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Human Bioavailability and Metabolism of Phenolic Compounds From Red Wine Enriched With Free or Nano-Encapsulated Phenolic Extract
Human Bioavailability and Metabolism of Phenolic Compounds From Red Wine Enriched With Free or Nano-Encapsulated Phenolic Extract
Human Bioavailability and Metabolism of Phenolic Compounds From Red Wine Enriched With Free or Nano-Encapsulated Phenolic Extract
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
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
A dealcoholised red wine was used as the vehicle for enrichment with both non-encapsulated
2016
The analysis of plasma only showed an increase in the concentration of syringic acid sul-
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
* 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.
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.
2.1.
2.2.
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
2.3.
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
W: 100 mL wine
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
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.
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.
2.6.
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
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.
3.
3.1.
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
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
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
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
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
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
(2A) Plasma
25
Phenolic acids
500
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
400
600
Urine excretion (mol)
Flavan-3-ols
500
400
300
200
100
60
120
600
180
240
300
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)
10
Stilbenes
0
Plasma concentration (nM)
15
40
500
Phenolic acids
20
360
600
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
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
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)
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).
89
Fig. 3 Proposed metabolic pathway of anthocyanins, and caftaric acid and fertaric acid.
90
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
91
Conflict of interest
All authors declare no conflict of interest concerning the content
of the manuscript.
92
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.
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