Antiadhesive Activity and Metabolomics Analysis of Rat Urine after

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Anti-adhesive Activity and Metabolomics Analysis of Rat Urine After Cranberry (Vaccinium macrocarpon Aiton) Administration Gregorio Peron, Pellizzaro Anna, Brun Paola, Elisabetta Schievano, Stefano Mammi, Stefania Sut, Ignazio Castagliuolo, and Stefano Dall'Acqua J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01856 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017

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Journal of Agricultural and Food Chemistry

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Anti-adhesive Activity and Metabolomics Analysis of Rat Urine after Cranberry (Vaccinium

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macrocarpon Aiton) Administration

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Gregorio Peron†, Anna Pellizzaro‡, Paola Brun‡, Elisabetta Schievano§, Stefano Mammi§,

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Stefania Sut†, Ignazio Castagliuolo‡, Stefano Dall’Acqua†*

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†

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5, 35131 Padova, Italy

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‡

Department of Molecular Medicine, University of Padova, Via Gabelli 63, 35121 Padova, Italy

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§

Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo

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*Corresponding author

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Address: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via

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Marzolo 5, 35131 Padova, Italy

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Phone number: +39 049 827 5332

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E-mail: [email protected]

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ABSTRACT

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Cranberry (Vaccinium macrocarpon Aiton) is used to treat non-complicated urinary tract

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infections (UTIs). A-type procyanidins (PAC-A) are considered the active constituents able to

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inhibit bacterial adhesion to the urinary epithelium. However, the role of PAC-A in UTIs is

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debated, because of their poor bioavailability, extensive metabolism, limited knowledge about

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urinary excretion and contradictory clinical trials. The effects of 35-day cranberry

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supplementation (11 mg/kg PAC-A, 4 mg/kg PAC-B) were studied in healthy rats using a UPLC-

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MS-based metabolomics approach. Microbial PAC metabolites, such as valeric acid and

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valerolactone derivatives, were related to cranberry consumption. An increased urinary excretion

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of glucuronidated metabolites was also observed. In a further experiment, urine samples were

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collected at 2, 4, 8 and 24 hours after cranberry intake and their anti-adhesive properties were

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tested against uropathogenic Escherichia coli. The 8 hours samples showed the highest activity.

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Changes in urinary composition were studied by UPLC-QTOF, observing the presence of PAC

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metabolites. The PAC-A2 levels were measured in all collected samples and the highest amounts,

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on the order of ng/mL, were found in the samples collected after 4 hours. Results indicate that the

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anti-adhesive activity against uropathogenic bacteria observed after cranberry consumption is

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ascribable to PAC-A metabolites rather than to a direct PAC-A effect, as the measured PAC-A

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levels in urine was lower than those reported as active in the literature.

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KEYWORDS

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Cranberry extract; anti-adhesive activity; E. coli; urine metabolomics; healthy rats.

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INTRODUCTION

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Cranberry (Vaccinium macrocarpon Aiton) juices, sauces and dried extracts are used as active

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ingredients of food supplements or nutraceuticals for the treatment of non-complicated urinary tract

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infections (UTIs). Cranberry fruits are characterised by the presence of phenolic acids, anthocyanins

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and A-type proanthocyanidins (PAC-A). The latter are oligomers of flavan-3-ol, featuring both C4β

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→ C8 and C2β → O7 interflavanoid bonds,1 and are considered the most important active

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compounds of the extract for the treatment of UTIs,1-5 because of their anti-adhesive properties

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against bacteria.1,

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activity are increasing. Some authors recently pointed out the role of other “simple” phenolic

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constituents, such as phenols and benzoic, phenylacetic and phenylpropionic acids, as anti-adhesive

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agents.3 Other researchers suggested that the effects of cranberry are related to the synergy of all its

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components and/or its metabolites rather than just PACs.8 Recent studies suggested that

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components of cranberry juice directly interact with P-fimbriae of uropathogenic bacteria, lowering

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their adhesive efficiency to urinary epithelium.9, 10 These anti-adhesive properties have caused many

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authors to consider cranberry as potentially useful to promote a healthy urinary tract,11-13 but a

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comprehensive understanding of its mode of action is still missing. Moreover, most of the studies

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on cranberry activity were performed in vitro, but many factors that may positively or negatively

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contribute to the in vivo effects were not taken into account; these factors are mainly related to

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pharmacokinetic processes such as absorption and metabolism of the phytocomplex. Recently,

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Feliciano and collaborators reported the identification and quantification of a large number of

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metabolites of polyphenols in urine and plasma of healthy men after the consumption of 450 mL of

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cranberry juice, showing extensive metabolism of the different phytochemicals.14 Previously,

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polyphenols and their metabolites were analyzed in human urine after cranberry-syrup

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consumption.15 Nevertheless, no activity data were related to the chemical evaluation of such

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metabolites.

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Nevertheless, because of poor bioavailability of PACs, doubts about their

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In a meta-analysis of clinical studies in women with recurrent UTIs, cranberry juice and cranberry

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dietary supplements were considered efficient to reduce symptomatic infections over a 12-months

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period.16 However, other reviews indicated that cranberry products are unlikely to prevent UTIs,

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except for specific patient sub-populations.17 These contrasting opinions can be related to different

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compositions of various cranberry preparations but also underline the need for new investigations

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on the mode of action of this phytochemical. In other studies, the effects of partially purified

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procyanidins from cranberry on urinary and plasmatic metabolome were monitored in rats using

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NMR- and UPLC-MS-based metabolomics.18, 19 These authors reported the presence of metabolites

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of cranberry constituents in plasma and urine after extract intake, demonstrating changes in these

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biofluids after cranberry administration.19 Moreover, they compared the changes of urinary

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composition induced by an oral administration of partially purified apple and partially purified

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cranberry procyanidins, showing that the consumption of cranberry procyanidins increased urinary

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excretion of hippurate but decreased the excretion of 3-(3′-hydroxyphenyl)-3-hydroxypropanoic

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acid, p-hydroxyphenylacetic acid and phenol compared to the apple preparation.18

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The same authors investigated the effects of cranberry or apple juices consumption on plasma and

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urine composition of young women,20 but again, none of these studies considered if the changes in

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biofluid composition could be responsible for changes in E. coli adhesive properties.

