in the plasma and urine, derived from phase-II 43 44 342 metabolism and microbial metabolism (Figure 1). Regarding phase-II 45 46 47 343 metabolites, peonidin-3-O-galactoside was detected in the urine resulting from 48 49 344 cyaniding-3-O-galactoside methylation by the action of COMT enzyme. 50 51 52 345 Methylation, as one of the first metabolic reactions of cyanidin glycosides, was 53 54 346 also reported by other authors in plasma and urine samples after the acute 55 56 57 347 intake of aronia berry extract (Xie et al. 2016), and also after the oral ingestion 58 59 348 of 500 mg of 13C-labelled cyanidin glucoside (De Ferrars et al. 2014). 60 61 62 63 64 14 65 349 Other cyanidin metabolites, based on the B-ring fission and cleavage of the 1 2 350 C-ring by the action of colonic enzymes (Mosele et al. 2015), were also 3 4 5 351 detected in our study. As a result, protocatechuic acid and 6 7 352 dihydroxyphenylpropionic acid (dihydrocaffeic acid) were respectively detected. 8 9 10 353 Protocatechuic acid might also have been formed by -oxidation of 11 12 354 dihydroxyphenylpropionic acid. Then, as proposed in Figure 1 (orange arrows), 13 14 15 355 protocatechuic acid could either be further degraded by the action of the 16 17 356 microbial flora to catechol metabolites (-oxidation), pyrogallol metabolites 18 19 20 357 (hydroxylation) and hydroxybenzoic acid (dehydroxylation), or methylated to 21 22 358 vanillic acid. 23 24 359 Despite B-ring fission and C-ring cleavage, phloroglucinol sulphate could 25 26 27 360 have been generated from A-ring fission. Nevertheless, this metabolite could 28 29 361 not be differentiated from pyrogallol sulphate due to the lack of commercially 30 31 32 362 available standards and because these two metabolites have the same 33 34 363 precursor (m/z 205) and product ions (m/z 125 and 83) (See Table 1). 35 36 37 364 Therefore, this metabolite could be tentatively identified as phloroglucinol 38 39 365 sulphate due to the A-ring fission (metabolic pathways not shown), or as 40 41 42 366 pyrogallol sulphate due to hydroxylation of catechol. 43 44 45 367 3.3.2. Other phenolic compounds 46 47 368 Chlorogenic acid (see Figure 1 green arrows). Caffeic acid would be the 48 49 50 369 first metabolite generated from chlorogenic acid by ester hydrolysis (Figure 1). 51 52 370 From this metabolite (caffeic acid), different reactions based on microbial 53 54 55 371 metabolism (dehydroxylation), and phase-I (dehydrogenation or reduction) and 56 57 372 phase-II (COMT) metabolism could occur resulting in the generation of 58 59 60 373 coumaric acid, dihydroxyphenylpropionic acid and ferulic acid, respectively. 61 62 63 64 15 65 374 Then, dihydroxyphenylpropionic acid and ferulic acid could be further degraded 1 2 375 to phenylpropionic acid, phenylacetic acid and vanillic acid. These metabolites 3 4 5 376 could be further degraded to such simpler phenolic compounds as 6 7 377 protocatechuic acid, p-hydroxybenzoic acid and catechol metabolites. 8 9 10 378 11 12 379 Vanillic acid hexoside (see Figure 1 blue arrows). Vanillic acid hexoside 13 14 380 was the second most abundant phenolic acid quantified in the red-fleshed apple 15 16 17 381 snack (Supporting Material Table S2). After deglycosylation of this phenolic 18 19 382 acid, vanillic acid could be formed and subsequently sulphated (vanillic acid 20 21 22 383 sulphate), glucuronided (vanillic acid glucuronide) and demethylated 23 24 384 (protocatechuic acid). Then, as has been commented before, protocatechuic 25 26 27 385 acid could also be further degraded by microbial activity to generate catechol 28 29 386 and pyrogallol metabolites. 30 31 32 33 387 The presence of the metabolites derived from these two phenolic acids 34 35 388 (chlorogenic and vanillic acids) was in agreement with the results reported in 36 37 389 the literature for the bioavailability study after the consumption of foods rich in 38 39 40 390 these phenolics, such as coffee (Monteiro et al. 2007; Renouf et al 2010; 41 42 391 Ludwig et al 2013;), cereals (Calani et al. 2014), olive oil enriched with thyme 43 44 45 392 phenols (Rubió et al. 2014) and apple juice (Kahle et al. 2011). 46 47 393 48 49 50 394 Flavan-3-ols (epicatechin and dimer B2) (see Figure 1 lilac arrows). 51 52 395 Flavan-3-ol metabolites included both phase-II and microbial catabolites. The 53 54 396 first metabolic step would be the hydrolysis of the proanthocyanidin dimer to 55 56 57 397 catechin and epicatechin, and these monomers were found in urine as 58 59 398 glucuronided ((epi)catechin glucuronide), sulphated ((epi)catechin sulphate), 60 61 62 63 64 16 65 399 and further methylated (methyl (epi)catechin sulphate) conjugates. On the other 1 2 400 hand, the flavan-3-ols monomers could also be metabolized by the gut 3 4 5 401 microbiota to dihydroxyphenyl-γ-valerolactone, detected in urine samples. 6 7 402 Similarly, other studies reported the valerolactones as specific flavan-3-ols 8 9 10 403 metabolites (Aura, 2008; Hackman et al. 2008; Serra et al. 2011; Mosele et al. 11 12 404 2015). Then, dihydroxyphenyl-γ-valerolactone could also be glucuronided, and 13 14 405 further sulphated and also dehydroxylated and further sulphated. 15 16 17 406 18 19 407 Quercetin derivatives (see Figure 1 pink arrows). Quercetin 20 21 22 408 galactoside/glucoside and arabinoside would firstly be deglycosylated, and then 23 24 409 the generated aglycone (quercetin) could enter epithelial cells by passive 25 26 27 410 diffusion and be absorbed. Nevertheless, in the present study, no phase-II 28 29 411 metabolites of quercetin were identified. On the other hand, quercetin 30 31 412 rhamnoside has been reported not to be metabolized in the small intestine and 32 33 34 413 to reach the colon where this is metabolized to dihydroxyphenylpropionic acid 35 36 414 (Arts et al. 2004; Aura, 2008; Serra et al. 2012; Mosele et al. 2015), and then 37 38 39 415 progressively metabolized to generate simple phenols, such as phenylacetic 40 41 416 and benzoic acids, down to catechol derivatives. In our study, only microbial 42 43 44 417 metabolites from quercetin derivatives were observed. 45 46 418 47 48 49 419 Phloretin (xylosyl) glucoside (see Figure 1 brown arrows). Phloretin 50 51 420 (xylosyl) glucoside could be firstly deglycosylated to generate phloretin. Then, 52 53 421 this dihydrochalcone could be glucuronided by UGT enzymes and further 54 55 56 422 sulphated with SULF enzymes. Regarding the literature, phloretin glucuronide 57 58 423 was also reported in plasma and urine samples after the oral consumption of 59 60 61 62 63 64 17 65 424 apple cider (Marks et al. 2009) and apple fruit (Saenger et al. 2017). This 1 2 425 compound could also be metabolized by the gut microbiota enzymes to 3 4 5 426 dihydroxyphenylpropionic acid and further metabolized to generate simpler 6 7 427 phenolic compounds, such as phenylacetic and benzoic acids down to catechol 8 9 10 428 derivatives. 11 12 13 429 14 15 16 17 430 3.4. Biomarkers for apple phenol consumption 18 19 431 As evidenced in the metabolic pathways proposed in Figure 1, a large 20 21 432 number of metabolites are generated from the five main phenolic groups 22 23 24 433 (anthocyanins, phenolic acids, flavan-3-ols, flavonols and dihydrochalcones) 25 26 434 present in the red-fleshed apple snack. Some of the metabolites identified in the 27 28 29 435 plasma and urine samples after the snack intake are common to several 30 31 436 phenolic groups. These compounds are mainly colonic metabolites, such as 32 33 34 437 phenylpropionic, phenylacetic, and benzoic acids, and catechol derivatives. 35 36 438 Additionally, these phenolic metabolites are common to other (poly)phenol-rich 37 38 439 foods. That is why, more attention was paid in this study to specific phenolic 39 40 41 440 compounds detected in plasma or urine that could be used as biomarkers for 42 43 441 red-fleshed apple intake. The identification of the specific food intake 44 45 46 442 biomarkers is of great importance to establish the relationship between 47 48 443 (poly)phenols intake and health benefits in human intervention studies. 49 50 51 444 The 37 phenol metabolites detected in the urine and plasma samples after 52 53 445 the red-fleshed apple snack intake (see Table 1, and Tables S3 and S4 in 54 55 56 446 Supporting Material) could be classified into two groups according to their 57 58 447 urine excretion (µmol) kinetic, as is shown in Figure 2. The first group would 59 60 448 include derivatives from phenylpropionic and phenylacetic acids, benzoic acids, 61 62 63 64 18 65 449 catechol and pyrogallol and hydroxycinnamic acids (see Figure 2A); and the 1 2 450 second group the derivatives from flavan-3-ols, valerolactones, 3 4 5 451 dihydrochalcones, and anthocyanins (see Figure 2B). The phenolic metabolites 6 7 452 from the first group are excreted at high concentration levels (µmol) in the 8 9 10 453 different interval times (0 to 24 h). However, these phenolic compounds 11 12 454 presented low specificity to be considered as intake biomarkers, as they were 13 14 455 also quantified under basal conditions (before the apple intake), and only a 15 16 17 456 slight but not significant increase in phenylpropionic/phenylacetic acids, benzoic 18 19 457 acids and catechol/pyrogallol derivatives was observed after the apple intake in 20 21 22 458 the urine excretion between 0 to 24 h. This fact was also observed in the 23 24 459 plasma samples and their concentration was also only slightly enhanced after 25 26 27 460 the intake of the red-fleshed apple snack (see Supporting Material Table S3). 28 29 461 So, these compounds could be considered “endogenous” phenolic metabolites 30 31 462 from the diet. 32 33 34 463 On the other hand, the phenolic metabolites from the second group were 35 36 464 excreted in urine at concentration levels (µmol) and significantly increased 37 38 39 465 (p<0.05) after the red-fleshed apple intake at the different interval times studied 40 41 466 (see Figure 2B). Phenolic metabolites from this group could be considered as 42 43 44 467 potential biomarkers for red-fleshed apple consumption, since they were 45 46 468 detected at trace levels or not detected in basal conditions. 47 48 49 469 Figure 3 shows the individual profile of the urinary excretion (μmol and 50 51 470 nmols for anthocyanins) of the potential biomarkers quantified after the red- 52 53 471 fleshed apple snack intake. These urinary biomarkers include cyanidin 54 55 56 472 galactoside (A1), cyanidin arabinoside (A2) and peonidin galactoside (A3) as 57 58 473 cyanidin derivatives (anthocyanins); phloretin glucuronide (B1), and phloretin 59 60 61 62 63 64 19 65 474 sulphate glucuronide (B2) as phloretin derivatives from dihydrochalcones 1 2 475 pathways, epicatechin sulphate (C1), methyl epicatechin sulphate (C2) and 3 4 5 476 epicatechin glucuronide (C3) as epicatechin derivatives, and dihydroxyphenyl-- 6 7 477 valerolactone (D1), dihydroxyphenyl--valerolactone sulphate glucuronide (D2), 8 9 10 478 dihydroxyphenyl--valerolactone glucuronide (D3) and hydroxyphenyl-- 11 12 13 479 valerolactone sulphate (D4) as phenyl--valerolactone derivatives from flavan-3- 14 15 480 ols pathways. 16 17 481 As shown in Figure 3 and in Table S3 (Supporting Material), cyanidin (A1- 18 19 20 482 A3), phloretin (B1 and B2) and epicatechin (C1-C3) derivatives were excreted in 21 22 483 the first hours after the apple intake, their maximum excretion being detected 23 24 25 484 from 2 to 4 h. Then, their urinary excretion decreased until 24 h. These results 26 27 485 are in agreement with other studies after apple consumption (DuPont et al. 28 29 30 486 2002; Mennen et al. 2006; Kahle et al. 2007; Marks et al. 2009; Kristensen et al. 31 32 487 2012; Saenger et al. 2017). 