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In vivo bioavailability of polyphenols from grape by-product extracts, and

effect on lipemia of normocholesterolemic Wistar rats: Grape by-product in
vivo bioavailability

Article  in  Journal of the Science of Food and Agriculture · April 2018

DOI: 10.1002/jsfa.9100

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Research Article
Received: 30 August 2017 Revised: 19 March 2018 Accepted article published: 24 April 2018 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.9100

In vivo bioavailability of polyphenols from

grape by-product extracts, and effect on
lipemia of normocholesterolemic Wistar rats
Raul Olivero-David,a María B Ruiz-Roso,b Nicola Caporaso,c,† Lourdes
Perez-Olleros,a Natalia De las Heras,b Vicente Laherab and Baltasar
Ruiz-Rosoa*

Abstract
BACKGROUND: The direct use of phenolic extracts from grape by-products can be useful when formulating functional food to
improve consumer health. The use of phenolic extracts instead of pure polyphenols as an ingredient is relevant in this context.
The present study investigated the bioavailability and absorption of polyphenols from grape by-product extracts and their
health effect on cholesterolemia, by adding the extract (GE) to Wistar rats diet (50 g kg−1 ) in vivo.

RESULTS: GE caused the appearance of (+)-catechin, myricetin and quercetic acid in plasma and liver. (+)-Catechin was the most
abundant compound (6 𝝁g mL−1 in plasma and 0.7 𝝁g mg−1 protein in liver), whereas no phenolic compounds were detected
in plasma or liver in the control group. Similarly, 3,4-hydroxyphenylacetic, a major product of polyphenol digestion, was
detected in the plasma, liver and urine of the GE-group only. GE-group had significantly lower cholesterol level and lower total
cholesterol/high-density lipoprotein ratio in plasma. Total bile acid content significantly increased in fecal matter after 24 h
administration of the GE-enriched diet.

CONCLUSION: Grape extract polyphenols are partially bioavailable and showed improvement in lipid metabolism. Thus, the
results suggest that GE is promising as a functional ingredient in the prevention of hypercholesterolemia.
© 2018 Society of Chemical Industry

Keywords: bioavailability; dietary polyphenols; grape extract; plasma lipids; food industry by-products; in vivo digestion

INTRODUCTION
Grape metabolism involves the production of a wide range of pri-
mary and secondary compounds that confer selective advantages
to the plant against ecological stresses. Among the products of
secondary metabolism, polyphenols represent a large family of
compounds that are involved in plant responses to biotic and abi-
otic stresses. These molecules have a physiological and ecolog-
ical function, including defence and protection against ultravio-
let radiation or pathogens. Phenolics in grape are classified into
two main groups: flavonoids and non-flavonoid compounds. The
flavonoid family includes the flavonols, which are found either as
sugar conjugates or aglycones, the flavan-3-ols, the anthocyanins,
dihydroflavonols and proanthocyanidins (PA).1–5
The chemical structure of polyphenols is a critical factor influenc-
ing their bioavailability. In foods, polyphenols are found as poly-
mers or in glycosylated forms and as aglycones. Most polyphenols
cannot be absorbed intact after consumption and undergo a series
of reactions by intestinal enzymes or the colonic microbiota. The
latter group hydrolyzes glycosides into aglycones and degrades
them to simple phenolic acids. This activity is of great importance
for the biological action of polyphenols with respect to producing
a number of specific metabolites with functional properties.6,7 It
has been estimated that 90–95% of the dietary polyphenols are
not absorbed in the small intestine and pass directly to the colon.
In the colon, they can be catabolized by the microbiota, absorbed
into the enterocytes via passive diffusion and, subsequently, reach
the liver to be further metabolized by phase II enzymes.8 Phase
II metabolites are then absorbed or eliminated through urine,
whereas non-absorbed polyphenol metabolites are excreted in
the feces or reach the colon again via enterohepatic circulation.9
Polyphenols are potent antioxidant compounds in terms of
their ability to donate electrons and eliminate free radicals.10 In
∗ Correspondence to: B Ruiz-Roso, Departamento de Nutrición y Bromatología I
(Nutrición). Facultad de Farmacia. Universidad Complutense de Madrid, Plaza
Ramón y Cajal S/N 28040-Madrid, Spain. E-mail: ruizrojo@ucm.es
† Current address: Department of Primary Production and Processing, Campden
BRI, Chipping Campden, UK
a Department of Nutrition and Bromatology I (Nutrition), Faculty of Pharmacy,
Complutense University of Madrid, Madrid, Spain
b Department of Physiology, Faculty of Medicine, Complutense University of
Madrid, Madrid, Spain
c Division of Food Sciences, School of Biosciences, University of Nottingham,
Leicestershire, UK