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Metabolomics is a useful tool to obtain information about dietary interventions or to assess the

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effects of food supplements on animal models and human subjects,21, 22 so it could be a valuable

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approach to study cranberry activity in vivo. In metabolomic studies, a great number of variables is

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considered allowing the complete metabolic profiling of biological samples. Moreover, the

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unbiased selection of variables that are significantly altered with a specific treatment allows one to

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discriminate between metabolites that are directly derived from the treatment and the endogenous

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ones.23 Metabolomic analysis of urine has several advantages compared to that of other biological

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fluids. First of all, urine contains metabolic end-products from the organism intended for excretion; 4 ACS Paragon Plus Environment

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therefore, compared to blood, it is more prone to biological variation.24 Furthermore, urine sampling

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is scarcely invasive and sample preparation is more straightforward because of the lower

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complexity and the minimal protein/peptide content.24

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In the present study, we used a metabolomic approach to evaluate the mode of action of cranberry

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against uropathogenic E. coli. Healthy Sprague-Dawley rats were orally supplemented with a

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standardized cranberry extract (100 mg/kg of extract, containing 15% of total PACs, every day) for

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35 days, to mimic a prolonged consumption of cranberry by healthy subjects. 24-hour urinary

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outputs were collected weekly at days 0, 7, 14, 21 and 35 during the experiment, and samples were

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subjected to UPLC-ESI-QTOF analysis using an untargeted approach. In a second experiment, a

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single dose of cranberry (oral gavage of 100 mg/kg of extract containing 15% of total PACs) was

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administered to animals (n = 6) and the changes of urinary composition at 2, 4, 8 and 24 hours after

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extract administration were monitored. Anti-adhesive properties of all the urine samples were

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studied. Furthermore, the markers related to cranberry intake were discovered using a multivariate

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data analysis approach. Among the markers, no unmodified PAC-A, but PACs intestinal

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metabolites were detected as significant variables at 8 hours after cranberry consumption. A specific

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chromatographic method was developed for the measurement of unmodified PAC-A in urine, and

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their amounts were in the range of ng/mL.

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RESULTS

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Metabolomic profiling of 24-h urine by UPLC-ESI-QTOF. 24-h urine samples collected during

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the 35 days of cranberry supplementation were analyzed by UPLC coupled with ESI-QTOF-MS,

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and urinary metabolite profiles of both treated and control animals were obtained. Mass

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spectrometric data were acquired both in positive and negative ion modes, in order to consider the

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largest number of metabolites, and two different datasets were obtained and used for multivariate

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analysis. Exploratory data analysis by PCA on urine samples collected at days 0, 7, 14, 21 and 35 5 ACS Paragon Plus Environment


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resulted in partial grouping of treated versus controls (Figure 1), supporting the changes of urine

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composition after oral administration of cranberry extract.

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Data modeling was subsequently performed by Partial Least Squares Discriminant Analysis (PLS-

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DA). The PLS-DA model for the “positive ion mode” dataset could explain 71% of the data

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variance using 3 components and the variance predicted was 36%. Y-axis intercepts after

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permutation test were R2 = (0.0, 0.408) and Q2 = (0.0, 0.216). On the other hand, the PLS-DA

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model built from the “negative ion mode” dataset could explain 80% of the data variance using 3

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components and the variance predicted was 53%. Y-axis intercepts after permutation test were R2 =

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(0.0, 0.468) and Q2 = (0.0, 0.308). Representative PLS-DA score scatter plots for the positive and

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negative datasets are reported in Figure 2 and 3, respectively. The plots show that urine samples

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collected during treatment and controls are grouped in two clusters, although they partially overlap.

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Time x mass variables contributing to group separation were selected on the basis of their variable

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importance on projection (VIP) value. Only variables bearing VIP values > 2.0 were considered

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significant in differentiating the two groups and were subsequently putatively identified. The

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selected variables are listed in Table 1A and 1B. Among the time x mass variables related to dietary

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intervention, none of them was identified as intact PAC-A, suggesting that only negligible amounts

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of such compounds are present in urine of the treated animals. However, two compounds, increased

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in urine after cranberry supplementation, were putatively identified as 4-hydroxy-5-(3'-

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hydroxyphenyl)-valeric acid-3'-O-sulfate and 5-(hydroxyphenyl)-gamma-valerolactone-O-sulfate,

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which can be related to the intestinal metabolism of PACs.25

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Other variables related to treatment were putatively identified as glucuronidated isoflavones,

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namely daidzein 4’-O-glucuronide and genistein 5-O-glucuronide, whose urinary amounts were

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significantly increased after cranberry supplementation. Conversely, lower amounts of free

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genistein and daidzein (introduced with the standard diet) were detected in treated rat urine as

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compared to controls. Genistein and daidzein were provided mainly by soy from dietary sources, as 6 ACS Paragon Plus Environment

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confirmed by HPLC-MS/MS analysis of fodder (data not shown). The same trend was observed for

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other two markers, namely 5-hydroxy-6-methoxyindole and 2,8-dihydroxyquinoline, a quinoline

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derivative found in urine of rats fed with fodder containing corn.26 Our data regarding induction of

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glucuronidation are in agreement with a previously published paper that demonstrated

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glucuronidation induction in ex vivo experiment.27

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Catechol sulfate, benzoyl glucuronide28 and hippuric acid29 are considered biomarkers of

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polyphenols intake formed by intestinal microbiota, and they have been reported in urine. Our data

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showed a significant increased amount of the first and the last metabolite after treatment, while

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values were not statistically significant for benzoyl glucuronide.

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Urinary amounts of tryptophan metabolites, namely xanthurenic and kynurenic acids and indoxyl

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sulfate, were influenced by treatment. Indoxyl sulfate, derived from tryptophan metabolisation by

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colonic bacteria,30 showed significant decrease suggesting an influence of cranberry consumption

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on intestinal microbial activity.

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Anti-adhesive properties of urine from animals treated with cranberry extracts. In order to

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study the anti-adhesive effects of urine samples after a single intake of cranberry, 6 healthy

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Sprague-Dawley rats were fed with a 100 mg/kg dose of cranberry extract (containing 15% total

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PACs) and urinary outputs were collected at 2, 4, 8 and 24 hours after treatment. Results showed a

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time dependent ability to decrease bacterial adhesion to epithelial cells (Figure 4). The adhesion of

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uropathogenic P-fimbriated E. coli incubated with urine samples collected at 2 and 4 hours after

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cranberry intake was not significantly changed compared to control urine. However, urine samples

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collected 8 hours after cranberry ingestion caused a significant reduction of E. coli adhesion to HT-

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29 cells. A 47% reduction in E. coli binding was still evident by incubating bacteria in urine

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collected 24 hours after cranberry consumption.

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Metabolomic profiling of urinary output at 2, 4, 8 and 24 hours after a single dose of

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cranberry. In order to evaluate the changes of urinary composition after a single intake of

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cranberry (100 mg/kg dose), the urinary outputs collected at 2, 4, 8 and 24 hours after treatment

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were analyzed by UPLC-QTOF. Control urine samples were collected from the same rats before

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treatment. Exploratory PCA analysis revealed limited differences, as reported in Figure 5 (R2 =

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0.306; Q2 = 0.202). Considering the significant anti-adhesive activity measured for the 8 hour

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samples, data modeling was performed by PLS-DA comparing control samples vs 8 hour collection.

