Vous êtes sur la page 1sur 14

Journal of Functional Foods 25 (2016) 8093

Available online at www.sciencedirect.com

ScienceDirect
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff

Human bioavailability and metabolism of


phenolic compounds from red wine enriched
with free or nano-encapsulated phenolic extract
Maria-Jos Motilva a,*, Alba Maci a, Maria-Paz Romero a,
Laura Rubi a, Merc Mercader b, Carolina Gonzlez-Ferrero c
a

Food Technology Department, Universitat de Lleida AGROTECNIO Center, Alcalde Rovira Roure 191, 25198
Lleida, Spain
b
Bodegas Miguel Torres S.A., Miquel Torres i Carb 6, 08720 Vilafranca del Peneds, Spain
c
Food Ingredients R&D&I, Centro Nacional de Tecnologa y Seguridad Alimentaria CNTA, Carretera NA134, km 53, 31570 San Adrin, Spain

A R T I C L E

I N F O

A B S T R A C T

Article history:

The impact of nano-encapsulation of a phenol extract from grape pomace on human plasma

Received 17 December 2015

pharmacokinetic parameters and urine clearance of phenolic metabolites was examined.

Received in revised form 17 May

A dealcoholised red wine was used as the vehicle for enrichment with both non-encapsulated

2016

and nano-encapsulated phenol extracts in a randomised cross-over pharmacokinetic study.

Accepted 24 May 2016

The analysis of plasma only showed an increase in the concentration of syringic acid sul-

Available online 31 May 2016

phate, catechin sulphate and catechin glucuronide, whereas urine data, especially at interval
of 06 hours, showed an increase in most of the metabolites from stilbenes, flavan-3-ols,

Keywords:

phenolic acids and anthocyanins after the intake of phenol-enriched wines compared with

Bioavailability

the control wine. The nano-encapsulation of the extract slightly enhanced the bioavailability

Encapsulation

of malvidin-3-O-glucoside, as observed in the higher urine excretion of its native form and

Metabolic pathways

its microbial metabolites syringic and gallic acids. Metabolic pathways of different pheno-

Phenolic compounds

lic groups were proposed, with special emphasis on novel pathways of hydroxytyrosol

Red wine

generation.
2016 Elsevier Ltd. All rights reserved.

1.

Introduction

The concept of the French paradox was first described by


Renaud and Lorgeril in 1992. Moderate wine drinking is part
of the Mediterranean Diet, together with abundant and variable plant foods, high consumption of cereals, olive oil as the
main (added) fat and a low intake of (red) meat. This healthy
diet pattern involves a Mediterranean way of drinking, which

is a regular and moderate wine consumption (up to two glasses


a day for men and one glass for women). This pattern of drinking increases longevity, reduces the risk of cardiovascular
diseases and does not appreciably influence the overall risk
of cancer (Giacosa et al., 2013). Moreover, it has been demonstrated recently that moderate consumption of red wine is
associated with a lower prevalence of the metabolic syndrome in an elderly Mediterranean population with a high
cardiovascular risk (Tresserra-Rimbau et al., 2015).

* Corresponding author. Food Technology Department, Universitat de Lleida AGROTECNIO Center, Alcalde Rovira Roure 191, 25198 Lleida,
Spain. Tel.: +34 973 702817; fax: +34 973 702596.
E-mail address: motilva@tecal.udl.es (M.-J. Motilva).
http://dx.doi.org/10.1016/j.jff.2016.05.013
1756-4646/ 2016 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 25 (2016) 8093

Phenolic compounds from wine can be classified into


flavonoids and non-flavonoids, their molecular weight
ranging from that of phenolic acids to highly polymerised
proanthocyanidins. The most abundant flavonoids in red wine
include flavan-3-ols (catechin and epicatechin), anthocyanins (malvidin-3-O-glucoside), flavonols (quercetin), as well
as lower proportions of flavanonols and flavones. The main
non-flavonoids in red wine are phenolic acids, such as
hydroxybenzoic acids (p-hydroxybenzoic and gallic acids) and
hydroxycinnamic acids (caffeic, caftaric and p-coumaric acids),
as well as other phenolic derivatives, including stilbenes
(resveratrol and its glucoside form piceid) (Woraratphoka,
Intarapichet, & Indrapichate, 2007). Wine production involves
the generation of huge amounts of by-products (Ayoub, de
Camargo, & Shahidi, 2016). These are a rich source of phenolic compounds and could be valorised for further use in the
production of functional food ingredients or supplements with
high nutritional value. Furthermore, this could also reduce the
environmental impact of wine making (Makris, Boskou, &
Andrikopoulos, 2007; Teixeira et al., 2014).
The sensorial characteristics of wines, including colour,
flavour, tastiness and body, as well as bitterness and astringency, are strongly affected by phenolic compounds. High
contents of these compounds in food could lead to rejection
by consumers. Moreover, due to low water solubility, phenolic compounds often have a poor bioavailability (Munin &
Edwards-Lvy, 2011) and are unstable in alkaline conditions,
such as those found in some biological fluids (Dube, Ng,
Nicolazzo, & Larson, 2010). In this sense, the emerging microencapsulation techniques would allow the stability and
bioavailability of the phenolic compounds to be improved and
the rate of active agent release to be controlled and could be
a way to avoid unpleasant taste.
Nano- or micro-encapsulation is the process by which core
materials enriched with bioactive compounds, such as antioxidants, enzymes, polyphenols and micronutrients, are packed
into wall materials to form capsules to be delivered to the controlled target site and to protect them from an adverse
environment (Gouin, 2004; Lee, Mijan, Ganesan, & Kwak, 2013).
Capsules ranging from 3 to 800 m in size are called microcapsules and the technology is called micro-encapsulation (Ahn,
Chang, & Kwak, 2010). If the particle size ranges from 10 to
1000 nm, these are called nanospheres and the technology is
termed nano-encapsulation (Lpez, Gavara, & Lagaron, 2006).
Nano- or micro-encapsulation technology is already well known
in the fields of medicinal, pharmaceutical and cosmetic
products and it is also emerging in the food industries (Huang,
Yu, & Ru, 2010). Over recent years, the main applications of encapsulation in the food industry have been focused on
improving the bioavailability of bioactive compounds, such as
vitamins A and E, isoflavones, phytosterols, lycopene, and lutein
(Gunasekaron & Ko, 2014).
Human bioavailability of flavanols and phenolic acids from
enriched cocoa-nut creams (Vitaglione et al., 2013), and
curcumin from enriched bread (Vitaglione et al., 2012), with free
or encapsulated polyphenols, has previously been reported. In
the case of wine, some studies have been performed regarding the encapsulation of grape by-products rich in phenolic
compounds, such as grape seeds (Zhang, Mou, & Du, 2007) or
grape pomace (Aizpurua-Olaizola et al., 2016). However, the

81

effect of nano-encapsulation on the bioavailability of wine polyphenols has yet to be studied. Moreover, although many studies
have focused on the bioavailability of specific phenolic compounds from wine, such as resveratrol or anthocyanins (Bell
et al., 2000; Urpi-Sarda et al., 2007; Vitaglione et al., 2005), a
complete pharmacokinetic study is needed that describes the
plasma and urine parameters, taking into account all the phenolic groups present in grape or wine products.
In the current study, the impact of the nano-encapsulation
of a phenol extract obtained from grape pomace on human
plasma pharmacokinetic parameters and urine clearance of
phenolic metabolites was investigated. For this purpose, a
dealcoholised red wine was used as the vehicle for enrichment with both non-encapsulated and nano-encapsulated grape
pomace extracts in a randomised cross-over human pharmacokinetic study. Additionally, a detailed study was performed
to identify new metabolites and elucidate the individual metabolic pathways of the different phenolic groups in red wine.

2.

Materials and methods

2.1.

Chemicals and reagents

Standards of catechol (as the internal standard (IS)),


p-hydroxybenzoic acid, protocatechuic acid, gallic acid, caffeic
acid, syringic acid, ferulic acid, epicatechin and catechin were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Quercetin, trans-resveratrol, dimer B2, cyanidin-3-O-glucoside and
malvidin-3-O-glucoside were purchased from Extrasynthese
(Genay, France) and p-coumaric acid and vanillic acid from Fluka
Co. (Buchs, Switzerland). Stock solutions of individual phenolic standard compounds were prepared by dissolving each
compound in methanol at a concentration of 1000 mg/L. These
were stored in amber glass flasks at 4 C. Standard stock mixes
of the phenolics were prepared weekly at a concentration of
50 mg/L dissolved in Milli-Q water.
Methanol (HPLC grade), acetonitrile (HPLC grade), acetone
and acetic acid were purchased from Scharlau Chemie
(Sentmenat, Barcelona, Spain). Ortho-phosphoric acid (85%) was
acquired from Panreac (Barcelona, Spain). Ultrapure water was
obtained from a Milli-Q water purification system (Millipore
Corp., Bedford, MA, USA).
The zein protein used for nano-encapsulation was supplied by CHEMOS GmbH & Co (Regenstauf, Germany). L-Lysine
was from Sigma-Aldrich and maltodextrin (MD) Glucidex 21D
was from Roquette Frres (Lestrem, France). Food grade ethanol
was purchased from Panreac.