33 34 35 488 Note that the native forms of some apple anthocyanins, such as cyanidin-3- 36 37 489 O-galactoside, cyanidin arabinoside, and peonidin-3-O-galactoside and also 38 39 490 phloretin glucuronide (a phase-II metabolite) were detected in the urine samples 40 41 42 491 from all the volunteers. Additionally, cyanidin-3-O-galactoside and phloretin 43 44 492 glucuronide were also detected in all the plasma samples. Observing the 45 46 47 493 plasma kinetic profile of these metabolites, they were rapidly absorbed in the 48 49 494 small intestine showing a maximum concentration at 2-3 h. After that, their 50 51 52 495 concentration decreased significantly until 24 h (see Figure 4 and Supporting 53 54 496 Material Table S4). 55 56 57 497 Regarding phenyl--valerolactone derivatives (Figure 3, D1-D4) from flavan- 58 59 498 3-ols metabolic pathways, their urinary excretion increased after the apple 60 61 62 63 64 20 65 499 snack intake showing a maximum excretion from 8 to 24 h. This trend indicates 1 2 500 intense colonic microbial metabolism, which could explain that these 3 4 5 501 compounds were not detected in the plasma. Nevertheless, in our previous 6 7 502 study in which we analyzed whole blood by sampling with dried blood spot 8 9 10 503 (DBS) cards from the same volunteers, hydroxyphenyl--valerolactone 11 12 504 glucuronide was detected after 12 h (Yuste et al. 2018). These differences 13 14 15 505 could indicate higher sensitivity in the detection of circulating valerolactones 16 17 506 from the analysis of whole blood instead of plasma. 18 19 507 From the results obtained in the present study, all the phenolic metabolites 20 21 22 508 shown in Figure 3 could be proposed as urinary markers for red-fleshed apple 23 24 509 consumption. Nevertheless, epicatechin and phenyl--valerolactone derivatives 25 26 27 510 were not considered in the present study as biomarkers since these compounds 28 29 511 are also present in other flavan-3-ol-rich foods, such as cocoa, wine and tea, 30 31 32 512 which form part of a regular diet. Epicatechin phase-II metabolites, such as the 33 34 513 sulphated, glucuronided and methylated derivatives found in the present study, 35 36 37 514 as well as the microbial derived phenyl--valerolactone metabolites, are 38 39 515 certainly good biomarkers for the correct assessment of intake and health 40 41 42 516 effects exerted by flavan-3-ol-rich diets (Van der Hooft et al. 2012; Urpi-Sardá et 43 44 517 al. 2015). However, when proposing intake biomarkers, more specific 45 46 47 518 metabolites must be sought. 48 49 519 On the one hand, as reported in previous studies (Mennen et al., 2006; 50 51 520 Saenger et al., 2017) we corroborate that phloretin derived metabolites, in this 52 53 54 521 study phloretin glucuronide as a phase-II metabolite was determined, are good 55 56 522 biomarkers for apple intake, as phloretin glucoside is an exclusively apple 57 58 59 523 dihydrochalcone (Richling, 2012). Moreover, phloretin glucuronide was 60 61 62 63 64 21 65 524 considered to be a good biomarkers as it was detected in all the plasma and 1 2 525 urine samples from all the volunteers and was not detected under basal 3 4 5 526 conditions. 6 7 527 On the other hand, cyanidin-3-O-galactoside, was considered also a 8 9 10 528 good intake biomarker as it is the main anthocyanin of red-fleshed apples (Guo 11 12 529 et al., 2016; Bars-Cortina et al., 2017), and apart from these apple varieties, it 13 14 530 can be only found in considerable amounts in chokeberry and lingonberry fruits. 15 16 17 531 Therefore, cyanidin-3-O-galactoside could be considered as good intake 18 19 532 biomarker for red-fleshed apple, and probably also for apples with white flesh 20 21 22 533 and red skin, although no data where found in the literature. This metabolite 23 24 534 was also detected in all the plasma and urine samples from all the volunteers 25 26 27 535 and was not detected under basal conditions. Peonidin-3-O-galactoside was 28 29 536 also quantified in the urine samples from all the volunteers, but at lower 30 31 537 amounts than cyanidin-3-O-galactoside. This methyl conjugate of cyanidin-3-O- 32 33 34 538 galactoside (peonidin-3-O-galactoside) could also be selected as a good urinary 35 36 539 biomarker for red-fleshed apple consumption jointly with phloretin glucuronide 37 38 39 540 and cyanidin-3-O-galactoside (Figure 4). 40 41 541 As pointed out, these three phenolic metabolites (phloretin glucuronide, 42 43 44 542 cyanidin-3-O-galactoside and peonidin-3-O-galactoside) are good intake 45 46 543 biomarkers although not strictly specific for red-fleshed apples. However, the 47 48 49 544 presence of both phloretin glucuronide and cyanidin galactoside metabolites 50 51 545 (cyanidin-3-O-galactoside and peonidin-3-O-galactoside) could be considered 52 53 546 as more specific intake biomarkers from apples with red-flesh. 54 55 56 547 Further, it is important to remark, as we previously reported (Bars-Cortina 57 58 548 et al., 2017), that the red-fleshed apples contains a higher concentration of 59 60 61 62 63 64 22 65 549 anthocyanins but a lower concentration of flavan-3-ols in its flesh in comparison 1 2 550 to the traditional white-fleshed varieties with red skin. This fact could be 3 4 5 551 explained by a competitive synthesis between anthocyanins and 6 7 552 proanthocyanidins in the flesh. Presumably this different ratio of 8 9 10 553 anthocyanins/flavan-3-ols reported in red-flesh apples, might be reflected in the 11 12 554 concentrations of the generated metabolites. So, in quantitative terms, after a 13 14 555 red-fleshed apples intake a lower amount of flavan-3-ols metabolites would be 15 16 17 556 expected in comparison to white-fleshed apples with red skin intake. To support 18 19 557 this hypothesis, the generated metabolites after an intake of red-fleshed and 20 21 22 558 traditional white-fleshed snack apples are being analyzed in an ongoing study. 23 24 559 25 26 560 27 28 29 561 Conclusions 30 31 562 In the present study, the different phenolic metabolites generated after the 32 33 34 563 intake of red-fleshed apple snacks were identified and tentatively quantified in 35 36 564 urine and plasma samples at different time intervals. Moreover, the metabolic 37 38 565 pathways of the phenolic metabolites generated from red-fleshed apple 39 40 41 566 phenolics were proposed, and these routes were based on phase-II and 42 43 567 microbial reactions. The results show that after the consumption of red-fleshed 44 45 46 568 apple snacks, (poly)phenols are extensively metabolized, resulting in the 47 48 569 production of a large number of compounds with different structure, all of which 49 50 51 570 should be considered when investigating the potential health effects of red- 52 53 571 fleshed apples. Among all the metabolites generated, phloretin glucuronide, 54 55 572 cyanidin-3-O-galactoside and peonidin-3-O-galactoside were proposed as the 56 57 58 573 best candidates as biomarkers after the intake of red-fleshed apple snack. 59 60 574 These two phenolic metabolites were not detected in basal samples and were 61 62 63 64 23 65 575 detected in the urine and/or plasma samples from all the volunteers. It is 1 2 576 important to highlight that these three phenolic metabolites are not strictly 3 4 5 577 exclusive to red-fleshed apple intake, since phloretin glucuronide is a common 6 7 578 biomarker for all apple fruits, and cyanidin-3-O-galactoside and peonidin-3-O- 8 9 10 579 galactoside could appear not only after the intake of red-flesh apple but also 11 12 580 after white-flesh with red-skin apples or other fruits such as chokeberry and 13 14 581 lingonberry. However, the presence of these three metabolites could be useful 15 16 17 582 as intake biomarkers in human intervention studies when studying the 18 19 583 bioactivity of red-fleshed apple. 20 21 22 23 584 ACKNOWLEDGEMENTS 24 25 26 585 This study was supported by the Spanish Ministry of Industry, Economy and 27 28 29 586 Competitiveness through the AGL2016-76943-C2-1-R and AGL2016-76943-C2- 30 31 587 2-R projects (co-funded by the European Social Fund, European Union); I.A.L. 32 33 34 588 enjoys a post-doctoral contract (2017PMF-POST2-19) from the European 35 36 589 Union's Horizon 2020 research and innovation programme under the Marie 37 38 590 Skłodowska-Curie grant agreement and from the Universitat Rovira i Virgili 39 40 41 591 (URV). S.Y. was supported by a grant from the University of Lleida. In addition, 42 43 592 the authors are grateful to NUFRI SAT (Mollerussa, Lleida, Catalonia, Spain) for 44 45 46 593 providing the red-fleshed apples. 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(2017). 11 12 774 Characterization of Wild Blueberry Polyphenols Bioavailability and Kinetic 13 14 775 Profile in Plasma over 24-h Period in Human Subjects. Molecular Nutrition 15 16 17 776 and Food Research, 61, 1700405. https://doi.org/10.1002/mnfr.201700405 18 19 777 20 21 22 778 23 24 779 25 26 27 780 28 29 781 30 31 782 32 33 34 783 35 36 784 37 38 39 785 40 41 786 42 43 44 787 45 46 788 47 48 49 789 50 51 790 52 53 791 54 55 56 792 57 58 793 Figure captions 59 60 61 62 63 64 32 65 794 1 2 795 Figure 1. Proposed metabolic pathways for the generation of phenolic 3 4 5 796 metabolites after the acute intake of red-fleshed apple snack. The metabolic 6 7 797 route for chlorogenic acid is in green, for vanillic acid hexoside in blue, for 8 9 10 798 anthocyanins in orange, from flavan-3-ols in lilac, from quercetin derivatives in 11 12 799 pink and from dihydrochalcones in brown. 13 14 800 Reactions: dH, dehydrogenation; SULT, sulphotransferase; UGT, glucuronosyl- 15 16 17 801 transferase; and COMT, catechol-O-methyltransferase; dOH, dehydroxylation; 18 19 802 dMe, demethylation; -oxidation, one decarboxylation; and -oxidation, two 20 21 22 803 decarboxylations. 23 24 804 Quercetin derivatives: quercetin glucoside, quercetin galactoside, quercetin 25 26 27 805 arabinoside and quercetin rhamnoside. 28 29 806 30 31 32 807 Figure 2. Phenolic metabolite excretion rate in urine A) phenylpropionic/ 33 34 808 phenylacetic acids, benzoic acids, catechol/pyrogallol and hydroxycinnamic 35 36 37 809 acids derivatives; and B) flavan-3-ols, phenyl--valerolactones, 38 39 810 dihydrochalcones, and anthocyanins derivatives. Except for anthocyanin 40 41 42 811 derivatives (nmol/h), data expressed as μmol/h as mean values ± standard error 43 44 812 of mean (n=10). Asterisks indicate significant differences (p0.05) in excretion 45 46 47 813 rate compared to basal conditions. 48 49 814 50 51 815 Figure 3. Urinary excretion of the proposed biomarkers for red-fleshed apple 52 53 54 816 consumption 0-24 h after acute intake of 80 g apple snack. A1-A3: Cyanidin 55 56 817 derivatives; B1-B2: Phloretin derivatives; C1-C3: Epicatechin derivatives; D1- 57 58 59 818 D4: Phenyl--valerolactone derivatives. Except for anthocyanin derivatives 60 61 62 63 64 33 65 819 (nmol/h), data expressed as μmol/h as mean values ± standard error of mean 1 2 820 (n=10). 3 4 5 821 6 7 822 Figure 4. Pharmacokinetic profile of the proposed biomarkers for red-fleshed 8 9 10 823 apple snack intake. In urine samples, data expressed as μmol as mean values 11 12 824 ± standard error of mean (n=10), except for anthocyanin derivatives (nmol/h). 13 14 825 15 16 17 826 18 19 827 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 34 65 Table 1