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The resulting model could explain 99% of the data variance using 3 components and the variance

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predicted was 73%. Y-axis intercepts after permutation test were R2 = (0.0, 0.992) and Q2 = (0.0,

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0.333).

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Variables contributing to differentiate urine at 8 hours after treatment from control were identified

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as intestinal degradation products of PACs,25 specifically vanillic acid 4-sulfate, epicatechin, 5-

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(hydroxyphenyl)-gamma-valerolactone-O-sulfate, 5-(3’-hydroxyphenyl)-gamma-valerolactone, 4-

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hydroxy-5-(3'-hydroxyphenyl)-valeric acid-3'-O-sulfate and 5-(3',4'-dihydroxyphenyl)-gamma-

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valerolactone (Table 2). None of the significant variables for the 8 hours samples was identified as

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intact PAC.

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Quantification of PAC-A in urinary output at 2, 4, 6, 8 and 24 hours after single dose of

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cranberry. In order to evaluate the amounts of intact PAC-A in urinary outputs at 2, 4, 6, 8 and 24

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hours after treatment, a targeted HPLC-MS/MS method was used. The chromatographic method

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allowed the detection of PAC-A dimers, trimers and tetramers in urine (a representative

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chromatogram is reported in Figure 6). Because of the lack of reference standards for trimeric and

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larger oligomeric PAC-A, the only available standard PAC-A dimer (PAC-A2) was used for the

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quantification of dimers and an estimation of the amount of other oligomeric derivatives. Before

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HPLC-MS/MS analysis, urine was purified from low molecular weight polyphenols,32 in order to 8 ACS Paragon Plus Environment

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increase mass spectrometric sensitivity in PACs detection; the method allowed us to detect PAC-A2

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down to 0.2 ng/mL in urine. Measurements on urine samples collected 24 hours after the

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administration of cranberry extract resulted in an average PAC-A2 concentration of 1.58 ± 0.44

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ng/mL. The highest concentration of intact PAC-A2, 4.04 ± 2.87 ng/mL, was found at 4 hours after

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cranberry administration while in the control group, PAC-A was not detectable. Figure 7

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summarizes the results. No other PAC-A or PAC-B were revealed.

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DISCUSSION

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In this study, the effects of supplementation of healthy rats with a cranberry extract containing 15%

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PACs were observed by monitoring the changes of urinary metabolome. In a first experiment,

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healthy rats were orally supplemented with 100 mg/kg of cranberry extract daily (11.3 mg/kg PAC-

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A; 4.3 mg/kg PAC-B) for 35 days and 24-h urinary outputs were collected weekly. The metabolic

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composition of urine was explored by a UPLC-QTOF metabolomic approach. We observed

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changes in urine composition after treatment using both unsupervised and supervised techniques

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and markers related to treatment were identified. Significant induction of glucuronidation was

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observed, as demonstrated by increased amounts of isoflavone glucuronides and decreased amounts

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of aglyconic isoflavones in urine samples. These findings are in agreement with a previously

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published paper,27 which describes the treatment of Wistar rats with cranberry extracts in doses of

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0.5 mg for 14 days or 1.5 mg of proanthocyanidins/kg/day for 1 day.27 Results showed that both

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cranberry dosages significantly enhanced microsomal UDP-glucuronosyl transferase activity, and

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long-term consumption of a regular dose had more pronounced effects on enzymatic activity than a

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short-term overdose27.

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Thus, our data confirm that the induced glucuronidation activity should be further investigated,

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considering the large use of cranberry in food supplements and because of the possible use by

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subjects during other pharmacological treatments. 9 ACS Paragon Plus Environment


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Changes in metabolites related to tryptophan, such as xanthurenic and kynurenic acids and indoxyl

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sulfate (this latter related to colonic bacteria catabolism) were also observed, suggesting a role of

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cranberry constituents on microbiota activity.

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Increased levels of cranberry-derived metabolites were also detected, namely 4-hydroxy-5-(3'-

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hydroxyphenyl)-valeric acid-3'-O-sulfate and 5-(hydroxyphenyl)-gamma-valerolactone-O-sulfate.

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Conversely, none of the variables describing the effect of treatment were identified as intact PAC-

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A.

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In a second experiment, rats were fed with a single dose of cranberry extract and urine samples

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were collected after 2, 4, 8 and 24 hours. These samples were used to perform adhesion assays, by

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treating uropathogenic E. coli, showing the highest activity 8 hour after cranberry ingestion. In

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order to establish the role of intact PAC-A on activity, samples were analyzed using a specific

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targeted LC-MS/MS method. Urinary PAC-A2 levels were measured in the collected urine samples

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(at 2, 4, 8 and 24 hours after cranberry administration) of the treated group and the results showed

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levels in the range of ng/mL (the highest concentration detected was 4.04 ± 2.87 ng/mL at 4 hours

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after cranberry administration). The LC-MS/MS results indicated negligible quantities of

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unmodified compound detectable in the urine. Those levels are far from the concentration used in

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previous in vitro tests demonstrating the anti-adhesive properties of PAC-A. Foo and collaborators

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described the anti-adhesive effects of some PAC-A oligomers isolated from cranberry using an in

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vitro model and the activity was observed with concentrations higher than 0.3 mg/mL.1 Our data

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show that a single administration of 100 mg/kg of extract (15 mg/kg of PACs) yielded in a

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maximum amount of 4.04 ± 2.87 ng/mL 4 hours after cranberry intake. This amount is six orders of

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magnitude lower than the previously published data.1 Gupta and collaborators observed a dose

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dependent anti-adhesive activity of cranberry in an in vitro model in the range of 5 to 74 µg/mL,

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still much higher than the values we measured.5 In a previously published paper, female Fischer-344

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rats were treated for 10 months with cranberry extract via gavage (1 g/kg body weight, 1 10 ACS Paragon Plus Environment

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gavage/day, five days a week) and urine samples were collected at 18, 25, 42, 48 hours after

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cranberry administration (1 gavage).33 The authors reported that intact PAC-A2 was detected at

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ng/mL, although the exact concentration was not specified.33

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In our experiment, an untargeted metabolomics approach revealed slight differences among urine

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samples collected at different times after cranberry administration. Furthermore, the results of

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adhesion assays, performed by treating uropathogenic E. coli with the same urine samples, showed

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the highest activity 8 hour after cranberry ingestion. Significant differences in the composition of

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urine samples collected at 8 hours with respect to control samples were observed. Among the

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identified metabolites, kaempferol glucuronide, equol glucuronide, daidzein and genistein

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glucuronides were the most discriminant ones, confirming the induction of glucuronidation

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observed in the first dataset. Furthermore, significant descriptors related to PACs and phenolic

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metabolites were identified, namely vanillic acid 4-sulfate, epicatechin, 5-(hydroxyphenyl)-gamma-

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valerolactone-O-sulfate,

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hydroxyphenyl)-valeric

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These latter metabolites derived from intestinal degradation of procyanidins,25 showing their

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extensive microbial degradation prior to absorption in the intestinal lumen.

