Cranberry Phenolic Compounds

Article pubs.acs.org/JAFC

Modulation of Strawberry/Cranberry Phenolic Compounds Glucuronidation by Co-Supplementation with Onion: Characterization of Phenolic Metabolites in Rat Plasma Using an Optimized μSPE−UHPLC-MS/MS Method Stéphanie Dudonné,†,‡ Pascal Dubé,†,‡ Geneviève Pilon,†,§ André Marette,†,§ Hélène Jacques,†,∥ John Weisnagel,¶ and Yves Desjardins*,†,‡ †

Institute of Nutrition and Functional Foods (INAF), Laval University, 2440 Boulevard Hochelaga, Québec (QC) G1V 0A6, Canada Research Center of Horticulture (CRH), Laval University, 2480 Boulevard Hochelaga, Québec (QC) G1V 0A6, Canada § Department of Medicine, Quebec Heart and Lung Institute (CRIUCPQ), Laval University, 2725 Chemin Ste-Foy, Québec (QC) G1V 4G5, Canada ∥ Department of Food and Nutrition Sciences, Laval University, 2425 Rue de l’Agriculture, Québec (QC) G1V 0A6, Canada ¶ Laval University Hospital Center (CHUL), Department of medicine, 2705 Boulevard Laurier, Québec (QC) G1V 4G2, Canada ‡

S Supporting Information *

ABSTRACT: Plant phenolic compounds are suggested to exert pharmacological activities in regards to obesity and type-2 diabetes, but their mode of action is poorly understood due to a lack of information about their bioavailability. This work aimed to study the bioavailability of GlucoPhenol phenolic compounds, a strawberry−cranberry extracts blend, by characterizing plasma phenolic profile in obese rats. A comparison was performed by co-supplementation with an onion extract. Using an optimized μSPE−UHPLC-MS/MS method, 21 phenolic metabolites were characterized, mostly conjugated metabolites and microbial degradation products of the native phenolic compounds. Their kinetic profiles revealed either an intestinal or hepatic formation. Among identified metabolites, isorhamnetin glucuronide sulfate was found in greater amount in plasma. Three glucuronidated conjugates of strawberry−cranberry phenolic compounds, p-hydroxybenzoic acid glucuronide, catechins glucuronide, and methyl catechins glucuronide were found in higher quantities when GlucoPhenol was ingested together with onion extract (+252%, +279%, and +118% respectively), suggesting a possible induction of glucuronidation processes by quercetin. This work allowed the characterization of actual phenolic metabolites generated in vivo following a phenolic intake, the analysis of their kinetics and suggested a possible synergistic activity of phenolic compounds for improving bioavailability. KEYWORDS: bioavailability, GlucoPhenol, μSPE, phenolic metabolites, UHPLC-MS/MS

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INTRODUCTION In recent years, phenolic compounds have been reported to possess various pharmacological actions, including antiobesity and antidiabetic actions.1 These compounds exert their effects through different mechanisms such as the reduction of oxidative stress and inflammatory processes, the improvement of glucose and lipid metabolism as well as sensitivity to insulin.1−3 Biological activities of phenolic compounds are known to be strongly dependent on their bioavailability, which is defined as the proportion of the nutrient that is digested, absorbed, and metabolized through normal pathways. Bioavailability differs greatly between the compounds, so that the ones most abundant in the diet are not necessarily those leading to the highest concentrations of metabolites circulating in the plasma. Moreover, the metabolites appearing in the circulation may not have the same bioactivity as that of parent compounds, often determined in vitro. The general metabolism of phenolic compounds is rather well understood.4,5 During food ingestion, phenolic compounds can be released from the food matrix by mastication and can be hydrolyzed in part by oral microbiota.6 In the stomach, most of © 2014 American Chemical Society

the phenolic compounds probably resist acid hydrolysis and arrive intact to the intestine, their major site of absorption.7 Mostly present as esters, glycosides, or polymers, phenolic compounds are very poorly absorbed in their native form and must be hydrolyzed. Phenolic glycosides are deglycosylated in the lumen by membrane-bound lactase-phlorhizin-hydrolase (LPH), and the released aglycones enter the epithelial cells by passive diffusion as a result of an increased lipophilicity.8 The phenolic glycosides may also be deglycosylated in epithelial cells by cytosolic β-glucosidase after they have been transported through the epithelium by sugar transporters such as SGLT1 (sodium-dependent glucose transporter).9 Once absorbed, the phenolic aglycones undergo methylation, glucuronidation, and sulfation.10 This conjugation process, which occurs mainly in liver, represents a metabolic detoxification process, which aims to facilitate the excretion of phenolic compounds by increasing their Received: Revised: Accepted: Published: 3244