2.2.

Dealcoholised red wine and phenolic extract

Dealcoholised red wine, which was used as a matrix for phenolic enrichment, and the phenolic extract, prepared from grape
pomace, were from Bodegas Miguel Torres, S.A. (Vilafranca del
Peneds, Barcelona, Spain). The phenolic extract was prepared from grape pomace by Bodegas Miguel Torres, S.A., and
the nano-encapsulation was performed by the CNTA (Centro
Nacional de Tecnologa y Seguridad Alimentaria, San Adrin,
Navarra, Spain). The phenolic extract was analysed by

82

Journal of Functional Foods 25 (2016) 8093

ultra-performance liquid chromatography coupled to tandem


mass spectrometry (UPLC-MS/MS), according to the method described in Section 2.5 below. Previously, this sample was filtered
through 0.22 m Nylon filters (Teknokroma, Barcelona, Spain).
The composition of the phenolic extract was made up of 52%
procyanidins (catechin, epicatechin, epigallocatechin,
epicatechin gallate, dimer and trimer), 22% other flavonoids
(mainly quercetin derivatives), 18% anthocyanins (malvidin,
delphinidin, peonidin and petunidin derivatives), 7% phenolic acids (mainly gallic, hydroxybenzoic, protocatechuic,
coumaric, coutaric, caffeic and caftaric acids) and 0.9% stilbenes (resveratrol and piceid). The lyophilised phenolic extract
was dissolved in the dealcoholised red wine at a proportion
of 1:3 (v/v).
The nano-encapsulation of the phenolic extract was performed according to the patent WO2012/007628 (Ageros Bazo
et al., 2012) developed by NUCAPS, a consortium of the CNTA,
the University of Navarra, Idifarma (Noin, Navarra, Spain) and
Cinfa (Huarte, Navarra, Spain) Laboratories. The nanoparticles
comprise a zein matrix and a basic amino acid. Briefly, zein
and L-lysine were dissolved in ethanol 70% (v/v). After
homogenisation, the phenolic extract, previously dissolved in
Milli-Q water, was added at the recommended mass ratio. The
magnetic agitation was maintained while enough Milli-Q water
to induce the desolvation process was added. Once the formation of the zein nanoparticles containing phenolic extract
had been induced by the addition of the water, the dispersion
was spray dried using a Mini-Spray Dryer B-290 (Bchi, Flawil,
Switzerland) coupled to a heat exchanger, an inert loop B-295
from the same company.The inlet temperature was set at 90 C,
the feed flow was approximately 3.5 mL/min (10% pump rate)
and the volume flow around 37 m3/h (100% aspirator rate).
The control dealcoholised red wine, phenol enriched wine,
and encapsulated-phenol enriched wine were filtered through
0.22 m Nylon filters (Teknokroma, Barcelona, Spain) and
analysed by ultra-performance liquid chromatography coupled
to tandem mass spectrometry (UPLC-MS/MS), according to the
method described in Section 2.5 below.

2.3.

Human intervention study and design

The protocol of the study was approved by the Ethical Committee for Clinical Research at the Arnau Vilanova University
Hospital, Lleida, Spain (Approval Number: CEIC-1326). The inclusion criteria used for participants were age (between 18 and
65 years) and health status. The volunteers were assessed with
a screening questionnaire (body mass index between 18.5 and
30.0 kg/m2). They had to have standard haematology and be
non-smokers. Anyone with a history of gastrointestinal problems, hyperacidity, diabetes, hyperlipidaemia, etc., and taking
any nutritional supplements at the time of study was excluded. Based on previous parallel bioavailability studies (Borges
et al., 2010; Vitaglione et al., 2005), twelve healthy volunteers
(six women and six men, aged between 19 and 50) were selected for the study. The men had a mean weight of 82.3 (SD
9.7) kg and the women, a mean weight of 71.1 (SD 14.2) kg. The
BMI of the men was 26.4 (SD 3.5) kg/m2, and 26.1 (SD 3.8) kg/
m2 for the women. They gave written informed consent before
starting the experiment protocol. The volunteers were asked
to follow a diet free from polyphenols and alcohol for a week

before the intervention day, and two weeks between the interventions. Thus, they were recommended to exclude all
polyphenol-rich foods and beverages, such as red wine, berry
fruit, coffee and tea, from their diet.
A randomised, controlled, crossover trial with three treatment conditions was designed administering the following: (a)
W: 100 mL of dealcoholised red wine; (b) PEW: dealcoholised
red wine with non-encapsulated phenolic extract (1.3 g phenolic extract); and (c) NPEW: 100 mL of dealcoholised red wine
with nano-encapsulated phenolic extract (9 g and these containing 1.3 g of phenolic extract). To omit the inter-individual
variability and the baseline values plasma, each subject participated in three one-day experimental sessions separated by
a 2-week wash-out period (Fig. 1). On the day of the experiment, the subjects reached the laboratory after fasting for 12
hours and were randomised to receive the respective wine in
a blind way. After each intervention, the volunteers received
a standard breakfast that consisted of white bread and cooked
turkey breast.
Blood samples were taken before (0 h) and after the intervention at 30, 60, 120, 240, and 360 min (Fig. 1). These samples
were collected in 4 and 6 mL Vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing
ethylene-diaminetetraacetic acid (EDTA) as an anticoagulant.
The tubes were protected from light with aluminium foil, and
centrifuged at 3416 g for 10 min (Hettich, Tuttlingen, Germany).
The plasma was then immediately separated from the cells
and stored at 80 C until the chromatographic analysis. Urine
samples were collected from each volunteer at 06, 612 and
1224 h after the three interventions, the urine volume was
measured and the samples were stored at 80 C until the chromatographic analysis.

2.4.
Plasma and urine samples clean-up and liquid
chromatography analysis
The phenolic metabolites from the plasma samples were extracted with the solid phase extraction (SPE) technique by using
OASIS HLB 60 mg cartridges (Waters Corp., Milford, MA, USA)
as reported by Serra et al. (2009). Briefly, the cartridges were
conditioned sequentially with 5 mL of methanol and 5 mL of
0.2% acetic acid. Twenty microlitres of phosphoric acid 85% and
50 L catechol (IS) at a concentration of 20 mg/L were added
to 1 mL of plasma sample. This solution was centrifuged at
8784 g for 5 min at room temperature. The supernatant was
loaded into the cartridge. The loaded cartridges were washed
with 3 mL of Milli-Q water and 5 mL of 0.2% acetic acid. The
retained phenolic compounds were then eluted with 4 mL of
solution acetone/Milli-Q water/acetic acid (70:29.5:0.5, v/v/v).
The elution solvent was evaporated to dryness under a nitrogen stream in an evaporating unit at 30 C (Pierce, Rockford,
IL, USA) and reconstituted with 100 L of elution solution.
Finally, the extract was filtered through a 0.22 m pore size,
4 mm diameter nylon syringe filter (Teknokroma, Barcelona,
Spain) and transferred to the autosampler vial before the chromatographic analysis. The injection volume was 2.5 L.
Regarding the urine samples, 5 mL per sample was freezedried with a Lyobeta 15 unit (ImaTelstar, Terrassa, Spain) and
then rehydrated with 1 mL of Milli-Q water for preconcentration.
The phenolic metabolites were then extracted with 30 m OASIS

83

Journal of Functional Foods 25 (2016) 8093

W: 100 mL wine

PEW: 100 mL wine + extract

NPEW: 100 mL wine + encapsulated extract

2nd ingestion

1st ingestion

3nd ingestion

Wash-out
(2 weeks)

n = 12

Wash-out
(2 weeks)

Blood (S)
Urine (O)
0h
S1
O1

30
S2

1h

2h

4h

6h

12h

24h

S3

S4

S5

S6
O2

O3

O4

Fig. 1 Study design.

HLB Elution plates (Waters Corp., Milford, MA). These were conditioned sequentially with 250 L of methanol and 250 L of
0.2% acetic acid. Fifty microlitres of phosphoric acid 4% and
50 L catechol (IS) at a concentration of 20 mg/L, which was
prepared in phosphoric acid 4%, was added to 100 L of the
rehydrated urine sample. This solution was centrifuged at 8784 g
for 5 min at 4 C. The supernatant was loaded onto the plate.
Then, the retained phenolic metabolites were eluted with
2 50 L of acetone/Milli-Q water/acetic acid (70:29.5:0.5, v/v/
v). The elution solution (2.5 L) was directly injected into the
chromatographic system. Three replicates of each sample were
performed and each replicate was analysed once.

2.5.