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In conclusion, metabolomics was a valuable tool to explore cranberry bioactivity. The changes

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induced by oral consumption of cranberry extract were explored in urine considering their entire

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metabolic composition. An untargeted approach showed that procyanidins from cranberry are

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poorly absorbed by the intestine and are extensively metabolized by gut microbiota, leading to the

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excretion of negligible amounts of intact PAC-A; considering this, PAC-A cannot be directly

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related to the anti-adhesive effects of cranberry in vivo. Furthermore, an influence on the

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glucuronidation of isoflavones was observed in the treated group, supporting an induction of

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glucuronidation after cranberry administration. The anti-adhesive activity of urine samples collected

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at different times after oral cranberry administration was studied and the highest anti-adhesive

5-(3’-hydroxyphenyl)-gamma-valerolactone, acid-3'-O-sulfate

and

4-hydroxy-5-(3'-

5-(3',4'-dihydroxyphenyl)-gamma-valerolactone.



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activity was observed for the sample collected 8 hours after gavage. Metabolomic analysis revealed

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the presence of polyphenol-derived microbial catabolites. These metabolites could play a crucial

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role against E. coli adhesion to epithelial cells and they can be, at least in part, responsible for

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cranberry activity.

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EXPERIMENTAL SECTION

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Materials. Cranberry dry extract was purchased from a local market. The powder extract used in

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both sets of experiments was a homogeneous batch. Standard PAC-A2, Sephadex LH-20 and

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epigallocatechin gallate were purchased from Sigma Aldrich. HPLC-grade acetonitrile and formic

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acid were purchased from Sigma Aldrich. Deionized water used in HPLC and UPLC analysis was

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filtered through a Milli-Q system equipped with a 0.22 µm cut-off filter (Millipore).

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Profiling of PACs in cranberry extract. 30 mg of dried cranberry extract were suspended in 5 mL

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of an acetone/water (70/30% v/v) mixture and were extracted using an ultrasound bath for 15 min.

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The sample was centrifuged at 3,500 rpm for 10 min and the liquid was collected in a flask. The

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solid residue was re-extracted with further 5 mL of solvent and sonicated for further 15 min. The

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extraction was repeated 4 times. The liquids were collected in a round bottom flask and the volume

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was reduced under vacuum at 30 °C to 5 mL. The liquid was then transferred to a volumetric flask

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and the volume was adjusted to 25 mL with methanol.

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Analyses of PAC-A and PAC-B were performed by HPLC-MS. The chromatographic system was

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composed by a Varian 212 binary pump coupled to a Varian 500-MS IT mass spectrometer (MS). A

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Tosoh TSKgel Amide-80 column (3.5 µm, 2.1 x 150 mm) was used as stationary phase. 0.5%

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formic acid in acetonitrile (A) and 2% formic acid in water (B) were used as mobile phase. Gradient

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elution was as follows: starting with 90% A, then in 20 min to 80% A, isocratic conditions (80% A)

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until 25 min, then to 35% A in 45 min and isocratic (35% A) until 51 min. Finally, the column was 12 ACS Paragon Plus Environment

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left to re-equilibrate to the initial conditions for 9 min. The flow rate was 200 µL/min and the

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injected volume was 10 µL. The MS parameters were as follows: spray chamber temperature, 50

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°C; nebulizer gas pressure, 25 psi; drying gas pressure, 25 psi; drying gas temperature, 385 °C;

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needle voltage, ± 4500 V; spray shield voltage, 600 V. Mass spectra were acquired in negative

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mode in the spectral range 150-2000 Da. For PAC-A, [M-H]- ions at m/z 575, 863 and 1151 were

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monitored for dimers, trimers and tetramers, respectively. For PAC-B, [M-H]- ions at m/z 577, 865

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and 1153 were monitored for dimers, trimers and tetramers, respectively. Because other trimeric or

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tetrameric procyanidins are not commercially available, PAC-A2 was used as standard compound

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for quantitative purposes. A calibration curve was built in the range 1-20 µg/mL, and all samples

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and calibration solutions were analysed in triplicate. The curve obtained was fitted with the linear

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function y = 0.6915x (R2 = 0.9989), showing a good linear response. Limit of detection (LOD =

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0.45 µg/mL) and limit of quantification (LOQ = 0.7 µg/mL) were determined following USP

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methods. For LOD, samples with known concentration of PAC-A2 were analysed and the minimum

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concentration at which the analyte could be reliably detected was established. On the other hand,

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LOQ was established by visual evaluation.

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The total PAC-A content was 11.3 ± 1.3% w/w of dry cranberry extract, whereas the total content

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of PAC-B was 4.3 ± 0.4% w/w. PAC-A dimers constituted the 4.5 ± 0.7% w/w of dry extract,

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trimers were 3.6 ± 0.2% w/w and tetramers 3.2 ± 0.2% w/w. PAC-B dimers constituted the 1.3 ±

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0.3% w/w of dry extract, trimers were 1.8 ± 0.1% and tetramers 1.2 ± 0.1% w/w.

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Animal procedures. All experimental protocols were approved by the Animal Care and Use Ethics

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Committee of the University of Padova (number 3112017PR) under license from the Italian

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Ministry of Health and they were in compliance with the National and European guidelines for

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handling and use of experimental animals. In the first experiment, twelve healthy Sprague-Dawley

325

rats (six males and six females) of 7 ± 2 weeks of age and weighing 173.0 ± 3.1 g, were used. 13 ACS Paragon Plus Environment


326

Animals were fed standard laboratory diet and had free access to water during the experiments. Rats

327

were maintained in a temperature- and photoperiod- controlled room (12-hrs light/dark cycle). Rats

328

were divided randomly in a cranberry treated group (three males and three females) and a control

329

group (three males and three females). No differences were observed between control and treated

330

groups at the beginning of the experiment. The treated group received, by intra-gastric gavage, a

331

daily dose of 100 mg/kg body weight of cranberry extract in 2 mL of water for a total of 35 days.

332

The control group received daily an identical volume of water by intra-gastric gavage. Rats were

333

housed individually in metabolic cages and 24-hrs urine output was collected and stored at -80 °C

334

to avoid chemical degradation. Urine samples were collected on days 0, 7, 14, 21 and 35 during the

335

experiment and subjected to UPLC-ESI-QTOF analysis.