November 6, 2013 March 3, 2014 March 14, 2014 March 14, 2014 dx.doi.org/10.1021/jf404965z | J. Agric. Food Chem. 2014, 62, 3244−3256

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hydrophilicity. Hepatic metabolites can then return to the intestinal lumen via the enterohepatic circulation and undergo further transformations. Then, luminal phenolic metabolites and phenolic compounds that have not been absorbed by the intestine reach the colon where they are subjected to microbial degradation.11 Microbial metabolites are absorbed from the colon and are also subjected to the conjugation processes in the liver, resulting in their glucuronidated and sulfated derivatives. Finally, the phenolic microbial metabolites are excreted from the body via urine as hepatic conjugates.4 The absorption, as well as the extent of hepatic conjugation and microbial deconjugation, is strongly affected by the structure of phenolic compounds.12 Although hepatic metabolism leads to the generation of numerous combinations of methylated, glucuronidated, and/or sulfated phenolic metabolites, colonic microbial metabolism follows a general pattern leading to a relatively small number of metabolites, mainly phenolic acids and derivatives of phenylpropionic and phenylacetic acids.5 Direct evidence of the bioavailability of phenolic compounds can be obtained by characterizing the profile of metabolites in the plasma. This requires the use of a sensitive analytical methodology such as UHPLC with tandem MS, associated with microelution SPE (μSPE) technology, which allows the characterization of numerous metabolites in biological fluids at low concentrations, in a small volume of sample and in a short time.13 Among foods rich in phenolic compounds, strawberry, cranberry, and onion have previously demonstrated some beneficial effects on blood glucose regulation in rodents,14−16 constituting a promising natural therapeutic approach in the prevention of obesity and type-2 diabetes. The purpose of the present work was thus to study the bioavailability of strawberry and cranberry phenolic compounds in a representative context of a diet-induced obesity model (rats fed a high-fat, high-sucrose diet) and compare it to a concomitant supplementation with an onion extract. Male Wistar rats were either administered a vehicle, a single intake of GlucoPhenol extract, or the same extract supplemented with an onion extract. Blood samples were collected pre- and postingestion, and plasma phenolic metabolites were characterized using an optimized UHPLC-MS/MS method, after their extraction with μSPE, and compared with the native phenolic composition of the studied extracts.