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 www.soci.org R Olivero-David et al.

vitro studies have demonstrated that several polyphenols provide

Table 1. Composition of the grape extract used in the present study
a higher antioxidant capacity than vitamins E and C. Because
of this antioxidant capacity, polyphenols have been studied in Grape extract
relation to several diseases, such as degenerative, inflammatory Component (dried weight)
and cardiovascular diseases.11–16 Several studies have reported on
the relationship between dietary polyphenol consumption and Energy (kcal kJ−1 ) 3.21/13.41
chronic pathologies such as cancer and diabetes.17–19 For other Moisture (g kg−1 ) 48
food products (e.g. polyphenols in green tea), previous studies Proteins (g kg−1 ) 45
have assessed their availability in humans, demonstrating that Fats (g kg−1 ) 20
green tea catechins are more readily available than previously Carbohydrates (g kg−1 ) 580
reported.20 Sugars (g kg−1 ) 41
As a result of the high polyphenol content of grape and grape Dietary fiber (g kg−1 ) 120
products, several epidemiological studies have suggested an Ash (g kg−1 ) 127
association between grape phenolic consumption and the pre- Total phenols (mg g−1 ) 257.47
vention of cardiovascular diseases. Previous studies have shown Soluble phenols (mg g−1 ) 11.07
the beneficial effects of grape polyphenol-rich diets on some Soluble-PA (mg g−1 ) 234.89
parameters related to cardiovascular risk, such as blood pressure Insoluble-PA (mg g−1 ) 11.51
and hyperlipemia.21,22 Very recent studies have used polyphe- (+)-Catechin (mg g−1 ) 6.28
nols extracted from red wine to assess their possible effect (−)-Epicatechin (mg g−1 ) 1.33
on obesity associated insulin resistance. Commercial purified Myricetin (mg g−1 ) 1.61
polyphenols were used at concentration of 600 mg day−1 in Quercetin (mg g−1 ) 1.18
humans, indicating that a significant improvement of insulin Sodium (mg g−1 ) 0.129
sensitivity was not observed, although whether this effect Calcium (mg g−1 ) 3.31
might be a result of only healthy subjects being used was
considered.23 Other studies have suggested that the polyphe-
nol concentration with a cardioprotective effect varies from 200 to
100% red grape (Vitis vinifera) variety Tempranillo, harvested from
1500 mg day−1 .21
vineyards located in the central region of Spain, specifically the
In terms of natural sources of grape phenolics, innovative meth-
ods for the purification of polyphenols from grape pomace have Ribera de Duero Designation of Origin, in Castilla y León. The
been proposed (e.g. by supercritical CO2 extraction), although extraction is based on a patented system using hydro-alcoholic
such methods require long process and expensive technologies.24 (50% v/v) distillation for 3–4 h at 40–55 ∘ C. The obtained liquid is
For these reasons, there is interest in assessing the effect of grape then stabilized using sodium alginate and concentrated by 50%
pomace from industry by-products without purification or extrac- its initial volume. Details of the procedure are reported by Moro.26