336

A second experiment was performed, involving six healthy Sprague-Dawley rats (three males and

337

three females), 6 ± 1 weeks of age and weighing 125 ± 7.4 g. They were orally given a single dose

338

of 100 mg/kg of 15% PACs cranberry extract suspended in 1 mL of water by gavage. Before

339

treatment, animals were individually positioned in metabolic cages to collect 24-h control urine.

340

After cranberry administration, urinary outputs at 2, 4, 6, 8 and 24 hours were collected following

341

the same procedure. Urine samples were then stored at -80 °C until used for UPLC-ESI-QTOF and

342

HPLC-MS/MS analyses and for adhesion studies, to avoid chemical degradation.

343 344

Metabolomic profiling of 24-h urine by UPLC-ESI-QTOF. To obtain a metabolic profiling of

345

urine, a UPLC-HRMS full-scan method was used. An Agilent 1290 Infinity UPLC system equipped

346

with a Waters Xevo G2 Q-TOF MS was employed. The detector was equipped with an electrospray

347

(ESI) ionisation source and was operating in both positive and negative resolution mode. The

348

sampling cone voltage was adjusted at 40 V, the source offset at 80 V. The capillary voltage was

349

adjusted to 2500 V. The nebulizer gas used was N2 at a flow rate of 800 L/h. The desolvation

350

temperature was 450 °C. The mass accuracy and reproducibility were maintained by infusing 14 ACS Paragon Plus Environment

Page 14 of 38

Page 15 of 38


+

= 556.2766 m/z and [M-H]

−

351

lockmass (leucine–enkephalin, [M+H]

352

Lockspray at a flow rate of 20 µL/min. Centroided data were collected for each sample in the mass

353

range 50 – 1200 Da, and the m/z value of all acquired spectra was automatically corrected during

354

acquisition based on lockmass.

355

An MSe experiment was simultaneously performed, setting the collision energy to 30 V. MSe

356

fragmentation spectra were subsequently used for variables putative identification.

357

An Agilent XDB C-8 column (2.1 mm x 150 mm, 3.5 µm) was used as stationary phase. The

358

mobile phase was composed of solvent A (0.1% formic acid in acetonitrile) and solvent B (0.1%

359

formic acid in water). Linear gradients of solvents A and B were used, as follows: 0 min, 8% A; 14

360

min, 48% A; 16 min, 100% A; 17 min, 100% A; 17.5 min, 8% A; 24 min, 8% A. The flow rate was

361

200 µL/min and the injection volume was 1 µL. Collected urine samples were centrifuged at 13,000

362

rpm for 10 min prior to analysis and directly injected in the UPLC. Quality control samples (QC, n

363

= 6) were prepared mixing 100 µL of every collected urine sample and they were centrifuged at

364

13,000 rpm for 10 min. Sequence of injection comprising pool, control and treated samples were

365

randomized in order to avoid any bias due to samples order.

366

Centroided and integrated chromatographic mass data were processed by MarkerLynx Applications

367

Manager version 4.1 (Waters) to generate a multivariate data matrix. An appropriate method for

368

data deconvolution, alignment and peak detection was created. The parameters used were retention-

369

time range 2.00–17.00 min, mass range 50–1200 Da, mass tolerance 0.01 Da. Noise elimination

370

level was set to 6.00, minimum intensity was set to 15% of base peak intensity, maximum masses

371

per RT was set to 6 and, finally, RT tolerance was set to 0.01 min. Isotopic peaks were excluded

372

from analysis. A list of the ion intensities of each peak detected was generated, using retention time

373

and the m/z data pairs as the identifier for each ion. The resulting three-dimensional matrix contains

374

arbitrarily assigned peak index (retention time-m/z pairs), sample names (observations), and ion

375

intensity information (variables). Variables having more than 30% of missing data in both groups 15 ACS Paragon Plus Environment

= 554.2620 m/z) thorough


376

were excluded. Data modelling was performed using SIMCA 13 software (Umetrics) applying

377

multivariate techniques based on projection. Principal Component Analysis (PCA) was used for

378

exploratory data analysis and for identification of outliers. Projection to Latent Structures by partial

379

least squares regression (PLS) was applied to investigate the relationships between the urine

380

composition and the cranberry administration. The measured variables and the matrix were used as

381

X-block and Y-block, respectively, in the PLS regression. Following good practice for model

382

validation, N-fold full cross-validation with different values of N (N = 6,7,8) and permutation test

383

on the responses (999 random permutations) were performed, in order to avoid overfitting and

384

prove the robustness of the obtained models as we used in other similar experiments.34

385

Biomarkers were identified calculating the molecular formula by high-resolution m/z values of

386

protonated and deprotonated pseudomolecular ions ([M+H]+ and [M-H]-), using MassLynx

387

Elemental Composition tool and searching for both the calculated molecular formula and accurate

388

m/z in freely available web databases (i.e. Human Metabolome Database, Metlin, MassBank).

389

Possible candidates were then screened comparing the MSe fragmentation spectra for variables

390

identification.

391 392

Metabolomic profiling of urinary outputs at 2, 4, 8 and 24 hours after cranberry

393

administration. To obtain a metabolic profiling of urine collected at 2, 4, 8 and 24 hours after

394

cranberry administration, a UPLC-ESI-QTOF full-scan method was used. The instrumentation

395

employed was the same described for metabolomic profiling of 24-h urine, as well as the settings of

396

the ESI-Q-TOF mass detector. An Agilent Zorbax Rapid Resolution High Definition (RRHD) SB-

397

C18 column (2.1 mm x 50 mm, 1.8 µm) was used as stationary phase and the mobile phase was

398

composed of solvent A (acetonitrile with 0.1% formic acid) and solvent B (water with 0.1% formic

399

acid). Linear gradients of solvents A and B were used, as follows: 0 min, 2% A; 1 min, 2% A; 8

400

min, 55% A; 10 min, 55% A; 10.5 min, 98% A; 11.5 min, 98% A; 12 min, 2% A and isocratic for 1 16 ACS Paragon Plus Environment

Page 16 of 38

Page 17 of 38


401

min. The flow rate was 300 µL/min and the injection volume was 1 µL. Samples were pre-treated in

402

the same way as for 24-h urine analysis and QC samples (n = 5) were prepared and randomly

403

injected in the UPLC.

404

Data extraction was performed as previously described, using MarkerLynx Applications Manager

405

version 4.1 (Waters) and setting time-range between 1.00–10.00 min; the obtained matrix was then

406

elaborated using SIMCA 13 software.

