An exploratory study of red raspberry (Rubus idaeus L.) (poly)phenols/metabolites in human biological samples

Xuhuiqun Zhanga, Amandeep Sandhua, Indika Edirisinghea and Britt Burton-Freeman*ab
aCenter for Nutrition Research, Institute for Food Safety and Health, Illinois Institute of Technology, IL, USA. E-mail: bburton@iit.edu; Fax: +708-341-7078; Tel: +708-341-7078
bDepartment of Nutrition, University of California, Davis, CA, USA

Received 16th June 2017 , Accepted 27th November 2017

First published on 21st December 2017


Red raspberry (Rubus idaeus L.) contains a variety of polyphenols including anthocyanins and ellagitannins. Red raspberry polyphenols absorbed in different forms (parent compounds, degradants or microbial metabolites) are subject to xenobiotic metabolism in the intestine, liver, and/or kidney, forming methylate, glucuronide, and sulfate conjugated metabolites. Upon acute exposure, (poly)phenol/metabolite presence in the blood depends mainly on intestinal absorption, enterohepatic circulation, and metabolism by resident microbiota. However, chronic exposure to red raspberry polyphenols may alter metabolite patterns depending on adaptions in the xenobiotic machinery and/or microbiota composition. Understanding the metabolic fate of these compounds and their composition in different biological specimens relative to the exposure time/dose will aid in designing future health benefit studies, including the mechanism of action studies. The present exploratory study applied ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole time-of-flight (QTOF) and triple quadrupole (QQQ) mass spectrometries to characterize red raspberry polyphenols in fruit and then their appearance, including metabolites in human biological samples (plasma, urine and breast milk) after the chronic intake of red raspberries. The results suggested that the most abundant polyphenols in red raspberries included cyanidin 3-O-sophoroside, cyanidin 3-O-glucoside, sanguiin H6 and lambertianin C. Sixty-two (poly)phenolic compounds were tentatively identified in the plasma, urine and breast milk samples after the intake of red raspberries. In general, urine contained the highest content of phenolic metabolites; phase II metabolites, particularly sulfated conjugates, were mainly present in urine and breast milk, and breast milk contained fewer parent anthocyanins compared to urine and plasma.


Introduction

Red raspberry (Rubus idaeus L.) is an ancient fruit that has been traced back to the Middle Ages when the wild fruits were gathered and used for medicinal purposes.1 In recent years, red raspberries have been acknowledged with many health benefits. In animal and human studies, the consumption of red raspberries has been associated with a decreased risk of developing several chronic diseases, including cardiovascular disease, type 2 diabetes mellitus (T2DM) and several types of cancer.2–6

In most studies, the health benefits of red raspberries have been associated with their polyphenol content, most notably their anthocyanin and ellagitannin (ET) content.1,5,6 Anthocyanins are a subgroup of flavonoids responsible for the bright red colour of red raspberries. The anthocyanins in red raspberries are the glycosylated (sophorosyl, glucosyl, rutinosyl) forms of their corresponding aglycones, cyanidin and pelargonidin7,8 (Fig. 1). ETs are unique to Rubus berries, pomegranates, muscadine grapes and some nuts.10 The ETs in red raspberries include free and conjugated ellagic acids and hydrolyzable tannins, the esters of hexahydroxydiphenoyl (HHDP) groups and galloyl groups to glucose cores7–11 (Fig. 2). Apart from anthocyanins and ETs, other phenolic compounds such as flavan-3-ols in single and polymer forms of (epi)catechin,8 flavonols in free and conjugated forms (quercetin and kaempferol),7–9 and glycosylated phenolic acids (caffeic and p-coumaric acid)8 are also reported in small quantities in red raspberries.


image file: c7fo00893g-f1.tif
Fig. 1 Main anthocyanins and proposed conversion mechanism into phenolic acids (adapted from ref. 1, 13 and 15).

image file: c7fo00893g-f2.tif
Fig. 2 Main ellagitannin and proposed conversion mechanism into urolithins (adapted from ref. 13–15).

After the ingestion of red raspberries, the polyphenols, or at least a portion of them, are bioavailable to systemic organs, through absorption, distribution, metabolism and excretion.1 Some of the anthocyanins can be absorbed intact in their glycosylated form, while others may be hydrolyzed to their aglycones, degraded to phenolic compounds or proceed to the colon where they are catabolized to phenolic acids by gut microbiota before absorption.12 The microbial catabolism of anthocyanins is performed by the cleavage of the heterocyclic flavylium ring (C-ring), followed by dehydroxylation or decarboxylation13–15 (Fig. 1). ETs are hydrolyzed to ellagic acid and also metabolized by the gut microbiota to produce urolithins. Ellagic acid is formed by two gallic acids through C–C coupling and condensation to the di-lactone. The transformation from ellagic acid to urolithin is initiated by the hydrolysis of one of two lactone moieties followed by the dehydroxylation to urolithin A, isourolithin A or urolithin B13–15 (Fig. 2). The absorbed phenolic compounds (parent compounds, degradants or microbial metabolites) are subject to phase I and II metabolism in the small intestine, liver, and/or kidney, forming methylate, glucuronide, and sulfate conjugated metabolites.16

Several methodologies have been described in the literature to qualify and quantify the red raspberry polyphenols, mainly based on the coupling of liquid chromatography with UV and MS detection.7–11,17–22 Most studies focused on the qualitative and/or quantitative analysis of the main anthocyanins (cyanidin and pelargonidin derivatives) and ETs (sanguiin H-6 and lambertianin C).7–9,17,18,20–22 However, the flavan-3-ols, flavonols, phenolic acids, peonidin-based anthocyanins and minor ETs may account for a considerable proportion of the total polyphenolic content of raspberries. A study on isolated raspberry ETs showed that the minor ETs (i.e., sanguiin H2, sanguiin H10 and sanguiin H6 derivatives) account for 20 to 27% of the total ETs in different raspberry cultivars.10 Although chemical hydrolysis has been widely used for the quantification of total ETs through the HPLC analysis of the hydrolyzed product, ellagic acid,17,18 the neglected sanguisorboate (galloyl-HHDP) and gallate moieties account for 46% of sanguiin H-6 and 39% of lambertianin C molecular mass, respectively. A considerable content of proanthocyanidins has also been reported in cultivars Mespi and Pima.18 Hence, red raspberries contain a number of polyphenols that likely contribute to their functional effects.

To understand the relationship between these components and their biological function, it is necessary to characterize their metabolic fate in biological samples. Plasma and urine are the most widely used human biological samples for qualitative and quantitative analyses,12,23–31 although feces, ileal fluid and colon tissue have also been studied for the bioavailability and health effects of polyphenols.23,25,27–29,31 The characterization of polyphenol metabolites over the years has not been easy due to their low concentration, limited availability of standards, and sensitivity limitations in instrumentation. Only a few studies have characterized raspberry polyphenol metabolites in plasma and urine.30 Breast milk is another excretion pathway of body fluids and has never been investigated for raspberry polyphenol metabolites. Therefore, this study aimed to characterize red raspberry polyphenols and their metabolites in human biological samples (plasma, urine and breast milk) after the intake of red raspberries for at least 1 week and to validate quantitative methods for further research purposes.

Materials and methods

Chemical and reagents

Standards of cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, pelargonidin 3-O-glucoside, pelargonidin 3-O-rutinoside, peonidin 3-O-glucoside, malvidin 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-rutinoside, quercetin 3-O-glucuronide, quercetin and gallic acid were purchased from Extrasynthese (Genay, France). Catechin, ellagic acid, 3-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 4-hydroxyphenylacetic acid, isovanillic acid, syringic acid, p-coumaric acid, caffeic acid, sinapic acid, dihydrocaffeic acid and hippuric acid were purchased from Sigma-Aldrich (St Louis, MO, USA). Ferulic acid and vanillic acid were purchased from Fluka (München, Germany). Urolithin A, urolithin B, urolithin A glucuronide, urolithin B glucuronide, urolithin A sulfate and urolithin B sulfate were provided by Dr Francisco A. Tomás Barberán (Murcia, Spain). Stock solutions of individual standards were prepared by dissolving 50 mg of each compound in 500 μL dimethyl sulphoxide (DMSO) and 9.5 mL of methanol to a final concentration of 5 mg mL−1. Standard stock solutions were prepared at a concentration of 400 ppm (μg mL−1) in methanol and working solutions were prepared by appropriate dilution of the standard stock solutions with methanol. All solutions were stored at −80 °C.