tion of the sole polyphenols. The use of such extract was recently The same extract has been used by Yubero et al.22 for the study
reported by Sanz-Buenhombre et al.25 who used in vitro gastroin- of low-density lipoprotein (LDL) cholesterol-lowering effects. Its
testinal digestion to assess the bioavailability of polyphenols from composition is reported in Table 1.
grape extract and polyphenol degradation during the simulated The composition of the grape extract was characterized using
digestion. specific methods. Water: internal method based on method num-
However, little is known about the in vivo absorption of grape ber 731 ‘Loss on Drying’ United States Pharmacopeia (USP) 34.27
polyphenols and extracts with a particular emphasis on their Sodium: internal method based on method number 851 ‘Spec-
effect on gut bacteria, as well as the possible influence of their trophotometry and Light-scattering’ United States Pharmacopeia
metabolites on lipemia. The use of extracts recovered from the (USP) 34.28 Calcium: internal method based on method number
wine-making industry or grape processing industry is of strong 851 ‘Spectrophotometry and Light-scattering’ United States Phar-
applicative interest because it can allow the valorization of a macopeia (USP) 34.28
food chain residue into a valuable ingredient and the eventual
formulation of functional food, or its use by the pharmaceutical
industry, as well as possibly also in feeds. Diet preparation
Therefore, the present study aimed to investigate the absorption, The diets were prepared using AIN-93G as the basal diet.29 The
metabolism and bioavailability of grape phenolic extracts, and dried grape extract (5% w/w) was mixed with the other ingredients
also whether the oral administration of grape by-product extract using a blade mixer for 8 min. The powders were vacuum-packed
affects lipemia and excretion of bile acids in normocholesterolemic and stored at 4 ∘ C. The diet composition (g kg−1 ) comprised: casein
Wistar rats. An additional objective was the investigation of pos- 200; L-cysteine 3; corn starch 317.5; maltodextrin 132; sucrose 100;
sible use of this winemaking by-product extract as a functional Dietary fibre: cellulose 100 or (cellulose 50 and grape extract 50);
ingredient with hypocholesterolemic properties. olive oil 100, t-butylhydroquinone 0.014; choline bitartrate 2.5;
mineral mixture 35 (composition in mg kg−1 of diet): Ca 5000; P
1561; K 3600; Na 1010; Cl 1571; S 300; Mg 507; Fe 35; Cu 6.0; Mn
MATERIALS AND METHODS 10.0; Zn 30.0; Cr 1.0; I 0.2; Se 0.15; F 1.0; B 0.50; Mo 0.15; Si 5.0;
Grape by-product extract Ni 0.5; Li 0.1; V 0.1. Vitamin mix composition (mg kg−1 ): thiamin
The grape extract used in this experiment was obtained from HCl 6.0; riboflavin 6.0; pyridoxine HCl 7.0; niacine 30.0; calcium
grape pomace. The extract used, trademarked as Eminol (ABRO- pantothenate 16.0; folic acid 2.0; biotin 0.2; vitamin B12 (0.1%) 25.0;
BIOTEC SL, Valladolid, Spain), was supplied by Grupo Matar- vitamin K 0.75. Vitamin A 4000 IU kg−1 ; vitamin D3 1000 IU kg−1 ;
romera (Valladolid, Spain).26 The pomace extract is produced from vitamin E 75 IU kg−1 .