407 408

HPLC-MS/MS determination of oligomeric PAC-A in treated animal urine. Urine samples

409

were purified prior to analysis to isolate PACs from lower molecular weight polyphenols and

410

increase sensitivity of mass spectrometric analysis. One mL of urine samples were added with 10

411

µL of a 10 µg/mL epigallocatechin gallate solution (equivalent to 100 ng of epigallocatechin

412

gallate), used as internal standard (ISTD). Samples were loaded on a 20 x 3 cm glass column pre-

413

packed with 400 mg of Sephadex LH-20, which was left to equilibrate in 70% MeOH in water for

414

30 min prior to sample loading. The loaded resin was then washed with 4 mL of 70% MeOH in

415

water to elute low molecular weight polyphenols. Subsequently, the resin was washed with 5 mL of

416

70% acetone in water to elute PACs. The acetone fraction was evaporated under vacuum to dryness

417

and the residue was dissolved with 100 µL of methanol for analysis. Calibration curves using

418

different amount of ISTD and PAC-A2 were obtained, namely adding to 1 mL of urine 100 ng of

419

ISTD and 2, 4, 8, 10, 20 ng of PAC-A2. Urine samples with added ISTD were purified through

420

Sephadex as described and final solutions used to build a calibration curve. Calibration curve (y =

421

quantity of ISTD/quantity of PAC; x = area ISTD/area PAC) was the following: y = 0.3683x (R2 =

422

0.9991). LOD and LOQ were 0.2 and 0.4 ng/mL, respectively, and they were established using USP

423

methods, as previously described.

424

An Agilent 1260 Infinity HPLC binary pump coupled to a Varian 320-MS TQD MS was employed.

425

The detector was equipped with an ESI ionisation source and was operating in negative mode. The 17 ACS Paragon Plus Environment


426

MS conditions were as follows: spray chamber temperature, 50 °C; nebulizer gas pressure, 25 psi;

427

drying gas pressure, 25 psi; drying gas temperature, 385 °C; needle voltage, ±4500 V; spray shield

428

voltage, 600 V. Mass spectra were acquired in negative mode in the spectral range 150-2000 Da.

429

MRM traces were used for quantitation purposes. For PAC-A dimer, the m/z transition 575-285 was

430

monitored, corresponding to the fragmentation of [M-H]- parent ion. m/z transitions monitored for

431

trimers and tetramers were 863-289 and 1151-285, respectively, corresponding to the

432

fragmentations of [M-H]- parent ions. A Tosoh TSKgel Amide-80 column (2.1 mm x 150 mm, 3.5

433

µm) was used as stationary phase. The mobile phase was composed of solvent A (0.1% formic acid

434

in water) and solvent B (0.1% formic acid in acetonitrile). Linear gradients of solvents A and B

435

were used, as follows: 0 min, 8% A; 14 min, 48% A; 16 min, 100% A; 17 min, 100% A; 17.5 min,

436

8% A; 24 min, 8% A. The flow rate was 200 µL/min and the injection volume was 10 µL.

437 438

Microbiology and in vitro adhesion assays. A freshly isolated uropathogenic P-fimbriated E. coli

439

sample from a woman with urinary tract infection (UTIs) was used in the adhesion assays. The

440

bacteria were cultured in trypticase soy broth, collected by centrifugation and adjusted to a

441

concentration of 107 CFU/mL. Then, bacteria were incubated in urine samples, from control or

442

animals treated with cranberry extract, at 105 CFU/mL final concentration for 4 h at 37 °C. Bacteria

443

were then harvested from the urine samples by centrifugation (2,500 rpm x 10 min), suspended in

444

DMEM at a concentration of 5 x 108 bacteria/mL and quantified by vital count assay seeding on LB

445

agar plates. The remaining microbes were added to human intestinal epithelial cells (HT-29 cells,

446

from ATCC) monolayers (MOI 1:10). Tissue cultures were incubated with E. coli for 30 min at 37

447

°C. Non-adhering microbes were rapidly removed by extensive washes with warm DMEM. Then,

448

to quantify the adhering E. coli to HT-29 cells, they were detached, collected and lysed. Living

449

bacteria were quantified by vital count assay. Serial dilutions were seeded on LB agar plates,

450

incubated at 37 °C for 24 hours and CFU enumerated. 18 ACS Paragon Plus Environment

Page 18 of 38

Page 19 of 38


451 452

AKNOWLEDGMENT

453

The authors gratefully acknowledge funding from the University of Padova (PRAT CPDA118080).

454 455

CONLICTS OF INTEREST

456

The authors declare no competing financial interests.

457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472



473 474

REFERENCES

475

1) Foo, L. Y.; Lu, Y.; Howell, A. B.; Vorsa, N. A-type proanthocyanidin trimers from

476

cranberry that inhibit adherence of uropathogenic P-fimbriated Escherichia coli. J. Nat.

477

Prod. 2000, 63, 1225-1228.

478

2) Feliciano, R. P. et al. Comparison of isolated cranberry (Vaccinium macrocarpon Ait.)

479

proanthocyanidins to catechin and procyanidins A2 and B2 for use as standards in the 4-

480

(dimethylamino)cinnamaldehyde assay. J. Agric. Food Chem. 2012, 60, 4578-4585.

481

3) De Llano, D. G. et al. Anti-adhesive activity of cranberry phenolic compounds and their

482

microbial-derived metabolites against uropathogenic Escherichia coli in bladder epithelial

483

cell cultures. Int. J. Mol. Sci. 2015, 16, 12119-12130.

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4) Guay, D. R. P. Cranberry and urinary tract infections. Drugs 2009, 69, 775-807.

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5) Gupta, K. et al. Cranberry Products Inhibit Adherence of P-Fimbriated Escherichia coli to

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Primary Cultured Bladder and Vaginal Epithelial Cells. J. Urol. 2007, 177, 2357-2360.

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6) Howell, A. B.; Vorsa, N.; Marderosian, A. D.; Foo, L. Y. Inhibition of the adherence of P-

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fimbriated Escherichia coli to uroepithelial-cell surfaces by proanthocyanidin extracts from

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cranberries. New Engl. J. Med. 1998, 339, 1085-1086.

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7) Foo, L. Y.; Lu, Y.; Howell, A. B.; Vorsa, N. The structure of cranberry proanthocyanidins

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which inhibit adherence of uropathogenic P-fimbriated Escherichia coli in vitro.

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Phytochemistry 2000, 54, 173-181.

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8) Vostalova, J. et al. Are high proanthocyanidins key to cranberry efficacy in the prevention of recurrent urinary tract infection? Phytother. Res. 2015, 29, 1559-1567.