Methanol, acetone and acetonitrile were purchased from Thermo Fisher (Waltham, MA, USA). Formic acid and acetic acid were from Sigma-Aldrich. Millipore water was used throughout this study. All chemicals and reagents were of HPLC grade.

Samples

Individually Quick Frozen (IQF) red raspberries (Rubus idaeus L. var. Wakefield) were obtained from Enfield Farms (Lynden, WA, USA), fresh red raspberries were purchased from a local grocery store (Driscoll's INC. brand, Watsonville, CA, USA), red raspberry purée (multiple varieties) was supplied by Northwest Berry Co-op (Everson, WA, USA) and freeze-dried red raspberry powder (multiple varieties) was supplied by Van Drunen Farms (Momence, IL, USA).

Plasma, urine and breast milk samples were obtained from two pilot human studies after the consumption of red raspberries. Studies were approved by the Institutional Review Board at the Illinois Institute of Technology and all volunteers gave their written informed consent. In the first study, 2 healthy volunteers consumed 1 cup (∼125 g) of red raspberry purée per day for 4 weeks. On the first and last days, subjects ingested 2 cups (∼250 g) red raspberry purée in the morning. Blood samples were collected in EDTA tubes before (0 h) and after red raspberry consumption at 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 5.5 h, 6 h, 7 h, 8 h, and 24 h and stored at −80 °C. Urine samples were collected before and after red raspberry ingestion starting 12 h before (−12–0 h) followed by collections after the red raspberry intake during time periods: 0–4 h, 4–8 h, 8–24 h, 24–36 h, 36–48 h and stored at −80 °C. In the second study, 1 healthy lactating volunteer consumed 1 cup (∼125 g) of fresh red raspberries per day for 1 week after 1-week washout from all berries. Breast milk samples were collected before and 2 h post red raspberry consumption on day 0 and day 7 and frozen immediately by the subject, then stored at −80 °C until analysis.

Sample extraction

Frozen red raspberry, fresh red raspberry or red raspberry purée samples (5 g) or red raspberry freeze-dried powder (0.5 g) samples were extracted with 5 mL of the extraction solvent, acetone[thin space (1/6-em)]:[thin space (1/6-em)]water[thin space (1/6-em)]:[thin space (1/6-em)]acetic acid (70[thin space (1/6-em)]:[thin space (1/6-em)]29.7[thin space (1/6-em)]:[thin space (1/6-em)]0.3, v/v/v), respectively, in triplicate. The samples were vortexed for 30 s, sonicated for 5 min, kept at room temperature for 20 min, sonicated for another 5 min, and then centrifuged at 8000g (Eppendorf, Hauppauge, NY, USA) for 10 min. The supernatant was decanted and the remaining seeds and tissue were further extracted with 5 mL extraction solvent. The second supernatant was combined with the initial extract, diluted to 15 mL and stored at −80 °C. The sample (50 μL) was evaporated under N2 and the residue was re-dissolved in 1 mL HPLC starting mobile phase, acetonitrile[thin space (1/6-em)]:[thin space (1/6-em)]water[thin space (1/6-em)]:[thin space (1/6-em)]formic acid (5[thin space (1/6-em)]:[thin space (1/6-em)]94[thin space (1/6-em)]:[thin space (1/6-em)]1, v/v/v) before analysis by UHPLC-QTOF and UHPLC-QQQ.

Human biological samples were extracted using solid-phase extraction (SPE) C18 cartridges (3 mL, 200 mg; Agilent Technologies, Santa Clara, CA, USA). SPE cartridges were pre-conditioned using 2 mL 1% formic acid in methanol, followed by 2 mL 1% formic acid in water. Breast milk samples were centrifuged twice at 11[thin space (1/6-em)]000g (Eppendorf, Hauppauge, NY, USA) for 10 min to obtain skimmed breast milk. The plasma sample (500 μL), urine sample (200 μL) or skimmed breast milk sample (1 mL) was diluted with 1% formic acid in water (3 times the sample volume), vortexed and loaded directly onto the SPE cartridges. The sample was drained under gravity and washed with 1 mL of 1% formic acid in water. Finally, the SPE cartridges were eluted with 500 μL 1% formic acid in methanol and then 500 μL 1% formic acid in acetone. The extract was evaporated under N2 and the plasma, urine or breast milk sample residue was re-dissolved in 100 μL, 1000 μL or 200 μL of the starting mobile phase, respectively.

Analysis by liquid chromatography coupled with mass spectrometry detectors

Two different analytical platforms were used for the characterization of red raspberry polyphenols and their metabolites. Generally, each platform consisted of a reversed phase liquid chromatography and a mass spectrometer detector. For each platform, two chromatographic methods were developed according to analytes.
UHPLC separation parameters. An Agilent 1290 Infinity UHPLC system was used for the separation of red raspberry polyphenols and their metabolites in biological samples.

The chromatographic separation of red raspberry polyphenols and their metabolites (except for phenolic acids) in human biological samples was achieved on a reversed-phase Poroshell C18 StableBond column (2.1 × 150 mm, 2.7 μm) at 35 °C. Acidified water (1% formic acid) and acetonitrile were used as mobile phases A and B, respectively, with a flow rate of 0.3 mL min−1. The linear gradient started with 5% of solvent B, reaching 15% solvent B at 10 min, 20% solvent B at 12 min, 50% solvent B at 20 min, and 90% solvent B at 23 min. The initial conditions were re-established at 25 min and isocratic conditions were maintained up to 30 min. The injection volume was 1 μL for each red raspberry sample and 5 μL for each biological sample.

The separation of phenolic acids and their derivatives in human biological samples were achieved on a Pursuit 3 PFP column (2.0 × 150 mm, 3 μm) at 40 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), with a flow rate of 0.4 mL min−1. The linear gradient started with 5% of solvent B, reaching 10% solvent B at 3 min, 15% solvent B at 7 min, maintained to 9 min, 20% solvent B at 10 min, maintained to 11 min, 25% solvent B at 12 min, 30% solvent B at 13 min, maintained to 14 min, and 95% solvent B at 15 min. The initial conditions were re-established at 16 min and isocratic conditions were maintained up to 20 min. The injection volume was 5 μL for each biological sample.

Mass spectrometer detectors. UHPLC-QTOF (MS/MS) was used for qualitative analysis, in order to obtain the accurate mass and fragmentation pattern of analytes. The UHPLC system was coupled with a 6500 Series Accurate-Mass Quadrupole Time-of-Flight (QTOF) (Agilent Technologies, Santa Clara, CA, USA). The coupling was done through an electrospray interface (ESI) with the following parameters: gas temperature 250 °C, gas flow 10 L min−1, nebulizer pressure 35 psi, sheath gas temperature 300 °C, sheath gas flow 11 L min−1, and capillary 4500 V for the positive mode and 3500 V for the negative mode. The QTOF was operated in positive mode for anthocyanin analysis and in negative mode for ETs, flavon-3-ols, phenolic acids, urolithins and other analytes. Spectra in positive mode were acquired in MS scan mode with the m/z range of 100–1100 and an acquisition rate of 1.5 spectra per s, and in MS/MS mode with the m/z range of 50–800, and an acquisition rate of 4 spectra per s. Spectra in negative mode were acquired in MS scan with the m/z range of 100–3000 and an acquisition rate of 1.5 spectra per s, and in MS/MS mode with the m/z range of 50–2000, and an acquisition rate of 4 spectra per s. The data were analyzed using the Mass Hunter Qualitative Analysis software (version B.06.00, Agilent Technologies, Santa Clara, CA, USA).

UHPLC-QQQ (MS/MS) was used for quantitative analysis, consisting of an UHPLC system coupled with a 6460 Series Triple Quadrupole (QQQ) (Agilent Technologies, Santa Clara, CA, USA). The ESI conditions were the same as those used in the UHPLC-QTOF analysis. All analytes were quantified in dynamic multiple reaction monitoring mode (dMRM) that provides high sensitivity and selectivity in the quantitative analysis of biological samples. For most analytes, two transitions, a quantifier and a qualifier, were optimized, by establishing the collision energy and fragmentor voltage. Standards were optimized using the Mass Hunter Opitimizer. The data were analyzed using the Mass Hunter Quantitative Analysis software (version B.07.00, Agilent Technologies, Santa Clara, CA, USA).