wileyonlinelibrary.com/jsfa © 2018 Society of Chemical Industry J Sci Food Agric (2018)

Grape by-product in vivo bioavailability
Animals and experiments
Fourteen male Wistar rats weighing approximately 200 g were
obtained from Harlan Laboratories Models (Harlan SL, Barcelona,
Spain). The animals were treated in the Animal Research Centre,
Complutense University of Madrid (Madrid, Spain), certified by the
Spanish Ministry of Agriculture ref. ES-28079-0000086. Rats were
placed in individual metabolic cages under a 12:12 h light/dark
cycle at 21 ∘ C and 55% relative humidity. All experiments were
performed in compliance with the Council Directive of November
24, 1986 on the protection of animals for experimental and other
scientific purposes (86/609/EEC).
During a 3-day adaptation period, 14 rats had ad libitum access
to commercial rat food (crushed pellets; Panlab, Barcelona, Spain)
and water. The rats were then randomly split into two groups of
seven rats each. The Control group was fed with the standard
semi-purified diet only (AIN-93G # 110113 Purified Diet). The grape
extract group was fed with the same diet enriched with 5% (w/w)
grape extract. Both groups of rats had ad libitum access to food
and water. The weight and the food intake were monitored over
12 days, from day 3 to day 15, and, during this period, feces and
urine were collected individually. On day 15 starting at 08.00 h, the
animals were anesthetized using pentothal. Rats were euthanized
by extracting blood from the descending aorta with a syringe.
Blood from the descending aorta was collected into heparinized
tubes. Plasma was separated from the whole blood within 30 min
of collection by centrifugation at 1500 × g for 20 min. Samples
were collected in tubes and stored at −80 ∘ C until analysis.
Analysis of phenolic compounds
Soluble phenolics (SP) and soluble proantocyanins (soluble-PA)
were extracted from grape extract and from rat feces using
sequentially methanol:water (50/50, v/v) and acetone:water
(70/30, v/v). The mixtures were sonicated in an ultrasound for
30 min at 4 ∘ C and stirred every 5 min. SP were determined at
700 nm following the Folin–Ciocalteau procedure.30 A fraction
of extract was treated with 5 mL of HCl-butanol mixture for 3 h
at 100 ∘ C for soluble-PA analysis. The remaining non-extracted
residues from the methanol/acetone/water extraction were
desiccated at 45 ∘ C in a vacuum oven and treated with 5 mL
of HCl-butanol (3 h, 100 ∘ C) for insoluble proanthocyanidins
(insoluble-PA). The products obtained were then filtered and mea-
sured at 550 nm in a GENESYS 20 spectrophotometer (Thermo
Scientific, Rochester, NY, USA).31–33
Total phenol concentration was calculated using the formula:
total phenols = SP + soluble-PA + insoluble-PA.
Percentage digestibility was calculated using the formula:
Digestibility (%) = [(mg ingested per day – mg excreted in feces
per day)/mg ingested per day)] × 100.
The % of the different polyphenol fractions remaining in feces
at 24 h was calculated using the formula: Poliphenols feces 24 h
(%) = (mg excreted in feces per day)/mg ingested per day) × 100.
Quantification of (+)-catechin [cat-(+)], (–)-epicatechin
[epic-(−)], myricetin (myr) and quercetin (quer) in plasma
and rat liver
Phenolic compounds extraction from plasma and liver was per-
formed using the method of Lee et al.34 with slight modifications.
Approximately 500 mg of liver was homogenized with 1 mL of
0.4 mol L−1 sodium phosphate buffer containing 6 mg of ascor-
bic acid and 0.5 mg of ethylenediaminetetraacetic acid (EDTA)
(pH of 6.5). After centrifugation at 16 000 × g for 5 min (Her-
aeus Multifuge 1S-R centrifuge; Thermo Electron Corporation,
J Sci Food Agric (2018) © 2018 Society of Chemical Industry
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Langenselbold, Germany), 400 𝜇L of the supernatant was used
to analyze the levels of cat-(+), epic-(−), myr and quer. A vol-
ume 200 𝜇L of 0.4 mol L−1 sodium phosphate buffer (pH 7.4) was
added to 400 𝜇L of samples (plasma or liver). The sample was
mixed with 2.5 𝜇L of Type HP-2 𝛽-glucuronidase (250 units) and
40 mL of Type H-1 sulfatase (20 units) and then incubated at
37 ∘ C for 45 min. The reaction was stopped by adding 500 𝜇L of
methylene chloride and 300 𝜇L of water. After a second extrac-
tion with methylene chloride and water, the organic phase was
discarded. The aqueous phase was extracted by ethyl acetate
thrice. The combined ethyl acetate solution was added to 10 𝜇L
