(C) Elsevier, 2019

Document downloaded from: http://hdl.handle.net/10459.1/65728 Copyright cc-by-nc-nd, (c) Elsevier, 2019 Està subjecte a una llicència de Reconeixement-NoComercial- SenseObraDerivada 4.0 de Creative Commons *Manuscript Clean version Click here to view linked References 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).

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