Table 1. SRM conditions used for quantification and metabolites detected in plasma and urine after acute intake of red-fleshed apple snack.

SRM Cone voltage (V) / a Phenolic metabolites Detected in Quantification Collision energy (eV)

Catechol and pyrogallol derivatives 1 Catechol sulphate 189  109 20 / 15 P (5), U (8) 2 Methyl catechol sulphate 203  123 20 / 15 P (5) U (7) 3 Methyl catechol glucuronide 299  123 40 / 15 U (6) 4 Pyrogallol sulphate or phloroglucinol sulphate 205  125 40 / 15 P (7) U (5)

Benzoic acid derivatives 5 Hydroxybenzoic acid 137  93 30 / 15 U (3) 6 Hydroxybenzoic acid sulphate 217  137 35 / 15 P (3), U (3) 7 Hydroxyhippuric acid 194  100 40 / 10 P (8) 8 Protocatechuic acid sulphate 233  153 35 / 15 P (4), U (5) 9 Vanillic acid sulphate 247  167 30 / 25 U (5) 10 Vanillic acid glucuronide 343  167 30 / 25 P (9) U (3)

Phenylacetic acid derivatives 11 Hydroxyphenylacetic acid 151  107 20 / 10 P (5), U (4) 12 Hydroxyphenylacetic acid sulphate 231  151 20 / 15 P (6), U (4) 13 Hydroxyphenylacetic acid glucuronide 327  151 20 / 15 U (2) 14 Dihydroxyphenylacetic acid sulphate 247  167 30 / 15 U (5) 15 Dihydroxyphenylacetic acid glucuronide 343  167 30 / 15 P (3), U (4)

Phenylpropionic acid derivatives 16 Hydroxyphenylpropionic acid 165  121 20 / 10 P (4), U (6) 17 Hydroxyphenylpropionic acid sulphate 245  165 35 / 15 U (7) 18 Hydroxyphenylpropionic acid glucuronide 341  165 40 / 25 U (4) 19 Dihydroxyphenylpropionic acid sulphate 261  181 40 / 15 P (2), U (5) 20 Hydroxymethoxyphenylpropionic acid 195  136 30 / 15 P (5), U (5) 21 Hydroxymethoxyphenylpropionic acid sulphate 275  195 35 / 15 P (2) U (2)