UPLC-ESI-MS/MS chromatographic analysis

The phenolic compounds and their generated biological metabolites were analysed with an Acquity Ultra-Perfomance
liquid chromatography (UPLC) system equipped with a binary
pump system (Waters). The analytical column was an Acquity
UPLC BEH C18 (100 2.1 mm, 1.7 m) from Waters. During the
analysis, the column was kept at 30 C. Two different mobile
phases were used to analyse anthocyanins and the rest of the
phenolic compounds. The anthocyanins were separated with
a mobile phase that consisted of 10% acetic acid (eluent A) and
acetonitrile (eluent B), and the rest of the phenolic compounds, with 0.2% acetic acid (eluent A) and acetonitrile (eluent
B). For the analysis of the anthocyanins, the flow-rate was
0.35 mL/min and the gradient elution was as follows: 010 min,
325% B; 1010.10 min, 2580% B; 10.1011 min, 80% B isocratic;
1111.10 min, 8083% B; 11.1012.50 min, 3% B isocratic. On the
other hand, for the analysis of the other phenolic compounds, the flow-rate was 0.3 mL/min, and the gradient elution
as follows: 05 min, 510% B; 510 min, 1012.40% B; 10
18 min, 12.4028% B; 1823 min, 2860% B; 2325 min, 60% B
isocratic; 2527 min, 6065% B; 2730 min, 5% B isocratic.

The tandem MS (MS/MS) analyses were carried out on a


triple quadrupole detector (TQD) mass spectrometer (Waters)
equipped with a Z-spray electrospray interface. The anthocyanins were analysed in the positive mode, and the rest of the
phenolic compounds in the negative mode, and the data were
acquired through selected reaction monitoring (SRM). The
ionisation source parameters were capillary voltage, 3 KV; source
temperature, 150 C; cone gas flow rate, 80 L/h and desolvation
gas flow rate, 800 L/h; desolvation temperature, 400 C. Nitrogen (>99% purity) and argon (99 % purity) were used as the
nebulising and collision gases, respectively. The SRM transitions and individual cone voltage and collision energy for each
phenolic compound were evaluated by infusion of a standard solution of 10 mg/L of each standard at a flow rate of 10 L/
min. Two SRM transitions were studied, the most sensitive
transition being selected for quantification and the second one
for confirmation purposes. Table S1 in the Supporting information shows the MS/MS transitions for quantification and
confirmation, as well as the cone voltage and collision energy
values optimised for each of the phenolic compounds determined in the control, phenol-enriched, and encapsulated
phenol-enriched wines, and the phenolic metabolites generated after each intervention in the plasma and urine samples.
The dwell time established for each transition was 30 ms. Data
acquisition was carried out with the MassLynx v 4.1 software.

2.6.

Instrumental quality parameters

The instrumental quality of the method, such as linearity, calibration curve, repeatability, accuracy, quantification limit (LOQ),
and detection limit (LOD) for the determination of the phenolic compounds studied in spiked blank plasma and urine
samples is shown in Table S2 in the Supporting information.
These parameters were determined as reported in our previous study (Mart et al., 2010). A pool of blank plasma and urine

84

Journal of Functional Foods 25 (2016) 8093

was obtained before the phenolic wine was ingested. The extraction recovery (%R) was higher than 75% and matrix effect
(%ME), lower than 17% for all the phenolic compounds studied
in the spiked urine and plasma samples (data not shown).
On the other hand, the quantification of the phenolic extract,
control dealcoholised red wine, phenol enriched wine, and
encapsulated-phenol enriched wine, was done by preparing the
phenolic compounds with the acetone/Milli-Q water/acetic acid
(70:29.5:0.5, v/v/v) solution at different concentration levels. The
instrumental quality parameters are not shown.

2.7.

To ensure the effectiveness of the nano-encapsulation of


the grape pomace extract, the mass ratio (zein/phenolic extract)
used in the present study was 3/1 (w/w). This ratio was
minimised to avoid incorporating a larger amount of exogenous material into the wine and to prevent any detrimental
interaction between the maize protein and such a complex food
matrix as wine. Despite that, due to the low concentration of
phenolic compounds in the initial wine extract, a high quantity of nanoparticles was incorporated into the enriched wine
when the extract was encapsulated and, as a result, slight turbidity appeared in the wine.

Statistical analysis and pharmacokinetic parameters

The statistical analysis was performed using the Statgraphics


plus V.5.1 software (Manugistics Inc., Rockville, MA). The data
were analysed via analysis of variance, excluding extreme outliers (ANOVA) followed by the post-hoc Tukeys test (intertreatment comparison). Values of p lower than 0.05 were
regarded as statistically significant.
The kinetic parameters of the main metabolites of the wine
phenolic compounds were calculated by means of pharmacokinetic (PK) functions (for Microsoft Excel). The area under
the plasma concentrationtime curve (AUC) was calculated using
the trapezoidal rule method. The peak plasma concentration
(Cmax) and time to maximum plasma concentration (tmax) were
the observed values. The data on the pharmacokinetic parameters are presented as mean values (n = 3) standard deviation.
The mean and standard deviation included all observations.

3.

Results and discussion

3.1.

Encapsulated phenol-enriched wine issues

To study differences in the bioavailability of phenolic compounds in a complex phenolic extract, zein nanoparticles were
selected as the coating material. This decision was based on
previous work carried out by the CNTA (Centro Nacional de
Tecnologa y Seguridad Alimentaria, San Adrin, Navarra, Spain)
on the nanoencapsulation of isolated compounds (patent
WO2012/007628 developed by NUCAPS, a consortium of the
CNTA, the University of Navarra, Idifarma and Cinfa Laboratories (Spain)). Some of these compounds were related to the
physical-chemical nature of the extract used in the present
study, such as resveratrol and quercetin. In our patented
nanocapsules, one of the main advantages from the use of zein
nanoparticles for encapsulating resveratrol and quercetin was
the improvement of the bioavailability compared with free compounds. Additionally, the encapsulation masked undesired
flavour, which was another advantage of the nanoencapsulation,
as commented previously. The total amount of encapsulated
phenols in the nanoparticles was based on the shell material/
core (w/w) ratio optimised according to the protocol described
in the patented process. The dose of encapsulated phenols to
be added to the wine was selected according to previous assays
in humans carried out by the company Bodegas Miguel Torres,
S.A. (Vilafranca del Peneds, Barcelona, Spain) with unencapsulated extract, in which a minimum active dose was set. The
dose of both the free phenols and encapsulated phenols was
the same in the present study.

3.2.
Plasma kinetics and urinary excretion of wine phenol
metabolites
The doses of phenolic compounds (mg phenol/100 mL wine)
ingested through the control wine (W) and the phenol-enriched
wines with non-encapsulated (PEW) and nano-encapsulated
(NPEW) phenolic extracts at equimolar dose are detailed in
Table 1. The total phenolics administered through the control
wine and phenol-enriched wines were 76.8 and 97.0 mg/
100 mL, respectively. The phenolic extract used for the
enrichment basically provided higher amounts of procyanidins
in the phenol-enriched wines.
The pharmacokinetic parameters Cmax and AUC, corresponding to the main phenolic metabolites detected in plasma
following a single dose of the three wines, are summarised in
Table 2. Fig. 2A shows the pharmacokinetic curves derived from
the sum of the phenolic metabolites of the different phenolic
groups (phenolic acids, stilbenes, flavan-3-ols, phenyl alcohols and anthocyanins) determined in the plasma samples from
0 to 360 min. As can be observed, most of the phenolic compounds studied underwent an intense phase II metabolism,
and the sulphated form predominated over the glucuronidated
one. After the three wine interventions, the main phenolic metabolites detected in the plasma samples were syringic acid
sulphate, resveratrol sulphate and hydroxytyrosol sulphate
(Table 2), but only catechin metabolites (sulphate and glucuronide conjugates) reached significantly higher Cmax (p < 0.05)
following the intake of PEW and NPEW (in the case of the catechin glucuronide) compared to the control wine (W). In terms
of Cmax, the nano-encapsulation of the phenolic extract did not
significantly enhance the bioavailability of phenols as no differences were observed in the Cmax values when PEW and NPEW
were compared.
Regarding the AUC results, apart from catechin metabolites (sulphate and glucuronide), syringic acid sulphate was
significantly increased after PEW (p < 0.05) when compared to
the control wine (W). It is remarkable that many other phenolic metabolites tended to increase after the administration
of phenol-enriched wines compared to the control wine
(Table 2), but these increases were not statistically significant. This fact was attributed to the high intervariability
observed among subjects, which has also been described in
other nutritional studies with a polyphenol-rich juice drink
(Borges et al., 2010) and phenol-enriched olive oils (Rubi et al.,
2012). Duthie (2011) proposed that the interindividual variability in phenol bioavailability could be explained by modulations
in the capacity of phase-II conjugating enzymes toward phenolic compounds exerted by polymorphisms, epigenetic and

Journal of Functional Foods 25 (2016) 8093

Table 1 Ingested dose of phenolic compounds through


the non-alcoholic wine and non-alcoholic enriched
wines with the free or nano-encapsulated phenolic
extract (n = 5). Results are expressed as mean
concentration standard deviation.
Phenolic compound