Method validation

A method validation was performed with UHPLC-QQQ for the quantitative analysis of polyphenols in different forms of red raspberries and their metabolites in plasma samples in terms of optimization, linearity, matrix effect, recovery, limit of detection (LOD), limit of quantification (LOQ), precision and stability.

Calibration curves were prepared in the starting mobile phase and in post-extraction blank plasma with at least 7 different levels, spiked with IS (malvidin 3-O-glucoside and syringic acid). The calibration curve was plotted by the peak area versus the nominal concentration of the analyte.

The matrix effect (ME) was evaluated, since the ionization of endogenous compounds in plasma could result in ion suppression or ion enhancement. The matrix effect for each standard was calculated comparing the slopes of calibration curves in the starting mobile phase (slopesol) or in post-extraction blank plasma (slopeplasma): % ME = (slopeplasma − slopesol)/slopesol × 100.

The recovery of the analyte during sample extraction was evaluated in triplicate using blank plasma spiked with a mixture of standards at a low level and a high level, extracted as described in sample extraction. Recovery was calculated by the responses of pre- and post-spiked samples.

The LOD and LOQ were obtained by injecting successively diluted standard solutions in the different matrixes. The LOD and LOQ were calculated by the IUPAC criterion of the signal to noise ratio (S/N) of 3 for the LOD and of 10 for the LOQ.27

The precision was calculated by injecting a mixture of standards prepared in the starting mobile phase three times in a single run and in three different runs.

Results

Analysis of polyphenols in red raspberries

A total of 13 anthocyanins, 14 free and conjugated forms of ellagic acid, 6 flavan-3-ols and proanthocyanidins, 10 flavonols and 7 phenolic acids were identified and quantified in 4 different forms of red raspberries. From these data the total polyphenol content ranged from 71.6 to 281.0 mg per 100 g of fresh weight red raspberries (Table 1).
Table 1 Red raspberry polyphenols identified by UHPLC-QTOF and quantified by UHPLC-QQQ
RT (min) Components QTOF qualification QQQ quantification
M+/− (m/z)a MS/MS fragments (m/z) MRM transition Concentration (mg per 100g) average ± SDc
Fresh RRB Frozen RRB Purée RRB Powder RRBb
a Molecular ion.b Calculated on fresh weight (assuming 85% moisture content in fresh red raspberries).c In triplicate.
6.15 Cyanidin 3,5-diglucoside 611.1642+ 287.0553, 449.1075 611/287 <LOD 0.14 ± 0.00 0.03 ± 0.00 0.30 ± 0.02
7.95 Cyanidin 3-O-sophoroside 611.1642+ 287.0615, 449.1078 611/287 9.64 ± 0.10 115.96 ± 0.43 41.93 ± 1.59 79.57 ± 1.28
8.38 Cyanidin 3-O-(2G-glucosylrutinoside) 757.2220+ 287.0572, 611.1608, 433.1120, 595.1663 757/287 2.92 ± 0.02 0.02 ± 0.00 2.26 ± 0.11 0.10 ± 0.00
8.73 Cyanidin 3-O-sambubioside 581.1530+ 287.0549 581/287 0.12 ± 0.00 2.04 ± 0.02 0.18 ± 0.01 0.41 ± 0.01
8.77 Cyanidin 3-O-glucoside 449.1099+ 287.0572 449/287 4.97 ± 0.04 23.26 ± 0.23 7.97 ± 0.41 22.76 ± 0.66
9.02 Cyanidin 3-O-(2G-xylosylrutinoside) 727.2113+ 287.0555, 581.1520, 433.1148 727/287 0.09 ± 0.00 <LOD 0.05 ± 0.00 <LOD
9.05 Pelargonidin 3-O-sophoroside 595.1686+ 271.062 595/271 0.40 ± 0.01 1.04 ± 0.01 1.71 ± 0.02 1.91 ± 0.11
9.34 Cyanidin 3-O-rutinoside 595.1686+ 287.0560, 449.1073 595/287 2.61 ± 0.04 0.02 ± 0.00 0.90 ± 0.04 0.05 ± 0.00
9.62 Pelargonidin 3-O-(2G-glucosylrutinoside) 741.2258+ 271.0603, 595.1652, 417.1159 741/271 0.80 ± 0.00 0.14 ± 0.00 0.80 ± 0.03 0.21 ± 0.00
10.06 Pelargonidin 3-O-glucoside 433.1145+ 271.0606 433/271 0.28 ± 0.00 0.31 ± 0.01 0.40 ± 0.02 0.57 ± 0.01
10.63 Pelargonidin 3-O-rutinoside 579.1719+ 271.0602, 433.1114 579/271 0.45 ± 0.01 0.10 ± 0.00 0.22 ± 0.01 0.16 ± 0.00
10.91 Peonidin-3-O-glucoside 463.1252+ 301.0706 463/301 0.01 ± 0.00 0.09 ± 0.00 0.04 ± 0.00 0.07 ± 0.00
11.40 Peonidin-3-O-rutinoside 609.1817+ 301.0501, 463.1252 609/301 0.02 ± 0.00 <LOD 0.02 ± 0.00 0.01 ± 0.00
  Total anthocyanins       22.30 ± 0.23 143.13 ± 0.70 56.51 ± 2.23 106.15 ± 2.10
5.42 Corilagin 633.0702 300.9973, 229.0158, 257.0077 633/301 0.42 ± 0.00 0.44 ± 0.01 0.47 ± 0.00 0.79 ± 0.01
6.65 Sanguiin H-10 isomer 1 [783.0690]2− 300.9988, 633.0715, 935.0731, 1235.0679 783/301 1.46 ± 0.02 2.19 ± 0.05 1.38 ± 0.02 5.23 ± 0.12
7.90 Sanguiin H-6 without gallic moiety [858.0677]2− 300.9974, 783.0701, 935.0680, 1235.0516 858/301 1.27 ± 0.01 1.43 ± 0.05 1.26 ± 0.02 2.56 ± 0.04
10.08 Sanguiin H-10 isomer 2 [783.0690]2− 300.9982, 633.0727, 933.0645, 1265.1347 783/301 1.92 ± 0.09 4.22 ± 0.19 2.09 ± 0.05 8.79 ± 0.37
11.44 Sanguiin H-10 isomer 3 [783.0690]2− 300.9986, 633.0727, 933.0607, 1265.1402 783/301 1.14 ± 0.03 1.50 ± 0.05 1.71 ± 0.02 2.80 ± 0.03
11.52 Sanguiin H-6 plus gallic moiety [1018.0726]2− 300.9980, 469.0035, 633.0720, 935.0749, 1567.1359 1018/301 1.35 ± 0.02 1.50 ± 0.03 2.96 ± 0.24 2.76 ± 0.07
11.88 Lambertianin C [1401.1060]2− 300.9985, 633.0729, 935.0770, 1867.1282 1401/301 1.85 ± 0.02 7.70 ± 0.60 1.96 ± 0.22 20.70 ± 2.80
12.41 Sanguiin H6 [934.0708]2−, 1869.1502 300.9983, 633.0734, 1235.0689, 1567.1416 934/301 19.61 ± 0.12 35.93 ± 2.68 19.16 ± 0.90 67.65 ± 5.07
12.48 Sanguiin H2 1103.0842, [551.0399]2− 301.9962, 469.0042, 633.0747, 935.0752 1103/301 0.74 ± 0.00 0.78 ± 0.01 0.87 ± 0.01 1.24 ± 0.03
12.94 Ellagic acid pentoside isomer 1 433.0417 300.9982, 229.0122, 257.0087 433/301 0.59 ± 0.00 0.89 ± 0.00 0.58 ± 0.02 2.32 ± 0.03
13.28 Ellagic acid pentoside isomer 2 433.0417 299.9903, 300.9971, 229.0140, 257.0080 433/301 0.34 ± 0.01 0.81 ± 0.04 0.46 ± 0.01 1.69 ± 0.02
13.61 Ellagic acid 300.9992 229.0131, 257.0088 301/301 0.30 ± 0.01 0.48 ± 0.03 7.45 ± 0.39 4.43 ± 0.13
15.02 Ellagic acid acetyl pentoside isomer 1 475.0525 300.9978, 229.0122, 257.0062 475/301 0.40 ± 0.00 0.43 ± 0.01 0.40 ± 0.01 0.88 ± 0.02
15.54 Ellagic acid acetyl pentoside isomer 2 475.0525 299.9904, 300.9973, 288.0066, 257.0043 475/301 0.33 ± 0.00 0.43 ± 0.00 0.36 ± 0.00 0.72 ± 0.04
  Total ellagic acid and ETs       31.71 ± 0.32 58.73 ± 3.73 41.09 ± 1.90 122.57 ± 8.80
7.79 Procyanidin B isomer 1 577.1355 289.0712, 125.0239, 407.0766, 245.0812 577/289 9.32 ± 0.34 4.35 ± 0.22 10.18 ± 0.17 23.33 ± 1.30
8.37 Procyanidin B isomer 2 577.1355 289.0717, 125.0244, 407.0765, 245.0820 577/289 1.23 ± 0.09 1.99 ± 0.12 0.37 ± 0.00 4.21 ± 0.27
9.43 Epicatechin 289.0719 125.0240, 245.0812, 109.0291 289/125 1.53 ± 0.04 2.08 ± 0.08 1.83 ± 0.21 2.83 ± 0.28
9.72 Proanthocyanidin dimer 561.1416 289.0714, 125.0243, 407.0756, 245.0813 561/289 2.15 ± 0.21 2.61 ± 0.18 20.87 ± 0.22 8.86 ± 0.48
9.83 Proanthocyanidin trimer isomer 1 849.2041 289.0704, 125.0240, 559.1242, 407.0774 849/289 ND <LOQ 0.10 ± 0.00 <LOQ
10.73 Proanthocyanidin trimer isomer 2 849.2041 289.0708, 125.0236, 407.0750, 525.0815 859/289 ND <LOQ 0.11 ± 0.01 <LOQ
  Total flavan-3-ols       14.24 ± 0.68 11.03 ± 0.60 33.45 ± 0.61 39.23 ± 2.32
10.77 Quercetin 3-O-glucosylrutinoside 771.1995 300.0264, 301.0324, 178.9973, 151.0027 771/301 ND ND 0.10 ± 0.02 ND
11.44 Quercetin 3-O-galactosylglucoside 625.1419 300.0267, 151.0023, 178.9959 625/301 0.11 ± 0.00 0.91 ± 0.05 0.27 ± 0.03 0.09 ± 0.01
11.68 Quercetin 3-sophoroside 625.1419 300.0264, 150.8107, 178.9972 625/301 ND 0.25 ± 0.03 0.04 ± 0.00 0.82 ± 0.07
12.88 Quercetin 3-O-galactosylrhamnoside 609.1466 301.0348, 151.0033, 178.9985 609/301 0.05 ± 0.01 0.08 ± 0.01 0.10 ± 0.02 0.54 ± 0.08
13.72 Quercetin 3-O-rutinoside 609.1466 301.0265, 151.0033, 178.9978 609/301 0.05 ± 0.01 ND 0.08 ± 0.00 ND
13.84 Quercetin 3-O-galactoside 463.0886 300.0269, 301.0332, 151.0033, 178.9983 463/301 0.21 ± 0.02 0.32 ± 0.02 0.33 ± 0.01 0.42 ± 0.03
14.08 Quercetin 3-O-glucoside 463.0886 300.0265, 301.0326, 151.0020, 178.9997 463/301 0.08 ± 0.00 0.20 ± 0.02 0.15 ± 0.01 0.88 ± 0.02
14.13 Quercetin 3-O-glucuronide 477.0679 301.0345, 151.0032, 178.9978 477/301 0.22 ± 0.00 0.37 ± 0.00 0.70 ± 0.02 2.18 ± 0.06
15.17 Kaempferol 3-O-glucuronide 461.0733 285.0391 461/285 0.07 ± 0.00 0.06 ± 0.00 0.14 ± 0.00 0.19 ± 0.00
17.45 Quercetin 301.0346 151.0032, 178.9981 301/151 0.16 ± 0.00 0.19 ± 0.00 0.34 ± 0.01 0.31 ± 0.00
  Total flavonols       0.95 ± 0.05 2.36 ± 0.13 2.15 ± 0.12 5.44 ± 0.27
2.35 Gallic acid 169.0141 125.0241 169/125 0.22 + 0.00 0.22 + 0.00 5.06 ± 0.19 0.80 + 0.01
4.04 3,4-Dihydroxybenzoic acid 153.0205 109.0292 153/109 ND ND 0.14 ± 0.00 0.07 ± 0.00
5.34 Caffeoyl hexoside isomer 1 341.0880 161.0238, 133.0292, 135.0448, 179.0345 341/161 1.29 ± 0.07 0.51 ± 0.02 3.71 ± 0.10 3.75 ± 0.03
6.75 Caffeoyl hexoside isomer 2 341.0880 179[thin space (1/6-em)]0352, 135.0452, 161.0241 341/179 0.20 ± 0.00 0.22 ± 0.01 1.12 ± 0.07 0.51 ± 0.02
7.32 p-Coumaryl hexoside isomer 1 325.0933 145.0294, 117.0345, 119.0500, 163.0396 325/145 0.46 ± 0.04 0.74 ± 0.01 3.39 ± 0.08 2.00 ± 0.06
8.02 p-Coumaryl hexoside isomer 2 325.0933 145.0293, 117.0345, 119.0494, 163.0396 325/145 0.14 ± 0.01 0.18 ± 0.01 0.82 ± 0.06 0.52 ± 0.02
11.30 p-Coumaric acid 163.0402 119.0496, 117.0337 163/119 ND 0.07 ± 0.01 1.02 ± 0.04 0.08 ± 0.01
  Total phenolic acids       2.31 ± 0.14 1.94 ± 0.06 15.25 ± 0.54 7.76 ± 0.15
  Total polyphenols       71.50 ± 1.43 217.21 ± 5.22 148.45 ± 5.41 281.16 ± 13.65