Hydroxycinnamic acid derivatives 22 Coumaric acid 163  119 35 / 10 P (2), U (5) 23 Coumaric acid sulphate 243  163 35 / 15 U (9) 24 Caffeic acid sulphate 259  179 35 / 15 P (2), U (2) 25 Ferulic acid sulphate 273  193 35 / 15 P (7), U (10)

Phenyl--valerolactone derivatives 26 Hydroxyphenyl--valerolactone sulphate 271  191 40 / 20 U (8) 27 Dihydroxyphenyl--valerolactone 207  163 40 / 15 U (8) 28 Dihydroxyphenyl--valerolactone glucuronide 383  207 40 / 20 U (8) Dihydroxyphenyl--valerolactone sulphate 463  287 40 / 20 U (7) 29 glucuronide

Flavan-3-ol derivatives 30 Epicatechin sulphate 369  289 40 / 20 U (6) 31 Epicatechin glucuronide 465  289 40 / 20 U (6) 32 Methyl epicatechin glucuronide 383  303 40 / 15 U (7)

Dihydrochalcone derivatives 33 Phloretin glucuronide 449  273 40 / 20 P (10), U (10) 34 Phloretin sulphate glucuronide 529  353 40 / 20 U (8)

Anthocyanin derivatives 35 Cyanidin-3-O-galactoside 449  287 40 / 20 P (7), U (10) 36 Cyanidin arabinoside 419  287 40 / 20 P (1). U (10) 37 Peonidin-3-O-galactoside 463  301 40 / 20 U (10)

aMetabolites detected in urine (U) and/or plasma (P). Figures in parenthesis indicate the number of samples (volunteers) in which the metabolite was detected. Supporting Material clean version

SUPPORTING MATERIAL

In vivo biotransformation of (poly)phenols and anthocyanins of red-fleshed apple

and identification of intake biomarkers

Silvia Yuste1+, Iziar A. Ludwig1,2+, Laura Rubió1, Maria-Paz Romero1, Anna Pedret2,3,

Rosa-Maria Valls2, Rosa Solà2,4, Maria-José Motilva1,5, Alba Macià1*

1Food Technology Department, XaRTA-TPV, Agrotecnio Center, Escola Tècnica

Superior d’Enginyeria Agrària, University of Lleida. Avda/ Alcalde Rovira Roure 191,

25198-Lleida, Catalonia, Spain

2Universitat Rovira i Virgili, Facultat de Medicina i Ciències de la Salut, Functional

Nutrition, Oxidation, and Cardiovascular Diseases Group (NFOC-Salut), C/Sant

Llorenç 21, 43201-Reus, Spain.

3Eurecat, Centre Tecnològic de Catalunya, Unitat de Nutrició i Salut, Reus, Spain.

4Hospital Universitari Sant Joan de Reus, Reus, Spain.

5Current address: Instituto de Ciencias de la Vid y del Vino-ICVV (CSIC-Universidad de

La Rioja-Gobierno de La Rioja), Finca “La Grajera”, Carretera de Burgos km 6,

26007-Logroño, Spain

+ SY and IAL contributed equally to the study.

*Corresponding author: E-mail: [email protected]

Phone: +34 973 702825

. Includes Supplementary Information on nutritional composition (fiber and

macronutrients) of the red-fleshed apple snack (Table S1); concentration of the

main phenolic compounds in red fleshed apple snack. Data are expressed as

mg/80 g portion dry weight (mean ± SD, n=3) (Table S2); total amounts of

phenolic compounds excreted in urine (Table S3); as well concentrations of

phenolic compounds detected in plasma (Table S4) after red-fleshed apple snack

intake. MS spectrum of the phenolic compounds and generated metabolites after

the acute intake of the red-fleshed apple snack. Collision energy applied was: a) 5

eV, b) 10 eV, c) 15 eV, d) 20 eV, and e) 25 eV (Figure S1).

. MATERIAL AND METHODS

- Nutritional facts. The samples were analysed for moisture, fat, protein, fibre

(soluble and insoluble), sugars (reducing and non-reducing sugars) and ash

content. All reagents used were of analytical grade. Moisture was estimated by

weight difference after drying. Total protein content was estimated by the

Dumas method and total fat was extracted with hexane from previously dried

samples using a Soxhlet extractor. Insoluble fibre was determined following the

method described by Van Soest et al. (1991), while soluble fibre was calculated

gravimetrically as the alcohol insoluble residue according to Maran (2015).

Sugar quantification (reducing and non-reducing sugars) was carried out by

titrimetry based on the Fehling reaction in alkaline media after acid hydrolysis of

non-reducing sugars. Ash content was determined by incineration at 550°C ±

10°C. Finally, total carbohydrates were calculated by difference. Results were

expressed as grams of each compound per 100 grams of sample (g/100 g).

. REFERENCES

- Van Soest, P.J., Robertson, J.B., Lewis, B.A. (1991). Methods for dietary fiber,

neutral detergent fiber, and nonstarch polysaccharides in relation to animal

nutrition. Journal of Dietary Sciences, 74, 3583-3597.

https://doi.org/10.3168/jds.S0022-0302(91)78551-2

- Maran, J.P. (2015). Statistical optimization of aqueous extraction of pectin from

waste durian rinds. International Journal of Biological Macromolecules, 73, 92-

98. https://doi.org/10.1016/j.ijbiomac.2014.10.050

Table S1. Nutritional facts of red-fleshed apple snack

Nutritional composition Per 100g Per portion (80g) Calories (KJ) 982.6 786.1 (Kcal) 234.8 187.9 Fat (g) 0.9 0.7 Total Carbohydrates (g) 93.6 74.9 - of which Sugars (g) 53.8 43.0 Protein (g) 3.0 2.4 g Fibre (total) (g) 16.5 13.2 - Soluble fibre (g) 7.6 6.1 - Insoluble fibre (g) 8.9 7.1 Minerals (g) 1.7 1.3

Table S2. Concentration of the main phenolic compounds in red fleshed apple snack. Data are expressed as mg/80 g portion dry weight (mean ± SD, n=3)

Phenolic compound Concentration (mg/80 g portion) Anthocyanins 42.3 ± 1.18 Cyanidin-3-O-galactoside 39.7 ± 1.03 Cyanidin arabinoside 2.60 ± 0.24 Phenoloc acids 88.0 ± 3.34 Protocatechuic acid 1.71 ± 1.06 Coumaric acid hexoside 0.77 ± 0.11 Ferulic acid hexoside 2.12 ± 0.26 Vanillic acid hexoside 4.28 ± 0.11 Chlorogenic acid 79.1 ± 2.75 Flavan-3-ols 13.8 ± 1.18 Epicatechin 5.58 ± 0.78 Dimer 6.92 ± 0.29 Trimer 1.30 ± 0.12 Flavonol 17.3 ± 1.97 Quercetin-3-O-arabinoside 3.67 ± 0.45 Quercetin-3-O-rhamnoside 9.26 ± 0.94 Quercetin-3-O-glucoside 4.41 ± 0.57 Flavanone 0.42 ± 0.02 Eriodictyol 0.42 ± 0.02 Dihydrochalcones 33.7 ± 3.08 Phloretin glucoside 21.7 ± 2.54 Phloretin xylosyl glucoside 11.7 ± 0.52 Hydroxyphloretin xylosyl glucoside 0.32 ± 0.03

TOTAL Phenols 196 ± 10.7

Table S3. Urinary excretion of phenolic compounds 0–24 h after acute intake of red-fleshed apple snack. Except for anthocyanin derivatives (nmol), data expressed in μmol as mean values ± standard deviation after subtraction of baseline excretion from each volunteer.