Anthocyanins
Cyanidin glucoside
Malvidin glucoside
Malvidin acetyl glucoside
Malvidin coumaroyl glucoside
Peonidin glucoside
Peonidin acetyl glucoside
Peonidin coumaroyl glucoside
Delphinidin glucoside
Delphinidin acetyl glucoside
Petunidin glucoside
Petunidin acetyl glucoside
Petunidin coumaroyl glucoside
Othersa
Total
Phenolic acids
Gallic acid
Gallic acid glucoside
Hydroxybenzoic acid
Protocatechuic acid
Coumaric acid
Coutaric acid
Coumaric acid ester hexoside
Caffeic acid
Caftaric acid
Fertaric acid
Syringic acid
Othersb
Total
Procyanidins
Catechin
Epicatechin
Epigallocatechin
Epicatechin gallato
Dimer
Trimer
Total
Other flavonoids
Quercetin
Quercetin glucoside
Quercetin glucuronide
Astilbin
Laricitrin glucoside
Syringetin glucoside
Total
Stilbenes
Resveratrol (trans + cis)
Piceid (trans + cis)
Total
Phenyl alcohol
Hydroxytyrosol
Tyrosol
Total
Total phenols (100 mL wine)
a

Non-alcoholic
wine
(mg/100 mL)

Non-alcoholic
phenol-enriched
wines (mg/100 mL)

0.18 0.00
11.3 0.02
3.61 0.13
0.94 0.02
1.43 0.01
0.62 0.02
0.24 0.01
1.12 0.03
0.35 0.02
1.34 0.03
0.47 0.03
0.12 0.00
0.44 0.00
22.1 0.32

0.04 0.00
14.4 0.37
4.77 0.15
1.61 0.05
1.61 0.07
0.62 0.01
0.45 0.00
0.32 0.02
0.22 0.01
1.26 0.05
0.43 0.01
0.19 0.00
0.42 0.00
26.3 0.74

4.29 0.36
1.23 0.00
1.58 0.03
3.98 0.02
0.38 0.03
1.77 0.01
0.55 0.01
3.68 0.23
3.42 0.05
0.52 0.02
0.18 0.00
0.65 0.07
22.2 0.83

7.52 0.03
1.96 0.02
0.14 0.01
3.43 0.01
0.23 0.01
1.52 0.24
0.46 0.04
3.56 0.00
3.57 0.41
0.63 0.06
0.29 0.01
0.77 0.06
24.1 0.90

3.08 0.09
1.88 0.04
0.66 0.02
n.d.
0.48 0.08
0.50 0.01
6.60 0.24

3.74 0.09
2.45 0.09
0.31 0.05
0.35 0.02
8.50 0.47
1.05 0.24
16.4 0.96

n.d.
0.16 0.01
13.88 1.54
2.17 0.02
2.60 0.02
1.70 0.08
20.5 1.67

1.03 0.03
0.13 0.00
17.87 1.23
2.18 0.04
2.48 0.15
1.11 0.04
24.8 1.49

0.96 0.02
2.25 0.09
3.21 0.11

0.92 0.01
2.33 0.08
3.25 0.09

0.33 0.02
1.90 0.11
2.23 0.13
76.8

0.31 0.04
1.80 0.14
2.11 0.18
97.0

Others: cyanidin acetyl glucoside, cyanidin coumaroyl glucoside, malvidin


arabinoside, malvidin glucoside vinilphenol, malvidin caffeoyl glucoside, malvidin glucoside acetaldehyde, delphinidin glucuronide, and
delphinidin coumaroyl glucoside.
Others: methyl gallate, ethyl gallate, vanillic acid, vanillic acid ester

hexoside, and ferulic acid.


n.d., not detected.

85

genetic individual factors. The similar phenolic dose administered with the control and phenol-enriched wines could also
partly explain the non-significant differences among wines in
the pharmacokinetic parameters.
The urine excretion of the same phenolic groups described in the plasma, together with valerolactone, at different
collection intervals (06, 612 and 1224 h) is represented in
Fig. 2B. In contrast to the plasma results, an improved
bioavailability, based on urine excretion, was observed after the
intake of the phenol-enriched wines compared to the control
wine in all the phenolic groups. This was mainly observed in
the first period of urine collection (0 to 6 h) in which the highest
amount of phenolic metabolites was excreted (Fig. 2B). Data
on the urinary molar excretion of individual phenolic metabolites were also expressed as mols/24 h (Table 3). Similarly to
what was observed in the plasma samples, the main phenolic compounds detected in the urine samples were phase-II
metabolites (sulphated and glucuronidated). Other metabolites, such as 3,4-dihydroxyphenylacetic acid and 5-(3,4dihydroxyphenyl)--valerolactone probably derived from the
colonic catabolism were also detected in the urine samples.
The highest urinary concentration after the consumption of
the three wines was observed for 5-(3,4-dihydroxyphenyl)-valerolactone, followed by 3,4-dihydroxyphenylacetic acid,
resveratrol sulphate and catechin sulphate. By comparing
these results with those reported in the literature, 5-(3,4dihydroxyphenyl)--valerolactone was also reported to be one
of the main microbial metabolites detected in urine samples
after a regular consumption of dealcoholised red wine
(Boto-Ordnez et al., 2013).
Regarding flavan-3-ols, glucuronide and sulphate conjugates of (epi)catechin and methyl (epi)catechin, and 5-(3,4dihydroxyphenyl)--valerolactone were the main phase-II and
microbial flavan-3-ols metabolites detected in the urine
samples. After phenol enrichment with the grape pomace
extract, the phenol-enriched wines mainly provided higher
amounts of procyanidins compared with the control wine. In
accordance with this, the most significant increase in the
urinary excretion of the phenolic metabolites was observed in
the group of flavan-3-ols. In this sense, the urine excretion of
catechin and methyl catechin (sulphate and glucuronide conjugates) was significantly higher (p < 0.05) after both phenolenriched wines (PEW and NPEW), while epicatechin and methyl
epicatechin (sulphate and glucuronide conjugates) urine excretion was only significantly higher after the NPEW. These
results are in line with the fact that flavonoids with different
stereochemistry can exhibit different bioavailability. (+)Catechin has been reported to have a higher bioavailability than
()-epicatechin, although they have the same molecular weight
(Baba et al., 2001; Ottaviani et al., 2011).
When comparing PEW and NPEW, the urine reflected a slight
enhancement of bioavailability with the encapsulation of the
phenolic extract, mainly reflected in the urinary molar excretion of the anthocyanin metabolites. Significant differences were
observed for syringic acid sulphate, syringic acid glucuronide
and malvidin-3-O-glucoside urine excretion, which was higher
(p < 0.05) after NPEW ingestion compared with PEW. As proposed in the next section, syringic acid could be formed by the
colonic metabolism of malvidin-3-O-glucoside, the most
common anthocyanin in red wine (Neveu et al., 2010). In

86

Journal of Functional Foods 25 (2016) 8093

Table 2 Plasma phenolic kinetic parameters expressed as Cmax (nM) and AUC (mol/L min1) determined after the intake
of 100 mL of the control wine (W) and the phenol-enriched wines containing an equimolar dose of free (PEW) or nanoencapsulated (NPEW) phenolic extracts (mean value standard deviation), n = 12.
Plasma phenols

Phenolic acids
Gallic acid sulphate
Gallic acid glucuronide
Syringic acid sulphate
Syringic acid glucuronide
Caffeic acid sulphate
Ferulic acid sulphate
Stilbenes
Resveratrol sulphate
Resveratrol glucuronide
Flavan-3-ols
Catechin sulphate
Epicatechin sulphate
Catechin glucuronide
Epicatechin glucuronide
Methyl catechin sulphate
Methyl epicatechin sulphate
Methyl catechin glucuronide
Methyl epicatechin glucuronide
Phenyl alcohols
Free hydroxytyrosol
Hydroxytyrosol sulphate
Anthocyanins
Malvidin glucoside

AUC (mol/L min1)

Cmax (nM)
Control
wine (W)

Phenolenriched
wine (PEW)

Encapsulatedphenol-enriched
wine (NPEW)

Control
wine (W)

Phenolenriched
wine (PEW)

Encapsulatedphenol-enriched
wine (NPEW)

76.8 33.3
n.d.
159 88.8
10.1 6.32
60.3 50.4
23.2 20.3

50.7 37.6
3.98 0.62
199 43.7
18.5 1.50
38.8 17.2
40.1 3.88

29.7 28.5
1.49 0.17
192 108
15.2 17.9
30.1 17.9
33.5 20.8

11.7 6.82
n.d.
19.3 6.68
1.22 0.53
5.77 2.29
4.54 2.27

11.4 5.88
0.41 0.12
29.2 10.5a
2.05 0.25
5.99 3.80
6.20 6.27

7.78 4.64
0.11 0.05
26.6 11.4
1.55 2.05
5.08 2.43
6.84 4.76

410 198
3.09 3.51

348 153
5.91 1.82

228 122
3.34 9.24

77.5 28.5
0.39 0.63

80.9 32.2
1.30 3.82

47.2 15.7a,b
0.92 2.95

62.5 23.4
63.4 21.9
n.d.
45.9 12.1
17.9 9.45
24.1 15.2
16.7 16.1
8.3 15.8

92.8 23.4a
71.2 35.8
9.96 5.21a
59.1 12.9
14.0 9.45
25.9 12.1
22.9 20.5
16.7 1.50

68.0 20.3
64.4 22.3
4.64 1.34a
63.4 28.9
10.8 9.62
15.2 16.7
24.4 22.1
23.3 18.7

9.01 4.40
11.8 5.81
n.d.
8.44 6.00
0.89 1.15
1.53 1.74
1.82 3.05
0.93 2.08

14.0 7.49a
16.2 9.12
0.71 1.18a
13.9 7.44
1.37 2.18
5.47 1.96
3.92 0.90
2.27 0.80

8.75 6.28
9.93 8.18
0.06 0.21
12.4 8.09
1.08 2.07
2.36 4.21
3.84 4.37
1.97 3.49

45.9 16.7
333 219

35.2 12.9
374 166

39.7 28.2
459 224

8.32 2.70
54.5 40.5

6.41 2.35
56.3 46.3

5.82 3.07
43.6 23.4

7.01 6.93

8.04 7.90

3.61 3.20a,b

1.02 0.66

1.38 1.50

0.55 0.24a,b

Mean values PEW or NPEW significantly different from W (p < 0.05).