Anthocyanins are glycosides of cyanidin, pelargonidin and peonidin, which constituted 31% to 66% of the total polyphenols, and predominantly consisted of cyanidin 3-O-sophoroside (43%–81%) and cyanidin 3-O-glucoside (14%–22%). The concentration of anthocyanins was highest in the frozen red raspberries (143.0 mg per 100 g) while it was lowest in the fresh red raspberries (22.3 mg per 100 g). ETs constituted 27%–44% of the total polyphenols, with sanguiin H-6 (47%–62%), the principal ET, followed by lambertianin C, sanguiin H-10 and sanguiin H-6 like ETs (plus or minus a gallic acid moiety). ETs were the highest in the freeze-dried red raspberry powder (122.7 mg per 100 g fresh weight, assuming 85% moisture content in fresh red raspberries) and the lowest in the fresh red raspberries (31.7 mg per 100 g). Flavan-3-ols accounted for 5%–23% of total polyphenols, consisting of different proanthocyanidin dimers and trimers, which were higher in the freeze-dried red raspberry powder (39.2 mg per 100 g fresh weight) and red raspberry purée (33.5 mg per 100 g) and lower in the fresh red raspberry (14.2 mg per 100 g) and the frozen red raspberry (11.0 mg per 100 g). Flavonols (<2%) were identified as free and glycoside forms of quercetin. Phenolic acids (<3%) were found in trace amounts in the frozen, fresh and freeze-dried powder, but constituted 10% of red raspberry purée polyphenols, including gallic acid, 3,4-dihydroxybenzoic acid, p-coumaric acid, p-coumaroyl hexosides and caffeoyl hexosides.