Phenolic metabolitea Basalb 0-2 h 2-4 h 4-8 h 8-24 h Catechol and pyrogallol derivatives Catechol sulphate (n=8) 21.7 ± 3.60 2.10 ± 0.90 1.86 ± 0.80 18.2 ± 8.44 30.5 ± 4.28 Methyl catechol sulphate (n=7) 16.6 ± 4.80 0.82 ± 0.18 0.10 ± 0.05 1.64 ± 1.33 34.0 ± 21.9 Methyl catechol glucuronide (n=6) 0.27 ± 0.05 0.03 ± 0.01 0.00 ± 0.00 0.03 ± 0.02 1.09 ± 0.75 Pyrogallol sulphate or phloroglucinol sulphate (n=5) 1.53 ± 0.40 0.05 ± 0.03 0.15 ± 0.07 1.20 ± 0.89 1.67 ± 0.31

Benzoic acid derivatives Hydroxybenzoic acid (n=3) 35.3 ± 10.3 18.3 ± 12.5 16.1 ± 8.01 15.4 ± 12.3 28.9 ± 21.1 Hydroxybenzoic acid sulphate (n=3) 125 ± 38.1 16.2 ± 9.17 8.17 ± 3.05 2.15 ± 2.15 131 ± 121 Protocatechuic acid sulphate (n=5) 16.0 ± 3.74 2.85 ± 0.88 11.5 ± 2.79 4.23 ± 1.83 8.85 ± 3.88 Vanillic acid sulphate (n=5) 36.3 ± 7.15 3.51 ± 1.74 14.5 ± 8.71 28.9 ± 28.9 90.3 ± 52.0 Vanillic acid glucuronide (n=3) 2.12 ± 0.74 0.15 ± 0.11 0.53 ± 0.15 1.65 ± 1.38 2.92 ± 0.91

Phenylacetic acid derivatives Hydroxyphenylacetic acid (n=4) 210 ± 26.3 8.59 ± 3.87 14.5 ± 5.59 73.5 ± 34.4 93.0 ± 37.9 Hydroxyphenylacetic acid sulphate (n=4) 52.0 ± 6.22 11.7 ± 5.32 6.20 ± 2.14 6.34 ± 3.68 47.4 ± 27.1 Hydroxyphenylacetic acid glucuronide (n=2) 2.03 ± 2.03 6.92 ± 0.66 3.69 ± 1.34 2.14 ± 0.18 4.53 ± 4.53 Dihydroxyphenylacetic acid sulphate (n=5) 24.6 ± 4.27 3.52 ± 1.30 2.01 ± 0.89 9.07 ± 4.16 20.7 ± 5.20 Dihydroxyphenylacetic acid glucuronide (n=4) 4.72 ± 0.26 0.90 ± 0.43 4.77 ± 2.62 5.37 ± 4.13 5.31 ± 2.81

Phenylpropionic acid derivatives Hydroxyphenylpropionic acid (n=6) 2.60 ± 0.88 1.86 ± 0.90 2.65 ± 0.98 3.79 ± 1.69 11.8 ± 4.35 Hydroxyphenylpropionic acid sulphate (n=7) 36.6 ± 11.1 4.35 ± 0.96 2.34 ± 0.52 13.4 ± 5.67 94.8 ± 42.2 Hydroxyphenylpropionic acid glucuronide (n=4) 1.57 ± 0.70 0.62 ± 0.41 0.13 ± 0.06 0.22 ± 0.06 3.40 ± 0.77 Dihydroxyphenylpropionic acid sulphate (n=5) 21.6 ± 2.83 2.86 ± 2.08 3.26 ± 1.61 22.4 ± 11.9 12.1 ± 3.75 Hydroxymethoxyphenylpropionic acid (n=5) 0.15 ± 0.04 0.02 ± 0.01 0.09 ± 0.04 0.29 ± 0.13 0.58 ± 0.22 Hydroxymethoxyphenylpropionic acid sulphate (n=2) 0.41 ± 0.16 0.09 ± 0.04 0.09 ± 0.01 0.48 ± 0.12 0.95 ± 0.42

Hydroxycinnamic acid derivatives Coumaric acid (n=5) 0.07 ± 0.05 0.32 ± 0.08 0.64 ± 0.12 1.28 ± 0.30 1.84 ± 0.43 Coumaric acid sulphate (n=9) 0.77 ± 0.26 0.49 ± 0.10 0.80 ± 0.17 1.48 ± 0.32 2.97 ± 0.63 Caffeic acid sulphate (n=2) 1.31 ± 0.91 0.51 ± 0.32 1.00 ± 0.74 1.30 ± 1.15 2.01 ± 0.92 Ferulic acid sulphate (n=10) 1.43 ± 0.41 0.29 ± 0.08 0.59 ± 0.11 0.74 ± 0.27 0.64 ± 0.24

Phenyl--valerolactone derivatives Hydroxylphenyl--valerolactone sulphate (n=8) 0.15 ± 0.15 0.02 ± 0.01 0.04 ± 0.02 1.21 ± 0.97 20.7 ± 17.1 Dihydroxylphenyl--valerolactone (n=8) 3.51 ± 2.25 0.98 ± 0.63 2.96 ± 1.91 15.9 ± 4.61 81.9 ± 32.0 Dihydroxylphenyl--valerolactone glucuronide (n=8) 0.01 ± 0.01 0.00 ± 0.00 0.01 ± 0.01 0.17 ± 0.07 1.12 ± 0.73 Dihydroxylphenyl--valerolactone sulphate glucuronide (n=7) 0.07 ± 0.07 0.06 ± 0.03 0.16 ± 0.08 1.34 ± 0.47 9.47 ± 5.97

Flavan-3-ol derivatives Epicatechin sulphate (n=6) 0.14 ± 0.14 0.40 ± 0.10 0.71 ± 0.11 0.52 ± 0.13 0.49 ± 0.06 Epicatechin glucuronide (n=6) n.d. 0.24 ± 0.07 0.37 ± 0.06 0.51 ± 0.28 0.32 ± 0.12 Methyl epicatechin glucuronide (n=7) 0.07 ± 0.07 0.23 ± 0.07 0.42 ± 0.08 0.36 ± 0.11 0.17 ± 0.06

Dihydrochalcone derivatives Phloretin glucuronide (n=10) n.d. 0.71 ± 0.10 1.26 ± 0.32 1.06 ± 0.25 0.15 ± 0.11 Phloretin sulphate glucuronide (n=8) 0.03 ± 0.01 0.12 ± 0.03 0.11 ± 0.03 0.09 ± 0.02 0.13 ± 0.04

Anthocyanin derivatives Cyanidin-3-O-galactoside (n=10) n.d. 2.49 ± 0.42 3.46 ± 0.63 2.88 ± 0.53 2.24 ± 1.38 Cyanidin arabinoside (n=10) 0.45 ± 0.07 0.74 ± 0.16 0.35 ± 0.11 0.35 ± 0.13 0.00 ± 0.00 Peonidin-3-O-galactoside (n=10) n.d. 0.82 ± 0.15 1.61 ± 0.25 1.22 ± 0.29 0.35 ± 0.17

a Figures in parenthesis next to compound names indicate the number of samples (volunteers) in which the metabolite was detected. b Content of urine collected for 12 h prior to supplementation and on an excretion per hour basis used to subtract from excretion values obtained after red-fleshed apple snack consumption to obtain the values cited in the Table. n.d.: not detected

Table S4. Phenolic compounds concentrations in plasma 0–24 h after acute intake of red-fleshed apple snack. Data expressed in nmol/L as mean values ± standard deviation

Phenolic compound a Basal (0 h) 0.5 h 1 h 2 h 4 h 6 h 24 h

Catechol and pyrogallol derivatives Catechol sulphate (n=5) 310 ± 59.2 367 ± 77.7 329 ± 62.2 389 ± 84.7 743 ± 243 1220 ± 434 373 ± 70.8 Methyl catechol sulphate (n=5) 133 ± 67.6 167 ± 79.3 195 ± 1075 159 ± 93.0 151 ± 64.5 260 ± 124 243 ± 71.3 Pyrogallol sulphate (n=7) 17.4 ±6.19 11.8 ± 4.84 11.6 ± 5.12 11.5 ± 5.26 17.1 ± 8.28 67.6 ± 21.0 16.3 ± 5.67

Benzoic acid derivative Hydroxybenzoic acid sulphate (n=3) 133 ± 44.1 179 ± 37.0 212 ± 52.4 132 ± 20.1 110 ± 21.3 144 ± 38.3 146 ± 52.5 Hydroxyhippuric acid (n=8) 13.5 ± 3.50 15.9 ± 5.68 21.3 ± 5.83 26.7 ± 7.57 27.8 ± 4.84 29.4 ± 7.33 15.8 ± 7.00 Protocatechuic acid sulphate (n=4) n.d. 19.5 ± 11.5 39.3 ± 3.15 29.2 ± 4.46 20.3 ± 10.7 16.4 ± 16.4 n.d. Vanillic acid glucuronide (n=9) 6.18 ± 2.52 10.7 ± 3.10 16.1 ± 2.61 23.6 ± 4.17 27.3 ± 3.25 23.9 ± 3.41 10.1 ± 2.54

Phenylacetic acid derivatives Hydroxyphenylacetic acid (n=5) 2178 ± 148 1946 ± 131 2049 ± 133 2577 ± 262 2788 ± 137 2993 ± 195 1820 ± 157 Hydroxyphenylacetic acid sulphate (n=6) 1016 ± 711 2047 ± 1120 2169 ± 1310 1398 ± 857 1607 ± 1166 1946 ± 1453 1523 ± 918 Dihydroxyphenylacetic acid glucuronide (n=3) 13.0 ± 13.0 23.3 ± 23.3 39.1 ± 20.3 63.2 ± 44.5 86.1 ± 17.6 84.6 ± 31.3 13.2 ± 13.2