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(Canada). Ultrapure water was obtained from a Millipore MilliQ water purification system (Billerica, MA). Determination of Strawberry−Cranberry and Onion Extracts Phenolic Composition. Characterization of Anthocyanins. The anthocyanin composition of GP extract was analyzed as previously described17 by reverse-phase analytical HPLC using an Agilent 1100 series system (Santa Clara, CA). The separation was performed with a flow rate of 1 mL/min using a Develosil C18 reverse phase column (250 mm × 4 mm, 5 μm particle size), protected with an Ultrasep C18 guard column (Phenomenex, CA). A binary gradient of 5% formic acid in ultrapure water (solvent A) and methanol (solvent B) was as follows for the anthocyanins separation: 0−2 min, 5% B; 2−10 min, 5−20% B; 10−15 min, 20% B; 15−30 min, 20−25% B; 30− 35 min, 25% B; 35−50 min, 25−33% B; 50−55 min, 33% B; 55− 65 min, 33−36% B; 65−70 min, 36−45% B; 70−75 min, 45− 53% B; 75−80 min, 53−55% B; 80−84 min, 55−70% B; 84−88 min, 70−5% B; 88−90 min, 5% B. Chromatographic data were acquired at 520 nm, and the quantification was performed using pelargonidin 3-glucoside as standard. Characterization of Procyanidins. The procyanidin composition of GP extract was analyzed as previously described18 by normal-phase analytical HPLC using an Agilent 1260/1290 infinity system (Santa Clara, CA) equipped with a fluorescence detector. The separation was performed at 35 °C with a flow rate of 0.8 mL/min using a Develosil Diol column (250 mm × 4.6 mm, 5 μm particle size), protected with a Cyano SecurityGuard column (Phenomenex, CA). The elution was performed using a solvent system comprising solvents A (acetonitrile/acetic acid 98/2 v/v) and B (methanol/ultrapure water/acetic acid 95/3/2 v/v/v) mixed using a linear gradient from 0% to 40% B for 35 min, 40% to 100% B for 5 min, 100% isocratic B over 5 min, and 100% to 0% B for 5 min. The fluorescence was monitored at excitation and emission wavelengths of 230 and 321 nm. Procyanidins with degrees of polymerization (DP) from 1 to >10 were quantified using an external calibration curve of epicatechin, taking into account their relative response factors in fluorescence.19 Determination of Total Ellagitannins. Total ellagitannins were estimated by reverse-phase analytical HPLC quantification of released ellagic acid following acid hydrolysis. Briefly, GP extract was dissolved in 25 mL of 50% methanol in ultrapure water containing ascorbic acid and hydrochloric acid (final concentrations of 10 mM and 1.2 M respectively). The mixture was heated to 85 °C in the dark for 2 h and then cooled in ice for 5 min. Ellagic acid separation was performed on an Agilent 1100 series HPLC system (Santa Clara, CA) using a Develosil C18 reverse phase column (250 mm × 4 mm, 5 μm particle size), protected with an Ultrasep C18 guard column (Phenomenex, CA, USA). The elution was performed at a flow rate of 1 mL/min using a solvent system comprising solvents A (methanol/ ultrapure water/acetic acid 10/88/2 v/v/v) and B (methanol/ ultrapure water/acetic acid 90/8/2 v/v/v) mixed using a gradient as follows: 0−15 min, 0−15% B; 15−25 min, 15−50% B; 25−34 min, 50−70% B; 34−35 min, 70−0% B. Chromatographic data were acquired at 250 nm, and the quantification was performed using ellagic acid standard. Characterization of Phenolic Acids and Flavonoids. Phenolic acids and flavonoids were characterized using a Waters Acquity UPLC-MS/MS equipped with an H-Class quaternary pump system, a flow through needle (FTN) sample manager system, and a column manager. The MS detector was a TQD mass spectrometer equipped with a Z-spray electrospray

MATERIALS AND METHODS

Plant Material. Strawberry-cranberry (Fragaria × ananassa, Duch. cv. authentique, and Vaccinium macrocarpon L.) extracts blend GlucoPhenol (GP) and onion (Allium cepa L.) extract were provided by Nutra Canada company (total phenolic content of 18% and 45% dry weight respectively, as determined by Folin−Ciocalteu method). Chemicals. The following phenolic standards were purchased from Sigma-Aldrich (St. Louis, MO): ellagic acid, 5caffeoylquinic acid, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, vanillic acid, gallic acid, quercetin, quercetin 3glucoside, kaempferol, myricetin, isorhamnetin, catechin, epicatechin, and rosmarinic acid. Ascorbic acid, citric acid, and hydrochloric acid were also purchased from Sigma-Aldrich (St. Louis, MO). Pelargonidin 3-glucoside was obtained from Extrasynthèse (France). Liquid chromatography grade solvents acetone, methanol, and acetonitrile were purchased from EMD Millipore Chemicals (Billerica, MA), and formic acid, glacial acetic acid, and phosphoric acid were obtained from Anachemia 3245

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Table 1. Description of Phenolic Supplementation Treatments: Dose of Extract, Equivalent Phenolic Content, and Total Phenolic Intake (mg/kg of Weight Body)a dose of extract (mg/kg)

a

group identification

GlucoPhenol

(1) control (2) 2.7 mg/kg GP (3) 5.4 mg/kg GP (4) 27 mg/kg GP (5) 36 mg/kg GP (6) 36 mg/kg GP + O

15 30 150 200 30

dose of phenolic compounds (mg/kg)

onion

GlucoPhenol

68

2.7 5.4 27 36 5.4

onion

total phenolic intake (mg/kg)

30.6

0 2.7 5.4 27 36 36

The experimental groups are numbered from (1) to (6).