Identification of polyphenol metabolites in human biological samples

According to the mass accuracy and fragmentation pattern generated by the UHPLC-QTOF platform, 62 metabolites were putatively identified in plasma, urine and breast milk samples following the intake of red raspberries, that is 8 anthocyanins, 3 urolithins, 1 flavan-3-ol conjugate, 1 flavonol, and 49 free and conjugated phenolic acids (Table 2).
Table 2 Identification and relative abundance of flavonoid metabolites and urolithins in plasma, urine and breast milk after red raspberry consumption
RT (min) Components Q-TOF qualification QQQ quantification Ref.
M+/− (m/z)a MS/MS fragments (m/z) MRM transition CE (eV)b Fragmentor (V) Relative abundancec
a Molecular ion.b Collision energy.c Detected in P, plasma; U, urine; BM, breast milk.d The relative abundance comparison was based on 2 h plasma, 2 h breast milk and 0–4 h urine samples after red raspberry ingestion.e Urolithins and phenylvalerolactone were not detected in 2 h plasma. Comparison based on 24 h plasma, 24 h breast milk and 8–24 h urine samples after red raspberry ingestion.
Anthocyanins
7.95 Cyanidin 3-O-sophoroside 611.1600+ 287.0550 611/287 +28 90 U > P > BMd 22 and 28
8.38 Cyanidin 3-O-(2G-glucosylrutinoside) 757.1545+ 287.0535 757/287 +28 90 U > P > BMd 22 and 28
8.77 Cyanidin 3-O-glucoside 449.1084+ 287.0551 449/287 +28 84 U > P > BMd 12, 22–24 and 27–29
9.05 Pelargonidin 3-O-sophoroside 595.1661+ 271.0605 595/271 +25 84 U > P > BMd 22 and 28
9.34 Cyanidin 3-O-rutinoside 595.1661+ 287.0506 595/287 +30 84 U > P > BMd 22 and 28
9.44 Cyanidin 3-O-glucuronide 463.1195+ 287.0800 463/287 +28 84 U > BM > Pd 12 and 23–24
9.75 Methyl cyanidin 3-O-sophoroside 625.1768+ 301.0712 625/301 +25 90 U > BM > Pd  
10.91 Peonidin 3-O-glucoside 463.1195+ 301.0754 463/301 +25 90 U > BM > Pd 23–24 and 29
Flavan-3-ols
8.79 Catechin glucuronide 465.1016 289.0709, 245.0827 465/289 −25 112 U > BMd 25
Flavonols
17.45 Quercetin 301.0365 151.1097, 257.0474 301/257 −20 85 U > BM > Pd  
Urolithins
12.24 Urolithin A glucuronide 403.0669 227.0358, 113.0247 403/227 −30 110 U > BM > Pe 22, 26 and 30
15.84 Urolithin B glucuronide 387.0759 211.0450 387/211 −35 110 U > BM > Pe 22, 26 and 30
16.71 Urolithin A 227.0347 198.0329, 182.0382 227/198 −35 110 BM > Ue 26 and 30
Benzoic acids
2.05 Gallic acid 169.0138 125.0244, 69.0333 169/125 −13 80 U > P > BMd 25
2.29 4-hydroxybenzoic acid glucuronide 313.0582 93.0342, 119.0508, 137.0218, 313/93 −25 87 U > BMd 24 and 27
2.86 3-hydroxybenzoic acid glucuronide 313.0562 93.0344, 113.0241, 137.0239, 313/93 −25 87 U > BMd  
2.95 Vanillic acid sulfate 246.9915 123.0451, 167.0353, 247/123 −30 90 U > BMd 23–25 and 27
2.98 Vanillic acid glucuronide 343.0666 152.0123, 123.0468, 167.0352 343/167 −20 87 U > BM > Pd 23–25 and 27
3.08 3,4-dihydroxybenzoic acid 153.0203 109.0290 153/109 −13 85 U > P > BMd 23–25, 27 and 29
3.32 3,4-dihydroxybenzoic acid sulfate 232.9761 153.0199, 109.0297 233/153 −25 87 U > BMd 23–25 and 27
3.59 Isovanillic acid glucuronide 343.0666 123.0457, 167.0355 343/167 −20 87 U > BM > Pd 23–25 and 27
3.94 3,4-dihydroxybenzoic acid sulfate 232.9761 109.0293, 153.0199 233/109 −30 87 U > BMd 24
3.92 Isovanillic acid sulfate 246.9915 123.0456, 167.0343 247/123 −30 90 U > BMd 24–25 and 27
4.24 4-hydroxybenzoic acid sulfate 216.9813 93.0344, 137.0242, 172.9914 217/93 −20 85 U > BMd 24, 27 and 29
4.26 4-hydroxybenzoic acid 137.0241 93.0342, 65.0392 137/93 −9 75 U > BM > Pd 23–24, 27 and 29
4.45 2,6-dihydroxybenzoic acid 153.0203 109.0284 153/109 −13 85 U > BM > Pd  
4.92 2,3-dihydroxybenzoic acid 153.0203 109.0291 153/109 −13 85 U > BM > Pd 27
5.23 Vanillic acid 167.0342 152.0118, 108.0217 167/152 −13 75 U > BMd 23–24, 27 and 29
5.28 2,4-dihydroxybenzoic acid 153.0203 109.0281 153/109 −13 85 Ud  
5.40 Isovanillic acid 167.0342 152.0115, 122.9431 167/152 −13 75 U > BMd 24 and 27
Benzoates
6.49 Methyl 4-hydroxybenzoate 151.0398 93.0342, 107.0484 151/93 −10 87 U > P > BMd  
8.00 Methyl-3,4-dihydroxybenzoate 167.0342 108.0221, 152.0094 167/108 −20 87 U > P > BMd 24 and 27
Benzaldehydes
2.03 3,4-dihydroxybenzaldehyde 137.0241 108.0211, 119.0137 137/108 −10 75 U > P > BMd 24 and 27
4.67 4-hydroxybenzaldehyde glucuronide 297.0632 121.0292 297/121 −15 87 Ud  
4.97 4-hydroxybenzaldehyde 121.0293 92.0268 121/92 −25 75 U > P > BMd 27
6.92 Phloroglucinaldehyde 153.0203 151.0039, 83.0140 153/151 −13 105 Pd 23–24 and 27
Phenylacetic acids
2.31 3,4-dihydroxyphenylacetic acid 167.0342 123.0439, 148.9014, 108.0246 167/123 −10 87 U > BM > Pd 23, 27 and 29
2.39 Homovanillic acid 181.0514 121.0293, 93.0340 181/121 −20 85 U > BM > Pd 23, 27 and 29
3.05 Isohomovanillic acid 181.0514 121.0295, 93.0343 181/121 −20 85 U > BM > Pd  
4.08 4-hydroxyphenylacetic acid 151.0406 107.0509 151/107 −10 87 U > BM > Pd 24
4.48 3-hydroxyphenylacetic acid 151.0406 107.0502 151/107 −10 87 U > BM > Pd  
4.71 2-hydroxyphenylacetic acid 151.0406 107.0512 151/107 −10 87 U > BM > Pd  
Phenylpropionic acids
4.79 Dihydroferulic acid glucuronide 371.0980 195.0661, 113.0241, 371/195 −30 87 U > BMd  
5.00 Dihydrocaffeic acid sulfate 261.0073 181.0510, 137.0605 261/181 −20 87 U > BMd  
5.56 3-hydroxyphenylpropionic acid sulfate 245.0123 121.0660, 165.0557, 119.0499 245/121 −20 85 U > BMd  
5.59 Dihydroferulic acid sulfate 275.0230 195.0674, 136.0532 275/195 −20 87 U > BMd  
6.12 3-hydroxyphenylpropionic acid 165.0192 121.0659 165/121 −13 87 U > BM > Pd  
Phenylcinnamic acids
5.17 p-coumaric acid glucuronide 339.0707 163.0390, 119.0489, 339/163 −20 75 U > P > BMd  
5.53 Caffeic acid 179.0350 135.0438, 119.9376 179/135 −10 87 Ud 24–25 and 27
6.16 Ferulic acid glucuronide 369.0821 178.0275, 134.0378, 193.0511 369/134 −30 87 U > P > BMd 25 and 29
6.34 Coumaric acid 163.0392 119.0503, 93.0327 163/119 −13 85 U > P > BMd  
6.34 Caffeic acid sulfate 258.9914 135.0452, 179.0355 259/135 −25 87 U > BM > Pd 25 and 29
6.67 p-coumaric acid sulfate 242.9969 163.0400, 119.0498 243/119 −17 75 U > BMd  
7.16 Ferulic acid sulfate 273.0074 134.0377, 193.0511, 178.0275 273/134 −35 110 U > BMd 25 and 29
7.18 p-coumaric acid 163.0392 119.0488, 93.0330 163/119 −13 85 U > P > BMd 23 and 27
8.47 Ferulic acid 193.0502 134.0386, 178.0276 193/134 −17 110 U > BM > Pd 23–25, 27 and 29
Phenylvalerolactone
3.42 5-(3′,4′,5′-trihydroxyphenyl)-r-valerolactone sulfate 303.0198 223.0617, 163.0403 303/223 −35 110 U > BMe  
5.18 5-(3′,4′-dihydroxyphenyl)-r-valerolactone 3-O-glucuronide 383.0977 163.0764, 207.0663 383/163 −40 110 U > BM > Pe 39
6.32 5-(3′,4′-dihydroxyphenyl)-r-valerolactone sulfate 287.0230 207.0668, 163.0765 287/207 −15 87 U > BM > Pe 39 and 40
6.62 5-(3′,4′,5′-trihydroxyphenyl)-r-valerolactone sulfate 303.0198 223.0616, 208.0379, 193.0150, 164.0482 303/223 −35 110 U > BMe  
Others
3.61 Phloroglucinol 125.0240 107.0100, 83.0142, 79.0183, 125/79 −40 85 Ud  
3.94 Hippuric acid 178.0511 134.0614, 77.0396 178/134 −9 75 U > BM > Pd 27 and 29