of a 20% ascorbic acid solution and then evaporated to dryness
under nitrogen flow.
Identification and quantification of individual polyphenols was
performed in accordance with the method of Suárez Valles et al.35
with slight modifications. External standards of cat-(+), epic-(−),
myr and quer (Sigma-Aldrich, Barcelona, Spain) were used to build
calibration curves with concentrations from 0 to 10 000 ng mL−1 .
The samples were analyzed using a high-performance liquid chro-
matography (HPLC) instrument (1100 Series; Agilent Technologies,
Madrid, Spain) with a DAD detector (1200 Infinity; Agilent Tech-
nologies) in conjunction with an ODS2 column of 250 × 4.6 mm
inner diameter (5.0 𝜇m particle size) (Waters, Milford, MA, USA)
and a sample loop of 20 𝜇L. The system was operated at 21 ∘ C. The
gradient and flow were: T1 = min 0, 2% B (flow = 0.6 mL min−1 );
T2 = min 50, 42% B (flow = 0.6 mL min−1 ); T3 = min 60, 50% B
(flow = 0.8 mL min−1 ); T4 = min80, 60% B (flow = 0.8 mL min−1 );
and T5 = min 85, 70% B (flow = 0.8 mL min−1 ). The eluent A
was a mixture of water:phosphoric acid to pH 2.8 and elu-
ent B was 100% methanol. The detection of cat-(+) and
epic-(−) was carried out at 280 nm, whereas myr and quer were
detected at 360 nm.
(3,4-Dihydroxy-phenylacetic acid) (DOPAC)
and 3,4-dihydroxy-phenyl propionic acid (DOPPR) analysis
in plasma, urine and rats liver
Extraction of DOPAC and DOPPR from plasma, urine and liver was
performed by modifying the method of Wang et al.36 Acid-washed
alumina (5 mg) was added to 350–700 𝜇L of sample (plasma,
urine or homogenized of liver) in 500 𝜇L of Tris-buffer (1.5 mol L−1 ,
pH 8.6) containing 0.07 mol L –1 EDTA. The mixture was vigorously
shaken for 30 min (Top Mix FB 15024; Fisher Scientific, Madrid,
Spain), then centrifuged at approximately 2000 × g for 2 min at
room temperature (Heraeus Multifuge 1S-R centrifuge; Thermo
Electron Corporation). The supernatant fraction was discarded.
The remaining alumina was washed three times with 1 mL of
glass-distilled water. The residue was dissolved with 200 𝜇L of
a mixture of 0.04 mol L−1 phosphoric acid/0.2 mol L−1 acetic acid
(20:80, v/v, pH 1.5–2.0). The eluate was analyzed by HPLC-DAD to
define the DOPAC and DOPPR concentrations.
The method of Iuvone et al.37 was used with slight modifications
to identify the DOPAC and DOPPR in plasma, urine and liver. To
identify and calculate the concentration of different metabolites
derivatives of soluble and insoluble PA fermentation, external stan-
dards of DOPAC and DOPPR (Sigma-Aldrich) were used. Stock solu-
tions (1 × 106 ng mL−1 ) of DOPAC and DOPPR were prepared in
methanol. An aliquot of each of the stock solutions was combined
and diluted with distilled water to produce an intermediate solu-
tion of 10 000 ng mL−1 in DOPAC and DOPPR. The resulting inter-
mediate solution was diluted with water to construct a curve with
concentrations from 0 to 1000 ng mL−1 . All solutions were stored
at −80 ∘ C in the dark.
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Figure 1. Growth rates in rats fed the control diet and the experimental diet enriched with grape phenolic extract. y = intercept (95% confidence
interval) ± slope (95% confidence interval) x; where y is the body-weight gain and x is the food consumption.
Samples were analyzed using an 1100 HPLC instrument (Agilent
Technologies) with a sample loop of 20 𝜇L. A DAD detector (1200
Infinity; Agilent Technologies) with a C18 reverse phase column of
250 × 4.6 mm inner diameter (5.0 𝜇m particle size) (Agilent Tech-
nologies) was used. Samples were isocratically eluted at a flow
rate of 1.0 mL min−1 at room temperature. The mobile phase was
a mixture of disodium EDTA (0.1 mmol L−1 ), sodium octylsulfate
(0.4 mmol L−1 ) in phosphoric acid buffer (0.1 mmol L−1 ) adjusted
to pH 2.30 and acetonitrile (92:8, v/v). The samples were detected
at 280 nm.
Protein analysis in rats liver
Liver protein concentration was obtained using bovine serum
albumin as standard compound in accordance with the method
of Bradford.38
Quantification of total cholesterol (total-chol),
high-density-cholesterol (HDL-chol), LDL-cholesterol
(LDL-chol) and triglycerides (TG) in plasma and rat liver
Total-chol, HDL-chol and TG in both plasma and liver were ana-
lyzed using standard enzymatic colorimetric tests (SPINREACT SA,
Sant Esteve de Bas, Girona, Spain). HDL-chol was calculated using
the Friedewald formula:
LDL-chol = total-chol – (HDL-chol + TG/5)