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9) Liu, Y.; Black, M. A.; Caron, L.; Camesano, T. A. Role of cranberry juice on molecular-

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scale surface characteristics and adhesion behavior of Escherichia coli. Biotechnol. Bioeng.

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2006, 93, 297-305.


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10) De Llano, D. G. et al. Anti-adhesive activity of cranberry phenolic compounds and their

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microbial-derived metabolites against uropathogenic Escherichia coli in bladder epithelial

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cell cultures. Int. J. Mol. Sci. 2015, 16, 12119-12130.

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11) Guay, D. R. P. Cranberry and urinary tract infections. Drugs 2009, 69, 775-807.

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12) Vasileiou, I.; Katsargyris, A.; Theocharis, S.; Giaginis, C. Current clinical status on the

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preventive effects of cranberry consumption against urinary tract infections. Nutr. Res.

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2013, 33, 595-607.

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13) Zirk, M. M.; Aluko, R. E.; Taylor, C. G. Cranberry (Vaccinium macrocarpon)

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proanthocyanidins and their effects on urinary tract infections. Curr. Top. Nutraceutical Res.

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2004, 2, 153-160.

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14) Feliciano, R. P. et al. Identification and quantification of novel cranberry-derived plasma and urinary (poly)phenols. Arch. Biochem. Biophys. 2016, 599, 31-41. 15) Iswaldi, I. et al. Identification of polyphenols and their metabolites in human urine after cranberry-syrup consumption. Food Chem. Toxicol. 2013, 55, 484-492.

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16) Wang, C. et al. Cranberry-containing products for prevention of urinary tract infections in

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susceptible populations: A systematic review and meta-analysis of randomized controlled

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trials. Arch. Intern. Med. 2012, 172, 988-996.

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17) Jepson, R. G.; Williams, G.; Craig, J. C. Cranberries for preventing urinary tract infections. Cochrane Database Syst. Rev. 2012, 10, 1-80.

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18) Liu, H.; Tayyari, F.; Edison, A. S.; Su, Z.; Gu, L. NMR-based metabolomics reveals urinary

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metabolome modifications in female Sprague-Dawley rats by cranberry procyanidins. J.

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Nutr. Biochem. 2016, 34, 136-145.

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19) Liu, H.; Garrett, T. J.; Tayyari, F.; Gu, L. Profiling the metabolome changes caused by

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cranberry procyanidins in plasma of female rats using 1H NMR and UHPLC-Q-Orbitrap-

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HRMS global metabolomics approaches. Mol. Nutr. Food Res. 2015, 59, 2107-2118. 21 ACS Paragon Plus Environment


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20) Liu, H.; Tayyari, F.; Khoo, C.; Gu, L. A 1H NMR-based approach to investigate

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metabolomic differences in the plasma and urine of young women after cranberry juice or

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apple juice consumption. J. Funct. Foods. 2015, 14, 76-86.

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21) Gibney, M. J. et al. Metabolomics in human nutrition: Opportunities and challenges. Am. J. Clin. Nutr. 2005, 82, 497-503. 22) Sut, S.; Baldan, V.; Faggian, M.; Peron, G.; Dall’Acqua, S. Nutraceuticals, a New Challenge for Medicinal Chemistry. Curr. Med. Chem. 2016, 23, 1-26.

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23) Lan, K.; Xie, G. & Jia, W. Towards polypharmacokinetics: Pharmacokinetics of

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multicomponent drugs and herbal medicines using a metabolomics approach. Evid. Based

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Complement. Altern. Med. 2013, 1-12.

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24) Kemperman, R. F. J. et al. Comparative urine analysis by liquid chromatography-mass

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spectrometry and multivariate statistics: Method development, evaluation, and application to

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proteinuria. J. Proteome Res. 2007, 6, 194-206.

536 537 538 539

25) Monagas, M. et al. Insights into the metabolism and microbial biotransformation of dietary flavan-3-ols and the bioactivity of their metabolites. Food. Funct. 2010, 1, 233-253. 26) Inagami, K.; Kaihara, M.; Price, J. M. The identification of 2,8-quinolinediol in the urine of rats fed a diet containing corn. J. Biol. Chem. 1965, 240, 3682-3684.

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27) Bártíková, H. et al. Effect of standardized cranberry extract on the activity and expression of

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selected biotransformation enzymes in rat liver and intestine. Molecules 2014, 19, 14948-

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14960.

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28) Pimpão, R. C.; Ventura, M. R.; Ferreira, R. B.; Williamson, G.; Santos, C. N. Phenolic

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sulfates as new and highly abundant metabolites in human plasma after ingestion of a mixed

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berry fruit purée. Br. J. Nutr. 2015, 113, 454-463.

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29) Lees, H. J.; Swann, J. R.; Wilson, I. D.; Nicholson, J. K.; Holmes, E. Hippurate: The natural

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history of a mammalian-microbial cometabolite. J. Proteome Res. 2013, 12, 1527-1546. 22 ACS Paragon Plus Environment

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30) Wikoff, W. R. et al. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 3698-3703. 31) Rzeppa, S.; Bittner, K.; Döll, S.; Dänicke, S.; Humpf, H. Urinary excretion and metabolism of procyanidins in pigs. Mol. Nutr. Food Res. 2012, 56, 653-665.

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32) Wallace, T. C.; Giusti, M. M. Extraction and normal-phase HPLC-fluorescence-electrospray

553

MS characterization and quantification of procyanidins in cranberry extracts. J. Food Sci.

554

2010, 75, 690-696.

555 556

33) Rajbhandary, R. et al. Determination of cranberry phenolic metabolites in rats by Liquid Chromatography–Tandem Mass Spectrometry. J. Agric. Food Chem. 2011, 59, 6682–6688.

557

34) Peron, G.; Uddin, J.; Stocchero, M.; Mammi, S.; Schievano, E.; Dall'Acqua, S. Studying the

558

effects of natural extracts with metabolomics: A longitudinal study on the supplementation

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of healthy rats with Polygonum cuspidatum Sieb. et Zucc. J. Pharm. Biomed. Anal. 2017,

560

140, 62-70.

561 562 563 564 565 566 567 568 569 570 571 572 23 ACS Paragon Plus Environment


573

FIGURE CAPTIONS

574 575

Figure 1. Exemplificative score scatter plots obtained from the PCA of UPLC-MS data recorded in

576

negative ion mode. The plots show cranberry extract treated group (red dots) and control group

577

(blue dots) at day 0, 7, 14, 21 and 35. Ellipsoid is not indicating 95% confidence interval.

578

579

Figure 2. Score scatter plot obtained from PLS-DA modeling of “positive” dataset. The plot shows

580

cranberry extract treated group (red dots) and control group (black squares). Ellipsoid is not

581

indicating 95% confidence interval.