(Milford, MA) were preconditioned using 250 μL of methanol and 250 μL of 0.2% acetic acid. Plasma samples (20 to 200 μL) were mixed with 4% phosphoric acid in ultrapure water (v/v) to disrupt phenol−protein binding prior loading into the plates. The loaded plates were washed with 200 μL of ultrapure water and 200 μL 0.2% acetic acid, and the retained phenolic compounds were then eluted with 2 × 50 μL of acetone/ ultrapure water/acetic acid solution (AWA) 70/29.5/0.5 v/v/v. Rosmarinic acid (1 μg/mL final concentration) was added in AWA elution solution and used as internal standard to check the homogeneity of the SPE extraction procedure. The eluted solutions, in the collecting plates, were directly injected in UHPLC-MS/MS for phenolic acids and nonanthocyanins flavonoids analysis (negative mode). The extracts were then lyophilized and dissolved in 30 μL of 20% methanol 0.1% acetic acid for anthocyanins analysis (positive mode). Characterization of Metabolites by UHPLC-MS. The analysis of plasma extracted phenolic metabolites was achieved with UHPLC-MS/MS using a previously developed methodology for the detection of anthocyanins and procyanidins,13 slightly modified to also allow the detection of phenolic acids and flavonols. The separation of phenolic acids and nonanthocyanins flavonoids was performed at 30 °C using an Agilent Plus C18 column (2.1 mm × 100 mm, 1.8 μm) (Santa Clara, CA). A flow rate of 0.4 mL/min was used with a mobile phase consisting of 0.2% acetic acid in ultrapure water and acetonitrile (solvent A and B, respectively) following this gradient elution: 0−8 min, 5− 50% B; 8−9.10 min, 50−90% B; 9.10−10 min, 90% B; 10−10.10 min, 90−5% B; 10.10−13 min, 5% B. The MS/MS analyses were carried out in negative mode using electrospray source parameters as follows: electrospray capillary voltage was 3.01 kV, source temperature was 150 °C, desolvation temperature was 400 °C, and cone and desolvation gas flows were 80 l/h and 800 l/h, respectively. The separation of anthocyanins was performed at 30 °C with a flow rate of 0.45 mL/min with a mobile phase consisting of 10% acetic acid in ultrapure water and acetonitrile (solvent A and B, respectively) using a gradient elution as follows: 0−3.50 min, 2− 50% B; 3.50−4 min, 50−90% B; 4−4.50 min, 90% B; 4.50−4.55 min, 90−2% B; 4.55−6 min, 2% B. The MS/MS analyses were carried out in positive mode using electrospray source parameters as follows: electrospray capillary voltage was 1.3 kV, source temperature was 130 °C, desolvation temperature was 350 °C, and cone and desolvation gas flows were 80 l/h and 900 l/h, respectively. Cone voltage and collision energy parameters were optimized for each compound. The identification of metabolites was performed by comparing their retention times with those of available phenolic standards and/or analyzing their fragmenta-