The anthocyanins identified in plasma, urine and breast milk consisted of parent anthocyanins [cyanidin 3-O-sophoroside, cyanidin 3-O-(2G-glucosylrutinoside), cyanidin 3-O-glucoside, pelargonidin 3-O-sophoroside, and cyanidin 3-O-rutinoside] and anthocyanin conjugates [cyanidin 3-O-glucuronide, methyl cyanidin 3-O-sophoroside and peonidin 3-O-glucoside]. Urolithin A glucuronide and urolithin B glucuronide were identified in plasma, urine and breast milk, while urolithin A was only identified in urine samples. The free and conjugated phenolic acids identified differed in plasma, urine and breast milk, including benzoic acids, benzoates, benzaldehydes, phenylacetic acids, phenylpropionic acids, phenylcinnamic acids, phenylvalerolactones and their glucuronidated and sulfated derivatives. Analytes in various biological samples were compared based on their relative abundance at similar time points after red raspberry ingestion (Table 2).

Discussion

The primary aim of this study was to characterize red raspberry polyphenols and their metabolites in human biological samples (plasma, urine and breast milk) after the intake of red raspberries for at least 1 week and establishing methods for further research purposes. In addition, we were interested in characterizing the polyphenol composition in four red raspberry fruit preparations (frozen, fresh, puree, and freeze-dried powder) to better understand the product polyphenol variance for consideration in future trials.

In the present study, we found that different forms of red raspberries commercially available have a relatively similar polyphenol profile. They were characterized by the presence of cyanidin 3-O-sophoroside and sanguiin H-6 as the major components. The IQF red raspberries contained the most anthocyanins and fresh red raspberries had the lowest anthocyanin content. The freeze-dried powder had the highest amount of ETs compared to other fruit forms. Red raspberry purée contained more phenolic acids than other fruit forms. The observed compositional differences may be due to the processing methods. The freeze-dried red raspberry was milled with seeds to a powder, which would release more ETs compared to other forms. Purée processing includes mashing which may release enzymes and other cellular content that when mixed breaks down some polyphenols to phenolic acids, such as ETs to gallic acid. However, varietal differences, time of harvest, and storage conditions likely also played a role in the differences observed. These data highlight the importance of characterizing products during clinical trial design to ensure the delivery of the planned doses of total polyphenols or specific compounds of interest.

Red raspberry anthocyanins are predominately cyanidin-based molecules with some pelargonidin- and peonidin (methyl cyanidin)-based molecules. In positive ion ESI-MS, cyanidin-, pelargonidin- and peonidin-based anthocyanins contained a main aglycone fragment at m/z 287, 271 and 301, respectively. With the exception of commercially available standard anthocyanins (cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, pelargonidin 3-O-glucoside, pelargonidin 3-O-rutinoside and peonidin 3-O-glucoside), the identifications of the other anthocyanins were assisted by the accurate mass, and the fragmentation pattern of published data.7,8,17,21 However, MS/MS fragments cannot provide the absolute structure information. Therefore, some anthocyanins that share the same mass, aglycone, and fragmentation ions (e.g., cyanidin 3,5-O-diglucoside and cyanidin 3-O-sophoroside) can be misidentified. For example, the tri-glycosylated anthocyanin (m/z 757) has been identified in the literature as cyanidin 3-O-(6-p-coumaryl)glucoside-5-glucoside32 (Fig. 3A); however, an alternative structure has been proposed, cyanidin 3-O-sophoroside-5-rhamnoside, due to the presence of the fragment m/z 433, suggesting a rhamnosyl link to cyanidin 5C on the A ring.21,33 This latter structure is consistent with our MS/MS fragmentation pattern (Fig. 3C). More recent studies have assigned the m/z 757 to cyanidin 3-O-(2G-glucosylrutinoside)7,8,17–20,29,30 (Fig. 3B) and this structure was confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy in boysenberries (Rubus ursinus × R. idaeus).34 However, the glucosyl link to the cyanidin structure cannot explain the presence of m/z 433 (Fig. 3D). Similarly, although cyanidin 3-O-(2G-xylosylrutinoside) and pelargonidin 3-O-(2G-glucosylrutinoside) were confirmed in other Rubus fruits, the fragments m/z 433 and m/z 417 both suggested a rhamnosyl link to the aglycone structure. Hence, MS/MS technology provides a tremendous power for characterizing the chemical composition of fruits; however, there are caveats relative to absolute identification when standards are not available. Further studies to definitively identify some anthocyanins in red raspberries are needed.


image file: c7fo00893g-f3.tif
Fig. 3 Compounds m/z 757: tentatively proposed structures and MS/MS spectra. (A) Cyanidin 3-O-(6-p-coumaryl)glucoside-5-glucoside. (B) Cyanidin 3-O-(2G-glucosylrutinoside). (C) Cyanidin 3-O-sophoroside-5-rhamnoside. (D) MS/MS spectra of m/z 757.

ETs have been reported in red raspberries and are usually characterized by the fragment m/z 301 (HHDP).7–11 In negative ion mode ESI-MS, ETs gave a single-charged ion [M − H]1− and/or double-charged ion [M − 2H]2−. However, the concurrence of single and double charged ions may lead to repeated identification or underestimated quantification. Sanguiin H-6 had [M − 2H]2− at m/z 934 as a major peak and [M − H]1− at m/z 1869 as a minor peak (Fig. 4A). The double-charged ion m/z 934 was identified according to the spectrum spacing by 0.5 (at m/z 934.0715, 934.5732, 935.0748 and 935.5761) (Fig. 4B), large single-charged fragments m/z 1567.1395 (loss of HHDP) and m/z 1235.0683 (loss of galloylglucose) (Fig. 4C), as well as the same retention time and fragmentation pattern of m/z 934 and m/z 1869 (Fig. 4C & D). Thus, sanguiin H-6 was quantified according to the MRM transition ion 934/301, instead of 1869/301. Similarly, the double-charged ions were identified in most ETs, such as lambertianin C, sanguiin H-10 and sanguiin H-2, consistent with previous studies.8,10,11


image file: c7fo00893g-f4.tif
Fig. 4 MS and MS/MS spectra of sanguiin H-6: (A) chromatogram of m/z 934 and m/z 1869; (B) MS spectrum of m/z 934; (C) fragmentation pattern of m/z 934; (D) fragmentation pattern of m/z 1869.