Mean values significantly different between the two phenol-enriched wines, PEW and NPEW (p < 0.05).
n.d., not detected.
a

accordance with our results, recently, the stability of anthocyanins in in-vitro fermentation was increased by encapsulation
with cyclodextrins (CDs), which could ensure steady and sustained release of anthocyanins in the colon, and therefore,
improve their bioavailability in vivo (Flores et al., 2015).
Regarding the resveratrol metabolites, both the glucuronide and sulphate forms were detected in the urine samples
after the three wine interventions and the urine excretion was
significantly higher after the NPEW compared to control wine.
These resveratrol metabolites were also determined in the 24 hurine samples after a long-term consumption of red wine and
dealcoholised red wine for 28 days (Rotches-Ribalta et al., 2012),
both an acute and long-term (15-day) consumption of a functional beverage enriched with grape extract (Rotches-Ribalta,
Urpi-Sarda, Mercader Mart, Reglero, & Andrs-Lacueva, 2014),
after consumption of red wine (250 mL) (Urpi-Sarda et al., 2007)
and after an oral dose of resveratrol (1 g) (Boocock et al., 2007).
In our study, the sulphate conjugates predominated over the
glucuronide conjugates in all three wine interventions. After
consumption of the NPEW, the urinary excretions of resveratrol
sulphate and resveratrol glucuronide were 45.1 18.4 and
6.06 4.31 mols/24 h (Table 3), respectively. This agreed with
the results reported in the literature after the consumption of
resveratrol or red wine in which the sulphate forms were also
predominant (Boocock et al., 2007; Urpi-Sarda et al., 2007). Nevertheless, in other studies, the glucuronide forms were reported

to predominate over the sulphate forms excreted in 24 hurine after the moderate consumption of red wine or
dealcoholised red wine for 28 days (Rotches-Ribalta et al., 2012,
2014). Interestingly, the results of these studies showed no differences between the two interventions (red wine or
dealcoholised red wine), indicating that the bioavailability and
biotransformation of resveratrol is not affected by the alcoholic matrix of wine.

3.3.
Metabolic pathways of the phenolic metabolite
generation
The results of the phenolic metabolite quantification in the
plasma (Table 2) and urine samples (Table 3) showed that after
the three wine interventions, the phenolic compounds absorbed underwent an intense phase-II metabolism in
the intestinal epithelium and the liver, forming sulphate,
glucuronide and/or methylated metabolites through the
respective action of sulphotransferases (SULF), uridine-5diphosphate glucuronosyl-transferases (UGT), and catecholO-methyltransferases (COMT). A large proportion of the
polyphenols in wine, including proanthocyanidins, flavan-3ols and anthocyanins, are not absorbed in the upper part of
the gastrointestinal tract and can be metabolised by the gut
microbiota releasing smaller molecules before its absorption.
Apart from colonic metabolism, phase II metabolism was also

87

Journal of Functional Foods 25 (2016) 8093

(2A) Plasma
25

Phenolic acids

500

Urine excretion (mol)

Plasma concentration (nM)

600

(2B) Urine

400
300
200
100
0
0

60

120

180

240

300

5
0-6 h

300
200
100
0
0

60

120

180

240

300

360

Urine excretion (mol)

400

600
Urine excretion (mol)

Flavan-3-ols

500
400
300
200
100
60

120

600

180

240

300

Urine excretion (mol)

Phenyl alcohols

400
300
200
100
0
0

60

30

120

180

240

300

Anthocyanins

20
15
10
5

Stilbenes
*

20

10
0

0-6 h

50

6-12 h
*

12-24 h

Flavan-3-ols

40
30
20
10
0-6 h

6-12 h

12-24 h

Phenyl alcohols

50
40

30

*
*

20
10
0

360

25

12-24 h

30

360

500

6-12 h
*

0-6 h
Urine excretion (mol)

Plasma concentration (nM)


Plasma concentration (nM)

10

Stilbenes

0
Plasma concentration (nM)

15

40

500

Phenolic acids

20

360

600

Plasma concentration (nM)

6-12 h

12-24 h

Anthocyanins
0.12

0.08

0.04
0

0
0

60

120

180

240

300

0-6 h

360

6-12 h

12-24 h

W: Plasma
PEW: Plasma
NPEW: Plasma

Urine
Urine
Urine

Urine excretion (mol)

Time (minutes)
50

Valerolactone

40
30
20
10
0

0-6 h

6-12 h

12-24 h

Fig. 2 (A) Mean concentration (nM) of generated phenolic metabolites versus time (min) in the plasma samples after the
three wine interventions; (B): Urine excretion (mol) of phenolic metabolites versus range time (h), 06 h, 612 h, and 12
24 h after the three wine interventions. W, wine; PEW, phenol enriched wine; NPEW, nanoencapsulated phenols enriched
wine.

88

Journal of Functional Foods 25 (2016) 8093

Table 3 Urine molar excretion (24 h) expressed as mols/24 h observed after the intake of 100 mL of non-alcoholic wine
(W) and the phenol-enriched non-alcoholic wines at an equimolar dose added through the free (PEW) or nanoencapsulated phenolic extract (NPEW) (mean value standard deviation), n = 12.
Urine phenol excretion (mols/24 h)
Phenolic acids
Gallic acid sulphate
Gallic acid glucuronide
Syringic acid sulphate
Syringic acid glucuronide
Protocatechuic acid sulphate
Ferulic acid glucuronide
3,4-Dihydroxyphenylacetic acid
Stilbenes
Resveratrol sulphate
Resveratrol glucuronide
Flavan-3-ols
Catechin sulphate
Epicatechin sulphate
Catechin glucuronide
Epicatechin glucuronide
Methyl catechin sulphate
Methyl epicatechin sulphate
Methyl catechin glucuronide
Methyl epicatechin glucuronide
Phenyl alcohols
Free hydroxytyrosol
Hydroxytyrosol sulphate
Valerolactones
5-(3,4-Dihydroxyphenyl)--valerolactone
Anthocyanins
Malvidin glucoside
a
b

Control
wine (W)

Phenol-enriched
wine (PEW)

Encapsulated phenolenriched wine (NPEW)

2.42 1.18
0.43 0.33
3.10 1.15
3.25 0.83
3.31 1.31
2.28 1.43
21.1 8.08

6.88 3.92a
0.75 0.67
3.90 1.60a
4.44 2.30
6.35 4.10a
2.31 0.61
37.9 11.3

6.36 2.56a
0.76 0.43a
6.27 2.52a,b
6.42 3.03a,b
6.05 2.38
3.27 1.08
28.2 9.98

30.3 19.2
2.63 1.62

35.0 19.8
3.51 2.13

45.1 18.4a
6.06 4.31a

10.9 7.45
1.65 1.54
0.22 0.15
1.23 1.09
1.46 1.14
2.55 3.72
0.60 0.48
0.79 0.70

22.5 13.5a
5.07 3.78
0.66 0.47a
6.00 4.55
2.63 1.24a
5.21 3.46
1.27 0.87a
1.57 1.00a

32.3 13.0a
9.62 3.89a
0.93 0.70a
8.07 4.48a
3.71 1.88a
7.86 3.06a
1.66 1.15a
2.83 2.26a

2.17 1.14
7.48 4.25

3.01 3.67a
14.5 9.25a

1.86 1.86
10.6 7.44

31.5 18.7

76.9 24.8a

71.8 29.5a

0.06 0.03

0.08 0.05

0.14 0.08a,b

Mean values PEW or NPEW significantly different from the control W (p < 0.05).
Mean values significantly different from the phenol-enriched wines, PEW and NPEW (p < 0.05).

detected in the present study. To understand and explain how


these phenol biological metabolites from wine are generated, different metabolic pathways are proposed in the present
study, independently of the wine intervention.
Malvidin-3-O-glucoside was the most abundant anthocyanin consumed through the three wine interventions (Table 1).
However, it was detected at low concentration levels in its native
glycosylated form in both the plasma and urine samples, probably as a consequence of the intense metabolism during
gastrointestinal digestion. Based on this intense metabolism,
we have proposed the metabolic pathways of the main anthocyanins quantified in the control and phenol-enriched wines
(Fig. 3). Regarding the metabolism of anthocyanins, syringic acid
has been reported to be the corresponding acid derived from
B-ring cleavage by the gut microbiota (Fleschhut, Kratzer,
Rechkemmer, & Kulling, 2006), and was also the main microbial metabolite derived from the malvidin-3-O-glucoside
detected after dealcoholised red wine consumption
(Boto-Ordnez et al., 2013). Then, syringic acid might suffer
further metabolism by enzymatic demethylation of the B-ring
and generate gallic acid (Fig. 3), which is also present in wine.
In synthesis, the microbial metabolism of anthocyanins includes ring opening, deconjugation and cleavage (Aura, 2008;
Hidalgo et al., 2012), and therefore malvidin-3-O-glucoside might
be converted into syringic and gallic acids. Additionally, gallic
acid could result from the methylation of syringic acid.