Phenylpropionic acid derivatives Hydroxyphenylpropionic acid (n=4) 48.3 ± 48.3 55.4 ± 55.4 69.8 ± 69.8 265 ± 134 357 ± 186 897 ± 393 852 ± 432 Dihydroxyphenylpropionic acid sulphate (n=2) n.d. 8.52 ± 0.95 9.94 ± 1.10 18.3 ± 2.04 38.1 ± 4.23 30.4 ± 3.37 10.4 ± 1.16 Hydroxymethoxyphenylpropionic acid (n=5) 11.1 ± 3.58 12.9 ± 2.71 12.8 ± 2.17 25.5 ± 9.89 41.6 ± 8.77 40.5 ± 8.01 37.9 ± 17.3 Hydroxymethoxyphenylpropionic acid sulphate (n=2) n.d. 1.26 ± 1.26 0.99 ± 0.99 6.67 ± 0.98 11.2 ± 1.07 3.20 ± 3.20 2.87 ± 2.87

Hydroxycinnamic acid derivatives Coumaric acid (n=2) 143 ± 10.8 148 ± 24.8 171 ± 0.47 194 ± 24.2 165 ± 18.6 292 ± 30.5 157 ± 16.4 Caffeic acid sulphate (n=2) 8.21 ± 1.99 29.1 ± 14.3 37.5 ± 13.8 14.3 ± 8.06 11.8 ± 4.59 18.7 ± 4.78 13.0 ± 1.05 Ferulic acid sulphate (n=7) 18.2 ± 4.57 25.9 ± 4.76 25.9 ± 3.64 20.6 ± 3.09 24.8 ± 6.01 30.0 ± 3.45 17.7 ± 2.82

Dihydrochalcone derivatives Phloretin sulphate (n=2) n.d. 20.7 ± 16.3 18.4 ± 12.4 8.35 ± 5.01 8.43 ± 5.00 24.0 ± 4.18 10.9 ± 6.81 Phloretin glucuronide (n=10) n.d. 28.1 ± 3.54 46.7 ± 1.57 61.0 ± 6.82 53.9 ± 11.0 34.5 ± 11.7 1.16 ± 1.16

Anthocyanin derivatives Cyanidin-3-O-galactoside (n=7) n.d. 9.66 ± 2.26 9.15 ± 1.81 10.3 ± 1.50 6.86 ± 0.74 1.52 ± 0.82 0.30 ± 0.30 Cyanidin arabinoside (n=1) n.d. 2.60 n.d. n.d. n.d. n.d. n.d.

aFigures in parenthesis next to compound names indicate the number of samples (volunteers) in which the metabolite was detected. n.d.: not detected

Pyrogallol sulphate or phloroglucinol Catechol sulphate (189  109) Methyl catechol sulphate (203  123) Methyl catechol glucuronide (299  123) sulphate (205  125)

189.1 100 100 203.1 100 298.9 100 205.0 188.7 a) % a) a) a) 202.7 109.4 0 0 0 0 80 100 120 140 160 180 200 80 100 120 140 160 180 200 80 120 160 200 240 280 80 100 120 140 160 180 200 220

100 189.1 100 202.9 100 299.1 100 124.8 125.4 204.7 b) 123.2 b) b) 205.4 b) 109.10 188.6 202.6 81.0 113.0 124.5 0 0 0 0 80 100 120 140 160 180 200 80 100 120 140 160 180 200 80 120 160 200 240 280 80 100 120 140 160 180 200 220

100 109.1 100 123.1 100 124.9 113.2 298.7 100 125.3 125.0 122.8 85.1 108.8 c) c) c) 189.1 203.1 205.2 80.3 122.9 c)

0 0 0 205.4

Abundance Abundance (%)

Abundance Abundance (%) Abundance Abundance (%) 80 100 120 140 160 180 200 80 100 120 140 160 180 200 80 120 160 200 240 280 Abundance (%) 0 80 100 120 140 160 180 200 220 100 109.2 100 123.2 298.7 100 100 125.2 280.4 125.1 122.9 113.0 125.3 d) d) 280.1 d) 108.9 d) 205.2 80.2 0 0 0 0 80 100 120 140 160 180 200 80 100 120 140 160 180 200 80 120 160 200 240 280

100 109.0 280.3 100 123.1 100 100 124.9 e) 108.6 122.9 e) 125.0 e) e) 81.3 85.5 0 125.2 80 100 120 140 160 180 200 0 0 0 80 100 120 140 160 180 200 80 120 160 200 240 280 80 100 120 140 160 180 200 220 m/z m/z m/z m/z

Figure S1

Hydroxybenzoic acid (137  93) Hydroxybenzoic acid sulphate (217  137) Hydroxyhippuric acid (194  100) Protocatechuic acid sulphate (233  153)

137.1 100 100 217.0 100 100 233.3 193.9 232.6 a) a) a) a)

92.8 0 0 0 0 80 100 120 140 160 180 200 80 100 120 140 160 180 200 220 80 120 160 200 240 280 80 120 160 200 240 280

100 137.2 100 137.1 216.8 100 100 100.0 194.0 232.6 233.3 93.0 136.8 93.1 b) 217.4 b) b) b) 96.9 136.5 0 0 0 0 80 100 120 140 160 180 200 80 100 120 140 160 180 200 220 80 120 160 200 240 280 80 120 160 200 240 280 93.0 100 100 100 100 136.7 137.4 153.1 92.8 100.0 137.3 c) 93.2 c) c) c) 216.7

0 0 0 0

Abundance Abundance (%) Abundance (%) Abundance (%) Abundance Abundance (%) 80 100 120 140 160 180 200 80 100 120 140 160 180 200 220 80 120 160 200 240 280 80 120 160 200 240 280

100 93.1 100 137.0 100 100 93.3 92.8 152.8 92.7 98.2 109.3 92.9 d) d) d) d) 137.1 0 0 0 0 80 100 120 140 160 180 200 80 100 120 140 160 180 200 220 80 120 160 200 240 280 80 120 160 200 240 280 93.2 100 93.2 92.7 153.0 100 100 100 92.8 e) e) e) 109.1 e)

137.0 0 80 100 120 140 160 180 200 0 0 0 80 100 120 140 160 180 200 220 80 120 160 200 240 280 80 120 160 200 240 280 m/z m/z m/z m/z

Figure S1

Vanillic acid sulphate (247  167) Vanillic acid glucuronide (343  167) Hydroxyphenylacetic acid (151  107) Hydroxyphenylacetic acid sulphate (231  151) 151.1 100 100 100 100 231.1 247.2 343.3

a) a) % a) a) 150.9

0 0 0 0 80 120 160 200 240 280 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 80 120 160 200 240 280 320 360 400 151.2 100 100 100 100 230.8 247.1 166.8 166.9 103.1 123.0 b) 343.6 b) 107.1 150.9 b) 167.1 b)

0 0 0 0 80 120 160 200 240 280 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 80 120 160 200 240 280 320 360 400 107.3 100 100 100 100 166.8 123.2 167.1

123.3 112.96 107.0 123.2 c) c) c) 230.8 c) 166.7 247.3 123.3 151.1

0 0 0 0

Abundance Abundance (%)

Abundance Abundance (%) Abundance Abundance (%) Abundance Abundance (%) 80 120 160 200 240 280 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 80 120 160 200 240 280 320 360 400 107.3 100 100 100 100 167.1 166.9 123.0 106.9 152.2 123.4 d) d) d) 167.3 230.9 d) 166.7 123.0 151.7 106.8 0 0 0 0 80 120 160 200 240 280 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 80 120 160 200 240 280 320 360 400

123.3 167.5 100 100 100 107.3 100 123.3 98.8 152.3 e) e) 107.9 e) 122.9 152.0 106.9 e) 167.2 0 0 0 80 120 160 200 240 280 320 360 400 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 80 100 120 140 160 180 200

m/z m/z m/z m/z

Figure S1

Hydroxyphenylacetic acid glucuronide Dihydroxyphenylacetic acid sulphate (247  167) Dihydroxyphenylacetic acid glucuronide Hydroxyphenylpropionic acid (165  121) (343  167) (327  151) 227.2 246.7 165.1 100 100 100 100 343.2

a) a) a) % a) 164.9

0 0 0 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 327.3 165.2 100 167.0 100 100 246.2 100 166.9 246.7 343.0 167.2 b) 123.1 b) b) 121.1 b)

0 0 0 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 166.7 121.3 100 167.0 100 100 100 167.2

123.0 123.3 121.0 327.2 c) c) 123.2 c) c) 165.1

0 0 0 0

Abundance Abundance (%)

Abundance Abundance (%) Abundance (%) 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 80 120 160 200 240 280 320 360 400 Abundance (%) 80 100 120 140 160 180 200 121.2 100 100 100 166.7 100 167.2 123.1 120.9 123.2 d) 166.6 d) d) d) 123.2