582

583

Figure 3. Score scatter plot obtained from PLS-DA modeling of “negative” dataset. The plot shows

584

cranberry treated group (red dots) and control group (black squares). Ellipsoid is not indicating 95%

585

confidence interval.

586

587

Figure 4. Histogram representing the adhering ability of uropathogenic E. coli to HT-29 epithelial

588

cells after incubation in rat urine samples collected at 2, 4, 8 and 24 hours following oral treatment

589

with 100 mg/kg cranberry extract or from untreated animals (control). Significance was calculated

590

using t-test. The asterisk indicates a p-value 285), track B shows trimeric PAC-A (m/z transition

598

863 > 289) and track C shows tetrameric PAC-A (m/z transition 1151 > 285).

599 600

Figure 7. Urinary PAC-A2 amounts at 2, 4, 6, 8 and 24 h after cranberry extract administration to

601

rats. Values are reported as means ± SD, LOQ is indicated.

602 603

604 605 606 607 608 609 610 611 612 613 614 615 616 617 618

619 620 621 25 ACS Paragon Plus Environment


622 623

FIGURES

624

Figure 1

625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 26 ACS Paragon Plus Environment

Page 26 of 38

Page 27 of 38

640


Figure 2 50

40

30

20

t[2]

10

0

-10

-20

-30

-40

-50

-60 -70

641

-60

-50

-40

-30

-20

-10

0

10

20

30

40

t[1] MarkerLy nx XS 3 - PLS-DA 35 day s.usp (M2: PLS-DA) - 2016-11-14 17:38:39 (UTC+1)

642

643

644

645

646

647

648

649

650

651

652

653

654 27 ACS Paragon Plus Environment

50

60

70


655

Page 28 of 38

Figure 3

50

40

30

20

t[2]

10

0

-10

-20

-30

-40

-50

-60

-50

-40

-30

-20

-10

0

10

20

30

40

t[1]

656

MarkerLy nx XS 3 - PLS-DA 35 day s negativ e.usp (M3: PLS-DA) - 2016-11-14 17:42:07 (UTC+1)

657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 28 ACS Paragon Plus Environment

50

60

Page 29 of 38

678


Figure 4

679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694



695

Figure 5

696 697

698

699

700

701

702

703

704


Page 30 of 38

Page 31 of 38

705


Figure 6

706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 31 ACS Paragon Plus Environment


725

Figure 7

726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 32 ACS Paragon Plus Environment

Page 32 of 38

Page 33 of 38


741

TABLES

742

Table 1A. Variables related to treatment with cranberry extract, obtained from the PLS-DA model

743

for UPLC-MS data acquired in positive ion mode. R.T. (min)

Measured HR m/z

Significant fragments, m/z

VIP

Amount T vs C#

p-value

Putative identification

Compound ID (Database)

2.2

146.0687

112.3876

2.3

↑

0.890

Unknown

-

3.3

271.1205

166.0743, 138.0784, 114.0430

2.0

↑

0.042*

Unknown

-

2.3

282.1004

150.0538, 114.0426

2.2

↑

0.001*

1-Methyladenosine

HMDB03331 (HMDB)

5.5

297.1261

286.1209, 279.1148, 126.0316

2.4

↑

0.021*

Desmosflavone

HMDB38518 (HMDB)

*

2,8Dihydroxyquinoline -beta-Dglucuronide

HMDB11658 (HMDB)

6.4

338.0716

162.0314, 134.0363

5.4

↑

0.002

6.7

340.0869

164.0474, 113.0001

4.6

↑

0.290

5-Hydroxy-6methoxyindole glucuronide

HMDB10363 (HMDB)

7.0

431.0875

255.0444, 199.0519

4.3

↑

0.015*

Daidzein 4'-Oglucuronide

HMDB41717 (HMDB)

7.6

447.0835

271.0399, 91.0316

2.9

↑

0.044*

Genistein 5-Oglucuronide

HMDB41738 (HMDB)

8.0

146.0364

128.0260, 85.0284

3.0

↑

0.221

2-Keto-glutaramic acid

HMDB01552 (HMDB)

9.6

347.2061

121.0778, 105.0467, 91.0317

2.1

↑

0.050*

Corticosterone

HMDB01547 (HMDB)

10.3

701.5128

373.2208, 333.2260

2.0

↑

0.900

Unknown

-

5.3

154.0266

127.0155, 92.0268, 84.0586

3.2

↓

0.755

Unknown

-

6.3

206.0224

178.0267, 160.0156, 132.0208

4.3

↓

0.235

Xanthurenic acid

HMDB00881 (HMDB)

6.5

190.0268

162.0315, 144.0206, 116.0261

3.2

↓

0.664

Kynurenic acid

HMDB00715 (HMDB)

7.3

162.0316

144.0204, 134.0365

2.7

↓

0.006*

2,8Dihydroxyquinoline

66378 (Metlin)

7.5

164.0470

146.0360, 118.0411

4.4

↓

0.008*

5-Hydroxy-6methoxyindole

144618 (Chemspider)

8.7

255.0448

199.0528, 181.0418, 136.9998

4.2

↓

0.023*

Daidzein

HMDB03312 (HMDB)

8.7

285.0568

270.0324, 153.0461

2.5

↓

0.030*

Unknown

-



Page 34 of 38

9.6

271.0402

152.9947, 91.0318

4.2

↓

0.035*

Genistein

HMDB03217 (HMDB)

10.1

373.2597

355.2486, 213.1415, 159.0931, 145.0775

2.7

↓

0.096

Prostaglandin derivative

-

10.2

371.2441

353.2316, 211.1254, 145.0772

2.8

↓

0.020*

Prostaglandin derivative

-

*Significant level < 0.05, calculated using ANOVA. # The level of biomarkers in treated (T) and control (C) urine samples was evaluated by their normalized area. ↑: higher amounts in treated group compared to control; ↓: lower amounts in treated group compared to control.

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765

Table 1B. Variables related to treatment with cranberry extract, obtained from the PLS-DA model

766

for UPLC-MS data acquired in negative ion mode. R.T. (min)

Measured HR m/z

Significant fragments, m/z

VIP

Amount T vs C#

p-value

Putative identification

2.2

190.9951

161.9964, 144.9897, 110.9843

2.4

↑

0.900

Citric acid

HMDB00094 (HMDB)

5.4

188.9616

109.0049, 96.9363

2.9

↑

0.020*

Catechol sulfate

HMDB61713 (HMDB)

5.9

172.9663

160.0156, 151.9870, 93.0109

4.6

↑

0.600

Unknown

-

6.3

289.0184

209.0586, 135.0208, 112.9998

4.4

↑

Antiadhesive Activity and Metabolomics Analysis of Rat Urine after

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