interface. The analysis was achieved using an Agilent Plus C18 column (2.1 mm × 100 mm, 1.8 μm) (Santa Clara, CA). The separation was performed at 40 °C at a flow rate of 0.4 mL/min with a mobile phase consisting of 0.1% formic acid in ultrapure water and acetonitrile (solvent A and B, respectively) using a gradient elution as follows: 0−4.5 min, 5−20% B; 4.5−6.45 min, 20% B; 6.45−13.5 min, 20−45% B; 13.5−16.5 min, 45−100% B; 16.5−19.5 min, 100% B; 19.5−19.52 min, 100−5% B; 19.52− 22.5 min, 5% B. The MS/MS analyses were carried out in negative mode using electrospray source parameters as follows: electrospray capillary voltage was 2.5 kV, source temperature was 140 °C, desolvation temperature was 350 °C, cone and desolvation gas flows were 80 l/h and 900 l/h, respectively. Data were acquired through multiple reaction monitoring (MRM) using Waters Masslynx V4.1 software. Phenolic standards were analyzed using the same parameters and used for the quantification, when available. Otherwise, the phenolic compounds were quantified using their aglycone or the most similar phenolic structure. Animals and Plasma Preparation. Sixty male Wistar rats (Charles River, St. Constant, QC) were placed in temperatureand humidity-controlled rooms (21 ± 2 °C, 35−40%), with a daily 12h−12h light−dark cycle. Animal facilities met the guidelines of the Canadian Council on Animal Care, and the protocols were approved by the Animal Care Committee of Laval University (reference CPAUL-2011-111). Animals were acclimated to their environment for a minimum of 5 days and consumed a nonpurified rodent diet ad libitum (rodent chow no. 2918, Harlan Teklad, Madison, WI). All animals had continuous access to tap water. After the acclimation period, rats were fed a high-fat, high-sucrose (HFHS) diet containing 27% sucrose and 40% fat for 7−8 days. The day before the test, rats were fasted for 12 h. The animals were randomly allocated to 6 groups (9 to 12 per group, Table 1). A first control group (group 1) consisted of animals which only received the vehicle (0.1% citric acid in water). The animals of four groups (groups 2−5) were administered a single dose of GP extract corresponding to a phenol intake of 2.7, 5.4, 27, and 36 mg/kg, respectively. The animals of an additional experimental group (group 6) were administered a single dose of GP extract supplemented with onion extract, corresponding to a total phenol intake of 36 mg/kg (5.4 mg/kg from GP extract and 30.6 mg/kg from onion extract). Blood samples were collected from the saphenous vein with EDTA-containing syringes preingestion and at 30, 60, 120, 180, and 360 min postingestion. Plasma samples were obtained by centrifugation (3500 rpm, 10 min at 4 °C) and stored at −80 °C until analysis. Extraction of Plasma Phenolic Compounds. Phenolic compounds extraction was realized according to a previously described methodology.13 Waters OASIS HLB μelution plates 3246

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Table 2. Phenolic Standards, MRM Transitions, and Optimized MS Parameters Used for the Analysis of Native Phenolic Compounds and Their Metabolites by UHPLC-MS compound

standard for quantification

ionization mode

MRM

cone voltage (V)

collision energy (eV)

phenolic acids p-hydroxybenzoic acid p-hydroxybenzoic acid glucuronide dimethyl ellagic acid glucuronide protocatechuic acid p-coumaric acid

p-hydroxybenzoic acid p-hydroxybenzoic acid ellagic acid protocatechuic acid p-coumaric acid

negative negative negative negative negative

137 > 93 329 > 153 505 > 329 153 > 109 163 > 119

35 40 40 29 35

15 25 22 11 15

pelargonidin 3-glucoside peonidin 3-galactoside

pelargonidin 3-glucoside pelargonidin 3-glucoside

positive positive

433 > 271 463 > 301

40 40

25 25

catechin - epicatechin catechins glucuronide methyl catechins glucuronide methyl catechins sulfate

epicatechin epicatechin epicatechin epicatechin

negative negative negative negative

289 > 245 465 > 289 479 > 303 383 > 303

42 40 40 45

13 22 22 20

quercetin quercetin glucuronide quercetin diglucuronide quercetin sulfate isorhamnetin glucuronide isorhamnetin diglucuronide isorhamnetin sulfate isorhamnetin glucuronide sulfate myricetin glucuronide myricetin diglucuronide

quercetin quercetin 3-glucoside quercetin 3-glucoside quercetin 3-glucoside quercetin 3-glucoside quercetin 3-glucoside quercetin 3-glucoside quercetin 3-glucoside quercetin 3-glucoside quercetin 3-glucoside

negative negative negative negative negative negative negative negative negative negative

301 > 150 477 > 301 653 > 301 381 > 301 491 > 315 667 > 315 395 > 315 571 > 315 493 > 317 669 > 317