0 0 0 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200

167.2 100 100 123.3 100 100 121.2 123.3 167.3 e) e) 98.6 e) 122.7 120.8 e) 167.2 0 0 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 80 120 160 200 240 280 320 360 400 0 80 100 120 140 160 180 200

m/z m/z m/z m/z

Figure S1

Hydroxyphenylpropionic acid sulphate Hydroxyphenylpropionic acid glucuronide Dihydroxyphenypropionic acid sulphate Dihydroferulic acid (195  136) (245 165) (341 165) (261 181) 100 100 100 100 244.8 341.2 260.99 195.1 a) a) a) a) 165.5 245.4 165.2 0 0 0 0 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400

100 100 341.3 100 100 245.4 261.0 195.2

244.8 b) b) b) 136.1 b) 165.0 165.2 181.3 0 0 0 0 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400

100 165.0 100 100 180.9 100 136.1 165.3 194.7 165.5 245.2 341.2 c) c) 261.1 c) 135.7 195.3 c) 121.2 244.8 121.2 137.3

0 0 0 0

Abundance Abundance (%)

Abundance Abundance (%) Abundance (%) 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 Abundance (%) 80 120 160 200 240 280 320 360 400

100 100 100 100 121.2 165.2 121.1 165.1 136.9 136.2 181.3 120.7 120.9 d) d) d) 136.0 d)

0 0 0 0 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400

165.2 100 136.9 135.7 100 100 165.0 100 121.3 e) e) 121.9 e) e) 121.2 181.2 104.3 136.1

0 0 0 0 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 440 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400

m/z m/z m/z m/z

Figure S1

Dihydroferulic acid sulphate (275  195) Coumaric acid (163  115) Coumaric acid sulphate (243  163) Caffeic acid sulphate (259  179)

163.2 259.1 100 100 100 243.1 100 274.9 a) a) 243.3 a) a)

0 0 0 0 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 220 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400

259.0 100 100 119.0 100 100 275.4 243.0 119.3 163.4 274.9 b) b) 163.2 b) 135.1 b) 118.9 0 0 0 0 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 220 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 135.2 100 119.3 100 100 195.2 100 163.0 274.7 243.1 c) 162.9 c) 259.2 c) 163.2 c) 119.2 117.2

0 163.4 0 0

Abundance Abundance (%)

Abundance Abundance (%) Abundance Abundance (%)

80 120 160 200 240 280 320 360 400 Abundance (%) 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 220 117.1 100 100 192.8 195.0 119.0 100 119.1 100 119.3 163.2 135.3 176.9 275.3 d) d) d) d) 118.1 93.1 163.1 0 0 0 0 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 220 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400

135.7 100 100 119.0 100 100 119.3 119.3 e) e) e) 136.3 93.2 e) 0 119.3 0 0 80 120 160 200 240 280 320 360 400 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 100 120 140 160 180 200 220

m/z m/z m/z m/z

Figure S1

Ferulic acid sulphate (273  193) Hydroxyphenyl--valerolactone sulphate Dihydroxyphenyl--valerolactone (207  163) Dihydroxyphenyl--valerolactone glucuronide (271  191) (383  207) 100 273.2 100 271.2 100 100 382.8 207.3 383.2 272.6 a) 271.7 a) a) a) 382.3 206.8 207.5 (383, 207, 163) 0 0 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 0 80 120 160 200 240 280 320 360 400 100 193.3 272.4 100 191.2 271.1 100 207.0 100 383.2 272.9 271.3 382.7 b) b) 207.4 b) 383.4 b) 206.6 207.0 0 0 0 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400

100 193.2 100 191.1 100 163.1 100 382.5 162.9 382.9 272.8 271.3 206.8 121.5 207.0 134.2 c) 147.3 c) 207.5 c) c) 148.8

0 0 0 0

Abundance Abundance (%)

Abundance Abundance (%) Abundance (%) 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 Abundance (%) 80 120 160 200 240 280 320 360 400

191.4 100 100 100 100 133.8 192.6 163.1 149.2 147.2 206.6 382.8 d) d) d) 135.3 d) 178.4 162.8 206.7 0 0 0 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400

178.0 163.2 133.9 100 149.1 100 147.1 100 100 135.4 163.2 84.9 e) e) e) 206.9 e) 191.3 134.3 207.0 163.5 0 0 0 0 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400

m/z m/z m/z m/z

Figure S1

Dihydroxyphenyl--valerolactone sulphate Epicatechin sulphate (369  289) Epicatechin glucuronide (465  289) Methyl epicatechin sulphate (383  303) glucuronide (463  287) 463.0 369.1 465.1 383.0 100 100 100 100

369.4 463.2 a) a) a)463.3 a)

0 0 0 0 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 440 480

463.2 465.0 383.1 100 100 369.1 100 100

289.2 369.3 287.1 b) b) 289.0 b) 303.2 b)

0 0 0 0 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 440 480

289.3 287.3 289.2 100 100 100 100 303.3

369.2 245.1 289.1 207.0 463.1 c) 245.1 c) 465.1c) 383.2 c)

0 0 0 0

Abundance Abundance (%)

Abundance Abundance (%) Abundance (%) 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 440 480 Abundance (%) 80 120 160 200 240 280 320 360 400 440 480

100 100 289.4 100 100 207.2 245.3 289.2 245.2 245.2 d) d) 289.1 d) 303.1 d) 287.4 369.1 0 0 0 0 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 440 480

207.1 100 100 245.3 100 100 e) 245.4 245.1 289.2 e) e) e) 289.0 0 0 0 0 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 440 480

m/z m/z m/z m/z

Figure S1

Phloretin glucuronide (449 273) Phloretin sulphate glucuronide (529  353) Cyanidin galactoside (449  287) Cyanidin arabinoside (419  287) 529.3 449.1 449.1 419.0 100 100 100 100 a) 449.4 a) 449.3a) 419.3 a)

0 0 0 0 80 120 160 200 240 280 320 360 400 440 480 100 200 300 400 500 600 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 440 480

449.0 449.2 419.0 100 100 353.2 529.3 100 100

273.2 b) b) 287.0 b) 287.0 b) 0 0 0 0 80 120 160 200 240 280 320 360 400 440 480 100 200 300 400 500 600 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 440 480

353.3 100 273.1 100 100 100 287.2 449.3 287.1 419.2 273.2 449.2 c) 529.1 c) c) c)

0 0 0 0

Abundance Abundance (%)

Abundance Abundance (%) Abundance (%) 80 120 160 200 240 280 320 360 400 440 480 100 200 300 400 500 600 80 120 160 200 240 280 320 360 400 440 480 Abundance (%) 80 120 160 200 240 280 320 360 400 440 480

273.1 273.0 100 100 100 100 287.3 287.2 353.1 d) d) 449.1 d) 419.0 d)

0 0 0 0 80 120 160 200 240 280 320 360 400 440 480 100 200 300 400 500 600 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 440 480 273.4 273.3 100 287.0 287.1 100 100 100 e) e) e) e)

0 0 0 0 80 120 160 200 240 280 320 360 400 440 480 100 200 300 400 500 600 80 120 160 200 240 280 320 360 400 440 480 80 120 160 200 240 280 320 360 400 440 480

m/z m/z m/z m/z

Figure S1

Peoniidin galactoside (463  301)

463.1 100 a) 463.4

0 80 120 160 200 240 280 320 360 400 440 480

463.3 100

301.1 b)

0 80 120 160 200 240 280 320 360 400 440 480

100 301.3 463.2 c) 0

Abundance Abundance (%) 80 120 160 200 240 280 320 360 400 440 480

100 301.1

463.2 d)

0 80 120 160 200 240 280 320 360 400 440 480

301.2 100 e)

0 80 120 160 200 240 280 320 360 400 440 480

m/z

Figure S1

Figure 2 Click here to download Figure: Revised_Figure 2_Yuste_et_al_JFF.ppt

A) B) 100 12 12 11 11 90 * 10 10 80 9 * 9 70

8 8

60 7 7

50 6 6

nmol/h µmol/h µmol/h 5 40 5 4 4 30 3 * 3 20 * 2 * 2 * 10 1 * * 1 * 0 0 0 basal 0-2 h 2-4 h 4-8 h 8-24 h basal 0-2 h 2-4 h 4-8 h 8-24 h

Phenylpropionic/phenylacetic acid derivatives Benzoic acid derivatives Dihydrochalcon derivatives Flavon-3-ol derivatives Catechol/pyrogallol derivatives Hydroxycinnamic acid derivatives Anthocyanin derivatives Phenyl-γ-valerolactones derivatives

Figure 2 Figure 3 Click here to download Figure: Revised_Figure 3_Yuste_et_al_JFF.ppt