The metabolic fate of fruit compounds after ingestion, such as those found in red raspberries, has gained increasing attention over the last decade. The knowledge of the metabolites formed, their bioavailability and pharmacokinetic behaviour aids several research platforms in assessing health benefits and determining possible mechanisms of action with the goal of optimizing intake strategies to achieve their maximum benefits. In the present study, 62 red raspberry polyphenol metabolites were tentatively identified, some of which have been detected in earlier studies.12,23–31 However, some phenolic acid conjugates (e.g., dihydroferulic acid glucuronide, dihydroferulic acid sulfate and 5-(3′,4′,5′-trihydroxyphenyl)-r-valerolactone sulfate) were identified for the first time in plasma and urine, and this is the first report of breast milk as a pathway for red raspberry polyphenol metabolites. In general, parent anthocyanins were identified in plasma, urine and breast milk, including cyanidin 3-O-sophoroside, cyanidin 3-O-(2G-glucosylrutinoside), cyanidin 3-O-glucoside, pelargonidin 3-O-sophoroside, and cyanidin 3-O-rutinoside, which are naturally present in red raspberries. A previous red raspberry feeding study only identified a low amount of cyanidin 3-O-glucoside and no other parent anthocyanins in plasma and urine.30 Peonidin 3-O-glucoside was present only in trace amounts in red raspberries, although, peondin 3-O-glucoside and methyl cyanidin 3-O-sophoroside were both identified in plasma, urine and breast milk samples. Peonidin 3-O-glucoside was also identified in urine previously, but methyl cyanidin 3-O-sophoroside was not identified.30 The presence of peonidin or methyl cyanidin derivatives may be attributed to the methylation of cyanidin derivatives, which is supported by a recent bilberry study.35 The major anthocyanins in bilberries are glycosides of cyanidin and delphinidin, while the major anthocyanins detected in plasma and urine were the glycosides of peonidin and malvidin due to the methylation of cyanidin and delphinidin, respectively.35 The anthocyanin conjugate cyanidin glucuronide was tentatively identified in plasma, urine and breast milk samples. Di- or tri-glycosylated anthocyanins were considered unlikely to be absorbed without the cleavage of the sugar moieties.30 However, our results suggest that anthocyanins with di- or tri-saccharides can be absorbed intact in their glycosylated form, distributed to different organs and metabolized to methylated conjugates without sugar moiety cleavage.

In addition to anthocyanins maintaining the C6–C3–C6 structure, A-ring and B-ring degradation and microbial catabolites were detected in samples. Phloroglucinaldehyde (PGA) is the primary A-ring product of anthocyanins after C-ring fission.13,15,25,30 In the present study, PGA was detected in considerable amounts after raspberry ingestion in plasma, but not detected in urine. In a cyanidin 3-O-glucoside isotope-labelled study, PGA was detected in serum, but in much lower amounts in urine, suggesting that PGA was further metabolized prior to excretion.25 Decarbonylation in the kidney to phloroglucinol is one possible pathway of PGA metabolism;13,15 however, additional studies are required to elucidate the mechanism. Benzoic acids (e.g., 3,4-dihydroxybenzoic acid and 4-dihydroxybenzoic acid), phenyl acids (e.g., 4-hydroxyphenyl acetic acid and hydroxyphenyl propionic acid), and phenylcinnamic acids (e.g., ferulic acid and p-coumaric acid), the degradation products of the anthocyanidin B-ring,15,29 were detected in all samples studied, consistent with previous findings for plasma and urine23–26 and as newly reported in the current study for breast milk. Additionally, the data indicate that these products may have gone through phase I and phase II metabolism as is evident by the presence of various methylated (e.g., vanillic acid and methyl 4-hydroxybenzoate), glucuronidated (e.g., 3,4-dihydroxybenzoic acid glucuronide, vanillic and isovanillic acid glucuronides), sulfated (3,4-dihydroxybenzoic acid sulfate, vanillic and isovanillic acid sulfate), de/hydroxylated (e.g., 2,3-dihydroxybenzoic acid and 2-hydroxyphenylacetic acid), and decarboxylated (e.g., 3,4-dihydroxybenzaldehyde and 4-hydroxybenzaldehyde) metabolites in plasma, urine and breast milk samples.

Urolithins are the gut microbial metabolites of ellagic acid and ETs and have attracted much attention over the last decade owing to their antioxidant, anti-inflammation and anti-proliferative effects in animal and human studies, as well as association with reduced risk of prostate and colon cancer recently27,30,31,36–38 In the present study, the urolithins identified were in line with previous studies31 and their presence was confirmed by using standards. The quantitative analysis of 24 h plasma and 48 h urine results indicated that, after dietary washout, the urolithins were not detected until 24 h post red raspberry consumption, supporting the microbial metabolism of ellagic acid to urolithins. After 1-week intake of red raspberries, both their production and/or abundance increased in plasma, urine and breast milk samples.

Phenylvalerolactones and their derivatives, e.g., 5-(3′,4′-dihydroxyphenyl)-r-valerolactone 3-O-glucuronide, 5-(3′,4′-dihydroxyphenyl)-r-valerolactone sulfate and 5-(3′,4′,5′-trihydroxyphenyl)-r-valerolactone sulphate, are the major gut microbial metabolites of proanthocyanidins.39 They were identified for the first time in plasma, urine and breast milk after the red raspberry intake, presumably from red raspberry proanthocyanidin dimers and trimers. Their fragmentation patterns were consistent with recent animal (procyanidin B) and human (green tea) feeding studies.40–42

Overall, urine samples were characterized by more types and greater abundance of (poly)phenolic compounds, particularly phenolic acid conjugates, than plasma samples. An isotope labeling study of cyanidin 3-O-glucoside indicated that the recovery of total phase II conjugates of 3,4-dihydroxybenzoic acid in urine was more than 3 times that in serum.43 Most of the glucuronide and sulfated conjugates identified in urine were also present in breast milk. A soymilk and tofu feeding study indicated that isoflavones (which are also flavonoid compounds) occurred in human breast milk in the form of glucuronide and sulfated conjugates.44 In our study all sulfate conjugates, some glucuronide conjugates (e.g., 4-hydroxybenzoic acid glucuronide, 3-hydroxybenzoic acid glucuronide, dihydroferulic acid glucuronide, catechin glucuronide) and some free phenolic acids (vanillic acid, isovanillic acid) were detected in both urine and breast milk, but were absent from the plasma samples. The observation that sulfated phenolic acids were present in urine and breast milk but not in plasma suggests that sulfation conjugation occurred in the kidney and mammary gland prior to their excretion/elimination.

Conclusions

In conclusion, the present study characterized an array of polyphenols in different forms of red raspberries and a greater number of their phenolic metabolites in plasma, urine and breast milk. Apart from the phenolic metabolites reported in previous studies, such as the parent anthocyanins, urolithins, different forms of benzoic acids and cinnamic acids, phenylvalerolactone conjugates were identified for the first time in human biological samples after the red raspberry intake. The results of this study confirm and extend previous findings on red raspberry metabolites in urine and plasma and offer new information for understanding the metabolic fate of these compounds and their composition in different biological specimens.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We want to thank the volunteers who participated in these pilot studies and the Clinical Nutrition Research Center staff who helped conduct various parts of the research. We are grateful to Dr Francisco A. Tomás Barberán for providing the urolithin standards.