42 40 40 40 40 40 40 40 40 40

20 22 22 20 22 22 22 22 22 22

anthocyanins

flavan-3-ols

flavonols

is presented in Table 3. GP extract contained 0.8% of anthocyanins, with the main presence of pelargonidin 3glucoside. This extract also contained about 3% of total procyanidins with a large spectrum of degree of polymerization, the monomers catechin and epicatechin representing around 0.1%. The extract presented a total ellagitannins content of 1.8%, including 0.8% of free ellagic acid. About 1.3% of phenolic acids were also identified in this extract, mainly chlorogenic acid and pcoumaric acid, and 1.3% of flavonols with the main presence of quercetin 3-glucoside. Conversely, onion extract did not contain any anthocyanins, procyanidins, or ellagitannins and contained almost exclusively aglycone quercetin and protocatechuic acid (about 14% and 8%, respectively). Chromatographic profiles of anthocyanidins and procyanidins of GP extract are available as Supplemental Figures S1 and S2, respectively. Rat Plasma Phenolic Content. The evolution of the total phenolic concentration in rat plasma after ingestion of the extracts is presented in Figure 1A. The presence of phenolic compounds was detected in all treated groups, with a maximum phenolic concentration ranging from 0.5 to 8 mg/L in GP groups (groups 2−5; phenolic intake from 2.7 to 36 mg/kg) and 10 mg/ L for the onion supplemented GP group (group 6; phenolic intake of 36 mg/kg). The kinetic profile of plasma total phenolic metabolites strongly differed depending on the phenolic source. GP-fed animals presented a high phenolic content 30 min after ingestion of the extract, followed by a rapid elimination of the metabolites by the organism (>85% over 360 min). GP/onion fed animals also presented a high phenolic content 30 min after ingestion of the mix, but the metabolites concentration remained high over time. Interestingly, the groups 5 and 6, which have ingested both the same dose of phenolic compounds (36 mg/kg) but from different sources, had the same phenolic concentration 30 min postingestion (about 8 mg/L; p = 0.7829) but a 10-fold

tion. Data were acquired through the multiple reaction monitoring (MRM) mode, tracking the transition of parent product ion specific for each compound. The detected metabolites were quantified using standards when available. Otherwise, a relative quantitation was achieved using the calibration curve of their aglycone or most similar phenolic structure. The phenolic standards, the MRM transitions, and the optimized MS parameters used for the characterization of phenolic metabolites in plasma are listed in Table 2. Data Analysis. All phenolic characterizations were carried out in triplicate, and results were expressed as percentage of extract weight ± standard deviation (SD). Time 0 values being very close to 0, phenolic concentrations in rat plasma were expressed as variation of concentration from baseline (Δ mg/L for total phenolic content and Δ nM or Δ μM for individual phenolic compounds). Metabolites quantification was expressed as area under the plasma concentration (μM) time (min) curve (AUC), with AUC calculated according to the linear trapezoidal rule. Results were expressed as means ± standard error (SEM) of the mean. The main effect of phenol intake on AUC values was estimated by one-way analysis of variance (ANOVA) using the MIXED procedure in SAS 9.3 (Cary, NC). When necessary, log transformation was applied on raw data to meet the criteria for normality. A priori contrasts between selected groups were analyzed using the LSD method. The REG procedure of SAS was used to analyze if plasma metabolites concentrations (determined as AUC) followed a linear regression model with GP phenol intake and to obtain the determination coefficients (R2). Differences were considered to be significant at p < 0.05.

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RESULTS Phenolic Composition of Strawberry−Cranberry and Onion Extracts. The phenolic composition of the two extracts 3247

dx.doi.org/10.1021/jf404965z | J. Agric. Food Chem. 2014, 62, 3244−3256

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Table 3. Phenolic Composition of GlucoPhenol and Onion Extractsa content (% dry weight) total phenolic content phenolic acids ellagic acid chlorogenic acid protocatechuic acid p-hydroxybenzoic acid p-coumaric acid vanillic acid caffeoyl glucoside feruloyl glucoside coumaroyl glucoside flavonoids flavonols quercetin quercetin 3-glucoside quercetin diglucoside quercetin galactoside quercetin rhamnoside quercetin xyloside quercetin arabinoside kaempferol kaempferol glucoside/galactoside myricetin myricetin glucoside/galactoside isorhamnetin total anthocyanins cyanidin 3-galactoside cyanidin 3-glucoside cyanidin 3-arabinoside pelargonidin 3-glucoside peonidin 3-galactoside peonidin 3-glucoside peonidin 3-arabinoside pelargonidin 3-(malonoyl)-glucoside flavan-3-ols catechin epicatechin total procyanidins monomers dimers trimers tetramers pentamers hexamers heptamers octamers nonamers polymers (DP > 10) total ellagitanins a

GlucoPhenol extract

onion extract

18 1.32 ± 0.04 0.78 ± 0.00 0.16 ± 0.01 0.04 ± 0.00