Malvidin glucoside derivatives (such as malvidin


acetylglucoside, malvidin coumaroylglucoside, among others),
which are also present in the wine (Table 1), might be hydrolysed to malvidin-3-O-glucoside before being metabolised to
syringic and gallic acids (Fig. 3). The other anthocyanins present
in the wine, peonidin, petunidin, delphinidin and cyanidin
derivatives, which are present at low concentrations in comparison with malvidin glucoside, could also suffer degradation.
Delphinidin glucoside and petunidin glucoside can also be
converted into gallic acid, with petunidin glucoside previously suffering a demethylation. On the other hand, cyanidin
glucoside and peonidin glucoside can be converted into protocatechuic acid, with peonidin glucoside previously suffering
a demethylation (Fang, 2014; Hanske et al., 2013; Mosele,
Maci, & Motilva, 2015a; Vitaglione et al., 2007). Therefore,
the gallic acid sulphate detected in the plasma and urine in
our study could be generated from various anthocyanins from
the red wine (malvidin, delphinidin and petunidin derivatives) as well as from the gallic acid already present in the
wine. On the other hand, gallic acid can be dehydroxylated
to generate protocatechuic acid. Once absorbed, protocatechuic, gallic and syringic acids can be sulphated or
glucuronidated.
Other phenolic acid metabolites detected in the plasma
samples were caffeic acid and ferulic acid in its sulphate forms,
and ferulic acid glucuronide was detected in the urine samples.

Journal of Functional Foods 25 (2016) 8093

89

Fig. 3 Proposed metabolic pathway of anthocyanins, and caftaric acid and fertaric acid.

Besides the caffeic and ferulic acids quantified in the wines,


additional amounts could be formed respectively from the
caftaric and fertaric acids present in wine by the action
of esterases in the small intestine. Additionally, ferulic acid
could also be generated from the methylation of caffeic
acid (Fig. 3). Regarding the microbial metabolism of caffeic
acid, this has been reported to be catabolised to 3-(3,4dihydroxyphenyl)propionic acid, which is further metabolised
to 3,4-dihydroxyphenylacetic acid and protocatechuic acid by
- and -oxidation, respectively (Aura, 2008; Del Rio et al., 2013;
Mosele et al., 2014). So, the protocatechuic acid sulphate detected in urine sample in the present study could be derived
from this described pathway or from the protocatechuic acid
already present in wine.
Fig. 4 shows the proposed metabolic pathway of flavan-3ols (catechin, epicatechin and dimer) leading to their phaseII and microbial metabolites. As is shown, a large part of
catechin and epicatechin can be metabolised by the gut
microbiota to 3-(3,4-dihydroxyphenyl)propionic acid and this
produces 5-(3,4-dihydroxyphenyl)--valerolactone, which is a
specific microbial metabolite of flavan-3-ols (Aura, 2008; Del
Rio et al., 2013; Hackman et al., 2008; Serra et al., 2011). This
can be further metabolised into protocatechuic acid and then
sulphated.

Other metabolites detected in the plasma and urine samples


after the wine intakes were resveratrol in its sulphate and glucuronide forms. These metabolites could be formed from
resveratrol and piceid (resveratrol glucoside), as proposed in
Fig. 4. However, piceid was probably firstly deglycoslylated to
resveratrol by means of a glycosidase enzyme to be absorbed
in the intestine and, then, to undergo phase-II metabolism
(Rotches-Ribalta et al., 2012).
Apart from all the above mentioned metabolites,
hydroxytyrosol was also detected in its free and sulphated forms
in the plasma and urine samples after the three wines intake
and a significant increase in its sulphated form was seen after
PEW compared to the control wine. Initially, it was thought that
these metabolites could be formed through the metabolism of
tyrosol and hydroxytyrosol from the wine. However, the low
concentration of these in wine and the high concentrations
detected in the plasma and urine samples suggest possible generation as a consequence of the metabolism of other phenolic
compounds in the red wine. An increase in the urinary excretion of tyrosol and hydroxytyrosol was also observed after an
acute intake of red wine (De la Torre, Covas, Pujadas, Fit, &
Farr, 2006; Prez-Ma et al., 2015a, 2015b; Schrder et al., 2009)
and dealcoholised red wine (Prez-Ma et al., 2015b). These
authors reported that the presence of ethanol in the wine

90

Journal of Functional Foods 25 (2016) 8093

Fig. 4 Proposed metabolic pathway of flavan-3-ols and stilbenes.

could be responsible for a shift of dopamine and tyramine


metabolism from the oxidative to the reductive pathway,
leading to hydroxytyrosol and tyrosol formation, instead of
3,4-dihydroxyphenylacetic acid and 4-hydroxyphenylacetic
acid, respectively, and a biotransformation of tyrosol into
hydroxytyrosol (Prez-Ma et al., 2015b). Nevertheless, in our
study, neither tyrosol metabolites nor homovanillic acid was
detected in the plasma and urine samples, and the
dealcoholised wine contained a low percentage of ethanol
(0.5 %, v/v). Therefore, to describe the possible generation of
hydroxytyrosol and its presence in plasma and urine samples,
different metabolic pathways are suggested in the present
work (Fig. 5). We hypothesise that hydroxytyrosol generation
could come from other phenolic compounds present in the wine
such as flavan-3-ols (catechin, epicatechin and dimer), quercetin glucuronide, caftaric and fertaric acid, and anthocyanins,
to be the phenolic compounds most abundant in the wine
(Table 1). All of these phenolic compounds can be microbially
metabolised to 3-(3,4-dihydroxyphenyl)propionic acid and
further to 3,4-dihydroxyphenylacetic acid (Aura, 2008; Del Rio
et al., 2013; Gonzlez-Barrio, Edwards, & Crozier, 2011; Serra
et al., 2011, 2012). Therefore, the metabolic pathway proposed to explain the generation of hydroxytyrosol could be
by means of the reduction of these microbial metabolites
(dihydroxyphenylpropionic and dihydroxyphenylacetic acids).

Similarly, this reduction pathway was also observed when chlorogenic acid was fermented in vitro with human gut microbiota,
and the catabolic metabolite hydroxyphenylpropionic acid was
converted into tyrosol (Toms-Barbern et al., 2014). A study
by Xu and Sim (1995) in a rat model and in absence of ethanol
showed that the enzyme DOPAC reductase is specifically involved in the one-step conversion of 3,4-dihydroxyphenylacetic
acid (DOPAC) into hydroxytyrosol (dihydroxyphenylethanol) in
the central metabolism of dopamine.
Another metabolic pathway proposed for the generation of
hydroxytyrosol could be from the anthocyanin metabolism
(Fig. 5). After anthocyanin deglycosylation by the gut microbiota,
the released aglycones can be unstable at intestinal pH resulting in A- and B-ring cleavage forming phenolic acids, which
could lead to the generation of hydroxytyrosol. In accordance with this, hydroxytyrosol was detected in human faeces
after an acute and long-term intake of anthocyanin-rich fruit,
including raspberries (Gonzlez-Barrio et al., 2011) and pomegranate (Mosele et al., 2015b).

4.

Conclusions

After the three wine interventions, the main phenolic metabolites detected in the plasma samples were syringic acid

Journal of Functional Foods 25 (2016) 8093

91

Fig. 5 Proposed metabolic pathway of hydroxytyrosol.

sulphate, resveratrol sulphate and hydroxytyrosol sulphate.


However, only catechin metabolites (sulphate and glucuronide conjugates) reached significantly higher Cmax following the
intake of the two phenol-enriched wines (PEW and NPEW) compared with the dealcoholised red wine used as a control. In
terms of plasma Cmax, the nano-encapsulation of the phenolic extract did not significantly enhance the bioavailability of
phenols as no differences were observed in the Cmax values
when the PEW and NPEW were compared. In contrast, the
results of the present study showed that the human urinary
excretion of phenolic metabolites derived from several
grape phenolic compounds (stilbenes, flavan-3-ols, phenolic
acids and anthocyanins) was significantly higher after the
ingestion of the two phenol-enriched wines compared with
the dealcoholised red wine used as a control. The nanoencapsulation of the grape phenolic extract slightly enhanced
the bioavailability of malvidin-3-O-glucoside, observed by the
higher urine excretion of its native form and by its phase-II
conjugates syringic and gallic acids, which were proposed as
the main metabolites generated by microbial metabolism. Therefore, despite the slight increase observed in the effective

bioavailability of the encapsulated phenolic extract, the low


concentration of the crude extracts used in this study and the
turbidity after adding the encapsulated extract reduces the interest for use in a final commercial functional wine.
Additionally, a main contribution of this study is the proposal of the metabolic pathways of different phenolic subgroups
found in red wine in humans. In particular, a novel endogenous production pathway of hydroxytyrosol metabolites after
the ingestion of a dealcoholised red wine is suggested. Knowledge about these metabolic pathways provides relevant
information to complement nutritional intervention studies to
help unravel the complex interactions between wine phenolic compounds and potential in-vivo health effects in subjects
consuming red wine.