References

  1. B. M. Burton-Freeman, A. K. Sandhu and I. Edirisinghe, Adv. Nutr., 2016, 7, 44–65 CrossRef CAS PubMed.
  2. G. Noratto, B. P. Chew and I. Ivanov, Food Funct., 2016, 7, 4944–4955 CAS.
  3. R. Törrönen, M. Kolehmainen, E. Sarkkinen, K. Poutanen, H. Mykkänen and L. Niskanen, J. Nutr., 2013, 143, 430–436 CrossRef PubMed.
  4. P. Pan, C. W. Skaer, S. M. Stirdivant, M. R. Young, G. D. Stoner, J. F. Lechner, Y. W. Huang and L. S. Wang, Cancer Prev. Res., 2015, 8, 743–750 CrossRef CAS PubMed.
  5. H. Cho, H. Jung, H. Lee, H. C. Yi, H. K. Kwak and K. T. Hwang, Food Funct., 2015, 6, 1675–1683 CAS.
  6. Y. Wang, D. Zhang, Y. Liu, D. Wang, J. Liu and B. Ji, J. Sci. Food Agric., 2015, 95, 936–944 CrossRef CAS PubMed.
  7. W. Mullen, J. McGinn, M. E. Lean, M. R. MacLean, P. Gardner, G. G. Duthie, T. Yokota and A. Crozier, J. Agric. Food Chem., 2002, 50, 5191–5196 CrossRef CAS PubMed.
  8. I. Dincheva, I. Badjakov, V. Kondakova, P. Dobson, G. McDougall and D. Stewart, Int. J. Agric. Sci. Res., 2013, 3, 127–137 Search PubMed.
  9. W. Mullen, T. Yokota, M. E. Lean and A. Crozier, Phytochemistry, 2003, 64, 617–624 CrossRef CAS PubMed.
  10. M. Gasperotti, D. Masuero, U. Vrhovsek, G. Guella and F. Mattivi, J. Agric. Food Chem., 2010, 58, 4602–4616 CrossRef CAS PubMed.
  11. M. Kähkönen, P. Kylli, V. Ollilainen, J. P. Salminen and M. Heinonen, J. Agric. Food Chem., 2012, 60, 1167–1174 CrossRef PubMed.
  12. C. D. Kay, G. Mazza, B. J. Holub and J. Wang, Br. J. Nutr., 2004, 91, 933–942 CrossRef CAS PubMed.
  13. J. F. Stevens and C. S. Maier, Phytochem. Rev., 2016, 15, 425–444 CrossRef CAS PubMed.
  14. R. González-Barrio, C. A. Edwards and A. Crozier, Drug Metab. Dispos., 2011, 39, 1680–1688 CrossRef PubMed.
  15. J. Mosele, A. Macià and M. J. Motilva, Molecules, 2015, 20, 17429–17468 CrossRef CAS PubMed.
  16. G. M. Woodward, P. W. Needs and C. D. Kay, Mol. Nutr. Food Res., 2011, 55, 378–386 CAS.
  17. W. Mullen, M. E. Lean and A. Crozier, J. Chromatogr. A, 2002, 966, 63–70 CrossRef CAS PubMed.
  18. W. Mullen, A. J. Stewart, M. E. Lean, P. Gardner, G. G. Duthie and A. Crozier, J. Agric. Food Chem., 2002, 50, 5197–5201 CrossRef CAS PubMed.
  19. K. R. Määttä-Riihinen, A. Kamal-Eldin and A. R. Törrönen, J. Agric. Food Chem., 2004, 52, 6178–6187 CrossRef PubMed.
  20. J. Beekwilder, H. Jonker, P. Meesters, R. D. Hall, I. M. van der Meer and C. H. Ric de Vos, J. Agric. Food Chem., 2005, 53, 3313–3320 CrossRef CAS PubMed.
  21. X. Wu and R. L. Prior, J. Agric. Food Chem., 2005, 53, 2589–2599 CrossRef CAS PubMed.
  22. G. McDougall, I. Martinussen and D. Stewart, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2008, 871, 362–369 CrossRef CAS PubMed.
  23. R. González-Barrio, G. Borges, W. Mullen and A. Crozier, J. Agric. Food Chem., 2010, 58, 3933–3939 CrossRef PubMed.
  24. R. M. de Ferrars, A. Cassidy, P. Curtis and C. D. Kay, Mol. Nutr. Food Res., 2014, 58, 490–502 Search PubMed.
  25. R. M. de Ferrars, C. Czank, Q. Zhang, N. P. Botting, P. A. Kroon, A. Cassidy and C. D. Kay, Br. J. Pharmacol., 2014, 171, 3268–3282 CrossRef CAS PubMed.
  26. R. C. Pimpão, T. Dew, M. E. Figueira, G. J. McDougall, D. Stewart, R. B. Ferreira, C. N. Santos and G. Williamson, Mol. Nutr. Food Res., 2014, 58, 1414–1425 Search PubMed.
  27. M. A. Nuñez-Sánchez, R. García-Villalba, T. Monedero-Saiz, N. V. García-Talavera, M. B. Gómez-Sánchez, C. Sánchez-Álvarez, A. M. García-Albert, F. J. Rodríguez-Gil, M. Ruiz-Marín, F. A. Pastor-Quirante, F. Martínez-Díaz, M. J. Yáñez-Gascón, A. González-Sarrías, F. A. Tomás-Barberán and J. C. Espín, Mol. Nutr. Food Res., 2014, 58, 1199–1211 Search PubMed.
  28. R. M. de Ferrars, C. Czank, S. Saha, P. W. Needs, Q. Zhang, K. S. Raheem, N. P. Botting, P. A. Kroon and C. D. Kay, Anal. Chem., 2014, 86, 10052–10058 CrossRef CAS PubMed.
  29. G. J. McDougall, S. Conner, G. Pereira-Caro, R. Gonzalez-Barrio, E. M. Brown, S. Verrall, D. Stewart, T. Moffet, M. Ibars, R. Lawther, G. O'Connor, I. Rowland, A. Crozier and C. I. Gill, J. Agric. Food Chem., 2014, 62, 7631–7641 CrossRef CAS PubMed.
  30. I. A. Ludwig, P. Mena, L. Calani, G. Borges, G. Pereira-Caro, L. Bresciani, D. Del Rio, M. E. Lean and A. Crozier, Free Radical Biol. Med., 2015, 89, 758–769 CrossRef CAS PubMed.
  31. R. García-Villalba, J. C. Espín and F. A. Tomás-Barberán, J. Chromatogr. A, 2016, 1428, 162–175 CrossRef PubMed.
  32. L. Wada and L. B. Ou, J. Agric. Food Chem., 2002, 50, 3495–3500 CrossRef CAS PubMed.
  33. N. P. Seeram, L. S. Adams, Y. Zhang, R. Lee, D. Sand, H. S. Scheuller and D. Heber, J. Agric. Food Chem., 2006, 54, 9329–9339 CrossRef CAS PubMed.
  34. T. K. McGhie, D. R. Rowan and P. J. Edwards, J. Agric. Food Chem., 2006, 54, 8756–8761 CrossRef CAS PubMed.
  35. D. Mueller, K. Jung, M. Winter, D. Rogoll, R. Melcher and E. Richling, Food Chem., 2017, 231, 275–286 CrossRef CAS PubMed.
  36. L. Paudel, F. J. Wyzgoski, M. M. Giusti, J. L. Johnson, P. L. Rinaldi, J. C. Scheerens, A. M. Chanon, J. A. Bomser, A. R. Miller, J. K. Hardy and R. N. Reese, J. Agric. Food Chem., 2014, 62, 1989–1998 CrossRef CAS PubMed.
  37. A. González-Sarrías, J. A. Giménez-Bastida, M. T. García-Conesa, M. B. Gómez-Sánchez, N. V. García-Talavera, A. Gil-Izquierdo, C. Sánchez-Alvarez, L. O. Fontana-Compiano, J. P. Morga-Egea, F. A. Pastor-Quirante, F. Martínez-Díaz, F. A. Tomás-Barberán and J. C. Espín, Mol. Nutr. Food Res., 2010, 54, 311–322 Search PubMed.
  38. J. A. Giménez-Bastida, A. González-Sarrías, M. Larrosa, F. A. Tomás-Barberán, J. C. Espín and M. T. García-Conesa, Mol. Nutr. Food Res., 2012, 56, 784–796 Search PubMed.
  39. J. C. Marques, A. J. Pinheiro, J. G. Araúj, R. A. de Oliveira, S. N. Silva, I. C. Abreu, E. M. de Sousa, E. S. Fernandes, A. D. Luchessi, V. N. Silbiger, R. Nicolete and L. G. Lima-Neto, Planta Med., 2016, 82, 1463–1467 CrossRef PubMed.
  40. M. M. Appeldoorn, J. P. Vincken, A. M. Aura, P. C. Hollman and H. Gruppen, J. Agric. Food Chem., 2009, 57, 1084–1092 CrossRef CAS PubMed.
  41. N. Brindani, P. Mena, L. Calani, I. Benzie, S. Choi, F. Brighenti, F. Zanardi, C. Curti and D. Del Rio, Mol. Nutr. Food Res., 2017, 61, 1700077 Search PubMed.
  42. Y. Xiao, Z. Hu, Z. Yin, Y. Zhou, T. Liu, X. Zhou and D. Chang, Tech. Front. Pharmacol., 2017, 8, 231 CrossRef PubMed.
  43. C. Czank, A. Cassidy, Q. Zhang, D. J. Morrison, T. Preston, P. A. Kroon, N. P. Botting and C. D. Kay, Am. J. Clin. Nutr., 2013, 97, 995–1003 CrossRef CAS PubMed.
  44. A. A. Franke, B. M. Halm, L. J. Custer, Y. Tatsumura and S. Hebshi, Am. J. Clin. Nutr., 2006, 84, 406–413 CAS.

This journal is © The Royal Society of Chemistry 2018