A1) Cyanidin galactoside B1) Phloretin glucuronide C1) Epicatechin sulphate D1) Dihydroxyphenyl-- 2.5 1.0 0.5 8.0 valerolactone

2.0 0.8 0.4

6.0

/h 1.5 0.6 0.3 4.0

1.0 0.4 0.2

nmol

µmol/h

µmol/h µmol/h 0.5 0.2 0.1 2.0 0.0 0.0 0.0 0.0 0-2 h 2-4 h 4-8 h 8-24 h 0-2 h 2-4 h 4-8 h 8-24 h 0-2 h 2-4 h 4-8 h 8-24 h 0-2 h 2-4 h 4-8 h 8-24 h

0.5 A2) Cyanidin arabinoside B2) Phloretin sulphate glucuronide 0.3 C2) Methyl epicatechin sulphate D2) Dihydroxyphenyl-- 0.08 3.0 valerolactone sulphate

0.4 glucuronide

0.06

0.2 0.3 2.0 0.04

0.2

nmol/h µmol/h

0.1 µmol/h

µmol/h 1.0 0.1 0.02

0.0 0.00 0.0 0.0 0-2 h 2-4 h 4-8 h 8-24 h 0-2 h 2-4 h 4-8 h 8-24 h 0-2 h 2-4 h 4-8 h 8-24 h 0-2 h 2-4 h 4-8 h 8-24 h

A3) Peonidin galactoside C3) Epicatechin glucuronide D3) Dihydroxyphenyl-- 0.3 0.8 1.0 valerolactone glucuronide

0.8

0.2 0.6 0.6 0.4

nmol/h 0.4 µmol/h 0.1 µmol/h 0.2 0.2 0.0 0.0 0.0 0-2 h 2-4 h 4-8 h 8-24 h 0-2 h 2-4 h 4-8 h 8-24 h 0-2 h 2-4 h 4-8 h 8-24 h

0.10 D4) Hydroxyphenyl--

0.08 valerolactone sulphate

0.06

0.04 µmol/h Figure 3 0.02 0.00 0-2 h 2-4 h 4-8 h 8-24 h Figure 4 Click here to download Figure: Revised_Figure 4_Yuste_et_al_JFF.ppt

OH OH OH HO OH + HO O

Glucuronide-O O O-Galactoside OH Phloretin glucuronide Cyanidin galactoside

Red-fleshed 80 14

Apple snack 70 12 60 10 50 8 40 6 30 PLASMA O-CH3 20 4

OH Concetration (nM) Concetration Concetration (nM) Concetration 10 2 + 0 0 HO O 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 O-Galactoside Time (h) Time (h) OH Human Peonidin galactoside Acute Selected intake Intake biomarkers 0.9 2.5 0.8 1.0 2.0 0.7 0.8

0.6

1.5

0.5 0.6

mol nmol

µ 0.4 URINE 1.0 mol 0.3 n 0.4 0.2 0.5 0.2 0.1 0.0 0.0 0.0 basal 0-2 h 2-4 h 4-8 h 8-24 h basal 0-2 h 2-4 h 4-8 h 8-24 h basal 0-2 h 2-4 h 4-8 h 8-24 h Figure 1 Click here to download Figure: Figure 1_Yuste_et_al_JFF.ppt

OH UGT C-ring cleavage HO OH

HO CO2H O OH HO OH O O Phloretin glucuronide O O OH HO Glucuronide-O O Hydrolysis O OH (P,U) HO OH SULT HO-S-O HO O O-glycoside O OH O OH OH O HO OH O OH HO-S-O Dihydroxyphenylpropionic HO HO-S-O OH

HO HO Quercetin derivatives (A) HO O HO OH

O acid sulphate (P,U) O Chlorogenic acid (A) SULT Phloretin

Coumaric acid sulphate (U) Coumaric acid (P,U) Phloretin sulphate Glucuronide-O O glucuronide (U) Hydrolysis Glycoside-O O

O O dOH SULT

O Phloretin (xylosyl) glucoside (A) Ring fission Ring HO-S-O O fission Ring OH HO Colonic degradation O SULT HO OH O HO OH O OH Caffeic acid sulphate (P,U) HO dH HO Colonic degradation Caffeic acid Dihydroxyphenylpropionic acid UGT OH COMT COMT (dihydrocaffeic acid) O Dihydroxyphenyl-- O O OH O O COMT -oxidation valerolactone (U) HO-S-O SULT HO

OH dH O-glucuronide O OH dOH

CH3-O H3C-O Dihydroxyphenyl--valerolactone dOH Ferulic acid sulphate (P,U) Ferulic acid O glucuronide (U) HO O OH O O OH O O SULT HO SULT HO-S-O OH HO HO O O O O OH UGT OH SULT O H3C-O Hydroxyphenylpropionic Dihydroxyphenylacetic acid O HO-S-O O O-S-OH OH H3C-O acid (P,U)

O Dihydroferulic acid (P,U) Hydroxyphenyl--

O

Dihydroferulic acid

valerolactone CH -O O-glucuronide 3 sulphate (P,U)

Vanillic acid oxidation

- Dihydroxyphenyl--valerolactone

SULT SULT

UGT  sulphate (U) dOH SULT β-oxidation O sulphate glucuronide (U) O O O O HO OH OH HO-S-O OH Glucuronide-O OH Glucuronide-O OH OH O OH O O HO-S-O O O O O H3C-O Glucuronide-O HO HO HO H3C-O Hydrolysis O O UGT Dihydroxyphenylacetic Vanillic acid Vanillic acid Hydroxyphenylpropionic Hydroxyphenylpropionic Hydroxyphenylacetic Dihydroxyphenylacetic O-S-OH acid (P,U) acid sulphate (U) acid glucuronide (P,U) glucuronide (P,U) O acid glucuronide (U) acid sulphate (U) O Hydroxyphenyl--valerolactone Hexoside-O O -oxidation OH O-CH3 sulphate (U)

OH CH3-O OH OH COMT + Vanillic acid SULT HO O HO O Glucuronide O hexoside (A) p-Hydroxybenzoic acid (U)

OH OH

OH O-Galactoside dMe SULT O OH O dOH O OH OH HO-S-O HO-S-O Glucuronide-O O HO Peonidin Galactoside (U) O O (epi)catechin glucuronide (U)

p-Hydroxybenzoic acid Hydroxyphenylacetic Hydroxyphenylacetic Colonic degradation Glycination Glycination acid glucuronide (U) sulphate (P,U) acid sulphate (P,U) COMT O SULT O O OH OH OH OH O O-CH3 UGT O HO N OH OH B-ring HO-S-O O HO O OH HO O OH H O HO-S-O O OH + fission HO OH O HO O OH HO O OH O COMT SULT OH OH HO-S-O OH O HO O Hydroxyhippuric acid (P) OH HO OH O-Galactoside/ HO OH Hydrolysis Protocatechuic acid Protocatechuic acid HO HO Arabinoside HO OH OH sulphate (P,U) Methyl (epi)catechin sulphate (U) (epi)catechin sulphate (U) Epicatechin (A) OH Dimer (A) Cyanidin galactoside/arabioside UGT COMT

(A, U, P) Glucuronide-O Glucuronide-O oxidation - H3C-O  HO OH OH Catechol glucuronide HO SULT OH SULT HO COMT H3C-O Methyl catechol glucuronide (U) O HO HO O O HO-S-O HO-S-O HO-S-O HO HO O O O Pyrogallol sulphate (P,U) Pyrogallol Catechol Catechol sulphate (P,U) Methyl catechol sulphate (P,U) Figure 1 *Graphical Abstract

OH OH OH HO OH + HO O

Glucuronide-O O O-Galactoside OH Phloretin glucuronide Cyanidin galactoside

Red-fleshed 80 14

Apple snack 70 12 60 10 50 8 40 6 30 PLASMA O-CH3 20 4

OH Concetration (nM) Concetration Concetration (nM) Concetration 10 2 + 0 0 HO O 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 O-Galactoside Time (h) Time (h) OH Human Peonidin galactoside Acute Selected intake Intake biomarkers 0.9 2.5 0.8 1.0 2.0 0.7 0.8

0.6

1.5

0.5 0.6

mol nmol

µ 0.4 URINE 1.0 mol 0.3 n 0.4 0.2 0.5 0.2 0.1 0.0 0.0 0.0 basal 0-2 h 2-4 h 4-8 h 8-24 h basal 0-2 h 2-4 h 4-8 h 8-24 h basal 0-2 h 2-4 h 4-8 h 8-24 h *Conflict of Interest

CONFLICT OF INTEREST

On behalf of all authors, the corresponding author states that there is no conflict of

interest.

*Ethics Statement

ETHICS STATEMENT

The protocol of the study was approved by the Ethical Committee of the Human Clinical Research Unit at the Arnau Vilanova University Hospital, Lleida, Spain (Approval Number: 13/2016).