Conflict of interest
All authors declare no conflict of interest concerning the content
of the manuscript.

92

Journal of Functional Foods 25 (2016) 8093

Acknowledgements
This work was supported by the Project INCOMES (Bodegas
Torres, S.A.), co-funded by the Spanish Ministry of Economy
and Competitiveness (Centro para el Desarrollo Tecnolgico Industrial) and FEDER. The authors wish to thank Xenia Borrs
for her technical assistance in the recruitment of the volunteers and execution of the human intervention study. We also
thank all the staff of UDETMA (Unidad de Deteccin y
Tratamiento de Enfermedades Aterotrombticas) from the Department of Nephrology, Hospital Universitari Arnau de Vilanova
(Lleida, Spain) for their technical assistance in the execution
of the human intervention study.

Appendix: Supplementary material


Supplementary data to this article can be found online at
doi:10.1016/j.jff.2016.05.013.
REFERENCES

Ageros Bazo, M., Esparza Cataln, I., Gonzlez Ferrero, C.,


Gonzlez Navarro, C. J., Irache Garreta, J. M., & Romo Hualde,
A. (2012). Nanoparticles for encapsulation of compounds, the
production and uses thereof. ICT, CNTA. Priority: ES201031095
(16/07/2010). Intern. Appl. N : PCT/ES2011/070518 (15/07/2011).
Pub. N : WO2012/007628.
Ahn, S. I., Chang, Y. H., & Kwak, H. S. (2010). Optimization of
microencapsulation of Inonotus obliquus extract powder by
response surface methodology and its application into milk.
Korean Journal of Food Science and Technology, 30, 661668.
Aizpurua-Olaizola, O., Navarro, P., Vallejo, A., Olivares, M.,
Etxebarria, N., & Usobiaga, A. (2016). Microencapsulation and
storage stability of polyphenols from Vitis vinifera grape
wastes. Food Chemistry, 190, 614621.
Aura, A. M. (2008). Microbial metabolism of dietary phenolic
compounds in the colon. Phytochemistry Reviews, 7, 407429.
Ayoub, M., de Camargo, A. C., & Shahidi, F. (2016). Antioxidants
and bioactivities of free, esterified and insoluble-bound
phenolics from berry seed meals. Food Chemistry, 197, 221232.
Baba, S., Osakabe, N., Natsume, M., Muto, Y., Takizawa, T., &
Terao, J. (2001). In vivo comparison of the bioavailability of (+)catechin, ()-epicatechin and their mixture in orally
administered rats. The Journal of Nutrition, 131, 28852891.
Bell, J. R. C., Donovan, J. L., Wong, R., Waterhouse, A. L., German, J.
B., Walzem, R. L., & Kasim-Karakas, S. E. (2000). (+)-Catechin in
human plasma after ingestion of a single serving of
reconstituted red wine. The American Journal of Clinical
Nutrition, 71, 103108.
Boocock, D. J., Patel, K. R., Faust, G. E. S., Normolle, D., Marczylo, T.
H., Crowell, J. A., Brenner, D. E., Booth, T. D., Gescher, A., &
Steward, W. P. (2007). Quantification of trans-resveratrol and
detection of its metabolites in human plasma and urine by
high-performance liquid chromatography. Journal of
Chromatography B, 848, 182187.
Borges, G., Mullen, W., Mullan, A., Lean, M. E. J., Roberts, S. A., &
Crozier, A. (2010). Bioavailability of multiple components
following acute ingestion of a polyphenol-rich juice drink.
Molecular Nutrition and Food Research, 54(Suppl. 2), S268S277.
Boto-Ordnez, M., Urpi-Sarda, M., Queipo-Ortuno, M. I., Corella,
D., Tinahones, F. J., Estruch, R., & Andrs-Lacueva, C. (2013).
Microbial metabolomic fingerprinting in urine after regular

dealcoholized red wine consumption in humans. Journal


Agricultural and Food Chemistry, 61, 91669175.
De la Torre, R., Covas, I., Pujadas, M. A., Fit, M., & Farr, M. (2006).
Is dopamine behind the health benefits of red wine? European
Journal of Nutrition, 45, 307310.
Del Rio, D., Rodrguez-Mateos, A., Spencer, J. P. E., Tognolini, M.,
Borges, G., & Crozier, A. (2013). Dietary (poly)phenolics in
human health: Structures, bioavailability, and evidence of
protective effects against chronic diseases. Antioxidants &
Redox Signaling, 18, 18181898.
Dube, A., Ng, K., Nicolazzo, J. A., & Larson, I. (2010). Effective use
of reducing agents and nanoparticle encapsulation in
stabilizing catechins in alkaline solution. Food Chemistry, 122,
662667.
Duthie, S. J. (2011). Epigenetic modifications and human
pathologies: Cancer and CVD. The Proceedings of the Nutrition
Society, 70, 4756.
Fang, J. (2014). Bioavailability of anthocyanins. Drug Metabolism
Reviews, 46, 508520.
Fleschhut, J., Kratzer, F., Rechkemmer, G., & Kulling, S. E. (2006).
Stability and biotransformation of various dietary
anthocyanins in vitro. European Journal of Nutrition, 45, 718.
Flores, G., Ruiz del Castillo, M. L., Costabile, A., Klee, A.,
Guerdoletto, K. B., & Gibson, G. R. (2015). In vitro fermentation
of anthocyanins encapsulated with cyclodextrins: Release,
metabolism and influence on gut microbiota growth. Journal of
Functional Foods, 16, 5057.
Giacosa, A., Barale, R., Bavaresco, L., Gatenby, P., Gerbi, V.,
Janssens, J., Johnston, B., Kas, K., La Vecchia, C., Mainguet, P.,
Morazzoni, P., Negri, E., Pelucchi, C., Pezzotti, M., & Rondanelli,
M. (2013). Cancer prevention in Europe: The Mediterranean
diet as a protective choice. European Journal of Cancer
Prevention, 22(1), 9095.
Gonzlez-Barrio, R., Edwards, C. A., & Crozier, A. (2011). Colonic
catabolism of ellagitannins, ellagic acid and raspberry
anthocyanins: In vitro and in vivo studies. Drug Metabolism
and Disposition: The Biological Fate of Chemicals, 39, 16801688.
Gouin, S. (2004). Microencapsulation: Industrial appraisal of
existing technologies and trends. Trends in Food Science &
Technology, 15, 330347.
Gunasekaron, S., & Ko, S. (2014). Rationales of nano- and
microencapsulation for food ingredients. In H.-S. Kwak (Ed.),
Nano- and microencapsulation for foods (pp. 4364). United
Kingdom: John Wiley & Sons.
Hackman, R., Polagruto, J. A., Zhu, Q. Y., Sun, B., Fujii, H., & Keen,
C. L. (2008). Flavanols: Digestion, absorption and bioactivity.
Phytochemistry Reviews, 7, 195208.
Hanske, L., Engst, W., Loh, G., Sczesny, S., Blaut, M., & Braune, A.
(2013). Contribution of gut bacteria to the metabolism of
cyanidin-3-glucoside in human microbiota-associated rats.
The British Journal of Nutrition, 109, 14331441.
Hidalgo, M., Oruna-Concha, M. J., Kolida, S., Walton, G. E.,
Kallithraka, S., Spencer, J. P., & Pascual-Teresa, S. (2012).
Metabolism of anthocyanins by human gut microflora and
their influence on gut bacterial. Journal of Agricultural and Food
Chemistry, 60, 38823890.
Huang, Q., Yu, H., & Ru, Q. (2010). Bioavailability and delivery of
nutraceuticals using nanotechnology. Journal of Food Science,
75, R50R57.
Lee, Y. K., Mijan, M. A., Ganesan, P., & Kwak, H. S. (2013). The
physicochemical properties of yoghurt supplement with a
possible functional ingredient. International Journal of Dairy
Technology, 66, 417723.
Lpez, A., Gavara, R., & Lagaron, J. (2006). Bioactive packaging:
Turning foods into healthier foods through biomaterials.
Trends in Food Science & Technology, 17, 567575.
Makris, D., Boskou, G., & Andrikopoulos, N. (2007). Polyphenolic
content and in-vitro antioxidant characteristics of wine

Journal of Functional Foods 25 (2016) 8093

industry and other agri-food solid waste extracts. Journal of


Food Composition and Analysis, 20, 125132.
Mart, M. P., Pantalen, A., Rozek, A., Soler, A., Valls, J., Maci, A.,
Romero, M. P., & Motilva, M. J. (2010). Rapid analysis of
procyanidins and anthocyanidins in plasma by microelution
SPE and ultra-HPLC. Journal of Separation Science, 33, 28412853.
Mosele, J., Gosalbes, M. J., Maci, A., Rubi, L., VzquezCastellanos, J. F., Jimnez-Hernndez, N., Moya, A., Latorre, A.,
& Motilva, M. J. (2015b). Effect of daily intake of pomegranate
juice on fecal microbiota and feces metabolites from healthy
volunteers. Molecular Nutrition and Food Research, 59, 19421953.
Mosele, J., Maci, A., & Motilva, M. J. (2015a). Metabolic and
microbial modulation of the large intestine ecosystem by
non-absorbed diet phenolic compounds: A review. Molecules :
A Journal of Synthetic Chemistry and Natural Product
Chemistry. [electronic resource], 20, 1742917468.
Mosele, J., Martn-Pelez, S., Maci, A., Farrs, M., Valls, R. M.,
Cataln, U., & Motilva, M. J. (2014). Study of the catabolism of
thyme phenols combining in vitro fermentation and human
intervention. Journal of Agricultural and Food Chemistry, 62,
1095410961.
Munin, A., & Edwards-Lvy, F. (2011). Encapsulation of natural
polyphenolic compounds: A review. Pharmaceutics, 3(4), 793
829.
Neveu, V., Prez-Jimnez, J., Vos, F., Crespy, V., du Chaffaut, L.,
Mennen, L., Knox, C., Eisner, R., Cruz, J., Wishart, D., &
Scalbert, A. (2010). Phenol-Explorer: An online comprehensive
database on polyphenol contents in foods. Database (Oxford),
bap024.
Ottaviani, J. I., Momma, T. Y., Heiss, C., Kwik-Uribe, C., Schroeter,
H., & Keen, C. L. (2011). The stereochemical configuration of
flavanols influences the level and metabolism of flavanols in
humans and their biological activity in vivo. Free Radical Biology
& Medicine, 50, 237244.
Prez-Ma, C., Farr, M., Pujadas, M., Mustata, C., Menoyo, E.,
Pastor, A., Langohr, K., & de la Torre, R. (2015a). Ethanol
induces hydroxytyrosol formation in humans. Pharmacological
Research, 9596, 2733.
Prez-Ma, C., Farr, M., Rodriguez-Morato, J., Papaseit, E.,
Pujadas, M., Fit, M., Robledo, P., Covas, M. I., Cheynier, V.,
Meudec, E., Escudier, J. L., & de la Torre, R. (2015b). Moderate
consumption of wine, through both its phenolic compounds
and alcohol content, promotes hydroxytyrosol endogeneous
generation in humans. A randomized controlled trial.
Molecular Nutrition and Food Research, 59, 12131216.
Renaud, S., & Lorgeril, M. (1992). Wine, alcohol, platelets, and the
French paradox for coronary disease. Lancet, 339, 15231526.
Rotches-Ribalta, M., Urpi-Sarda, M., Llorach, R., Boto-Ordnez, M.,
Juregui, O., Chiva-Blanch, G., Prez-Garca, L., Jaeger, W.,
Guillen, M., Corella, D., Tinahones, F. J., Estruch, R., & AndrsLacueva, C. (2012). Gut and microbial resveratrol metabolite
profiling after a moderate long-term consumption of red wine
versus dealcoholized red wine in humans by an optimized
ultra-high-pressure liquid chromatography tandem mass
spectrometry method. Journal of Chromatography. A, 1265, 105
133.
Rotches-Ribalta, M., Urpi-Sarda, M., Mercader Mart, M., Reglero,
G., & Andrs-Lacueva, C. (2014). Resveratrol metabolic
fingerprinting after acute and chronic intakes of a functional
beverage in humans. Electrophoresis, 35, 16371643.
Rubi, L., Valls, R.-M., Maci, A., Pedret, A., Giralt, M., Romero, M.
P., de la Torre, R., Covas, M. I., Sol, R., & Motilva, M. J. (2012).
Impact of olive oil phenolic concentration on human
plasmatic phenolic metabolites. Food Chemistry, 135, 2922
2929.
Schrder, H., de la Torre, R., Estruch, R., Corella, D., MartnezGonzlez, M. A., Salas-Salvad, J., Ros, E., Ars, F., Flores, G.,
Civit, E., Farr, M., Fiol, M., Vila, J., Fernndez-Creuet, J., Ruiz-

93

Gutirrez, V., Lapetra, J., Sez, G., & Covas, M. I. (2009). Alcohol
consumption is associated with high concentrations of
urinary hydroxytyrosol. The American Journal of Clinical
Nutrition, 90, 13291335.
Serra, A., Maci, A., Romero, M. P., Angls, N., Morell, J. R., &
Motilva, M. J. (2011). Metabolic pathways of the colonic
metabolism of procyanidins (monomers and dimers) and
alkaloids. Food Chemistry, 126, 11271137.
Serra, A., Maci, A., Romero, M. P., Reguant, J., Ortega, N., &
Motilva, M. J. (2012). Metabolic pathways of the colonic
metabolism of flavonoids (flavonols, flavones and flavanones)
and phenolic acids. Food Chemistry, 130, 383393.
Serra, A., Maci, A., Romero, M. P., Salvad, M. J., Bustos, M.,
Fernndez-Larrea, J., & Motilva, M. J. (2009). Determination of
procyanidins and their metabolites in plasma samples by
improved liquid-chromatography-tandem mass
spectrometry. Journal of Chromatography B, 877,
11691176.
Teixeira, A., Baenas, M., Domnguez-Perles, R., Barros, A., Rosa, E.,
Moreno, D., & Garca-Viguera, C. (2014). Natural bioactive
compounds from winery by-products as health promoters: A
review. International Journal of Molecular Sciences, 15, 15638
15678.
Toms-Barbern, F., Garca-Villalba, R., Quartieri, A., Raimondi, S.,
Amaretti, A., Leonarndi, A., & Rossi, M. (2014). In vitro
transformation of chlorogenic acid by human gut microbiota.
Molecular Nutrition and Food Research, 58, 11221131.
Tresserra-Rimbau, A., Medina-Remn, A., Lamuela-Ravents, R.
M., Bull, M., Salas-Salvad, J., Corella, D., Fit, M., Gea, A.,
Gmez-Gracia, E., Lapetra, J., Ars, F., Fiol, M., Ros, E., SerraMajem, L., Pint, X., Muoz, M. A., & Estruch, R. (2015).
Moderate red wine consumption is associated with a lower
prevalence of the metabolic syndrome in the PREDIMED
population. The British Journal of Nutrition, 113, S121S130.
Urpi-Sarda, M., Zamora-Ros, R., Lamuela-Ravents, R., Cherubini,
A., Juregui, O., De la Torre, R., Covas, M. I., Estruch, R., Jaeger,
W., & Andrs-Lacueva, C. (2007). HPLC-tandem mass
spectrometric method to characterize resveratrol metabolism
in humans. Clinical Chemistry, 53, 292299.
Vitaglione, P., Donnarumma, G., Napalitano, A., Galvano, F., Gallo,
A., Scalfi, L., & Fogliano, V. (2007). Protocatechuic acid is the
major human metabolite of cyanidin-3-glucosides. The Journal
of Nutrition, 137, 20432048.
Vitaglione, P., Lumaga, R. B., Ferracane, R., Radetsky, I., Mennella,
I., Schettino, R., Koder, S., Shimoni, E., & Fogliano, V. (2012).
Curcumin bioavailability from enriched bread: The effect of
microencapsulated ingredients. Journal of Agricultural and Food
Chemistry, 60, 33573366.
Vitaglione, P., Lumaga, R. B., Ferracane, R., Schettino, R., Morell, J.
R., Reguant-Miranda, J., Shimoni, E., & Fogliano, V. (2013).
Human bioavailability of flavanols and phenolic acids from
cocoa-nut creams enriched with free or microencapsulated
cocoa polyphenols. The British Journal of Nutrition, 109, 1832
1843.
Vitaglione, P., Sforza, S., Galaverna, G., Ghidini, C., Caporaso, N.,
Vescovi, P. P., Fogliano, V., & Marchelli, R. (2005). Bioavailability
of transresveratrol from red wine in humans. Molecular
Nutrition and Food Research, 49, 495504.
Woraratphoka, J., Intarapichet, K., & Indrapichate, K. (2007).
Phenolic compounds and antioxidative properties of selected
wines from north east Thailand. Food Chemistry, 104, 1485
1490.
Xu, C. L., & Sim, M. K. (1995). Reduction of dihydroxyphenylacetic
acid by a novel enzyme in the rat brain. Biochemical
Pharmacology, 50, 13331337.
Zhang, L., Mou, D., & Du, Y. (2007). Procyanidins: Extraction and
micro-encapsulation. Journal of the Science of Food and
Agriculture, 87, 21922197.