This formula is commonly used in humans but, in our case, we
applied it in our model system for comparison purposes between
the group fed with grape extract and the control group.
For the liver analysis, 100 mg of liver tissue was homogenized
in ice-cold phosphate buffer (10 mmol L−1 , pH 7.4). It was then
centrifuged at 2800 × g at 4 ∘ C for 15 min and the supernatant
fraction was collected for the analysis as described previously.
All spectrophotometric measurements were carried out using a
GENESYS 20 spectrophotometer (Thermo Scientific).
Analysis of total bile acids (TBA) in feces at 24 h and rats liver
Feces at 24 h were collected and dried in a vacuum oven at 55 ∘ C
for 24 h. Feces (50 mg) were transferred to a 1.5-mL Eppendorf
tube and extracted with 1000 𝜇L of t-butanol:water (1:1) mixture
and then incubated for 30 min at 37 ∘ C. During the extraction
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R Olivero-David et al.
period, the Eppendorf tubes were vortexed every 5 min for 10 s.
Subsequently, the sample was centrifuged for 2 min at 10 000 × g
(Heraeus Multifuge 1S-R centrifuge; Thermo Electron Corporation)
and the supernatant was collected for TBA analysis.39
For the TBA extraction from liver tissue, 100 mg of liver was
homogenized in ice-cold with 1000 𝜇L of phosphate buffer
(10 mmol L−1 , pH 7.4), and centrifuged at 2800 × g at 4 ∘ C for
15 min to collect the supernatant fraction. TBA in feces and liver
was assessed using the Spinreact enzymatic colorimetric test
(SPINREACT SA). All spectrophotometric measurements were car-
ried out in a GENESYS 20 spectrophotometer (Thermo Scientific).
Statistical analysis
Statistical analysis was performed using SPSS version 19.0 (IBM
Corp., Armonk, NY, USA). All chemical analyses were performed in
triplicate. The effect of grape extract consumption was evaluated
using an unpaired Student’s t-test. The relationship between food
intake and body-weight gain was assessed using analysis of covari-
ance (ANCOVA). The regression model was created to describe the
weight gain of the rats in relation to the daily food intake, and
a separate regression was created for each group. In addition, a
Pearson correlation analysis was carried out to determine the rela-
tionship between the different fractions of phenolic compounds
and the parameters analyzed in the rats, namely TBA in feces at
24 h, TBA in the liver, plasma total cholesterol, plasma LDL and
HDL-cholesterol (and their ratio), trigycerides in plasma, and total
cholesterol over HDL ratio. The results of the correlation analysis
were considered statistically significant at P < 0.05 and highly sig-
nificant at P < 0.01.
RESULTS
Rats growth rate
Figure 1 shows the relationship between food intake and increase
in animal body weight for the control group and grape extract
group. No statistically significant difference was found with respect
to body weight gain between the two experimental groups.
The mathematical expression obtained to describe the growth
rate of the control group was: y = 0.321 (0.305; 0.337) – 1.179
(−2.337; −0.021) x; r2 = 0.978. The expression for the rat group fed
J Sci Food Agric (2018)
Grape by-product in vivo bioavailability www.soci.org

Figure 2. In vivo content and digestibility of phenolic compounds from grape extract in male Wistar rats weighing 200 g fed with a control diet and
a diet enriched with 5% of grape extract, over 12 days. (a) Intake and excretion (mg day−1 ) of different phenolic fractions and (b) their digestibility
rate. The results are the average of three replicates, and bars indicate the SD. Total phenolic (TP) concentration was calculated using the formula:
TP = SP + soluble-PA + insoluble-PA. The percentage digestibility was calculated using the formula: digestibility (%) = [(mg ingested per day – mg
excreted in feces perday)/mg ingested perday)] × 100.

with grape extract was: y = 0.338 (0.312; 0.363) – 0.262 (−2.213;
1.690) x; r2 = 0.939. The mean growth rate values were not signifi-
cantly different (P > 0.01) according to the ANCOVA test for control
versus grape extract groups.
Absorption and excretion of SP, soluble-PA, insoluble-PA
and total phenols
Figure 2(a) reports on the amount of individual polyphenols
groups ingested and excreted per day. At 24 h, the soluble-PA frac-
tion was the most abundant phenolic group in the feces, whereas
SP and insoluble-PA were found in significantly lower amounts
(Fig. 2a). In these animals, 61.8 ± 24.5% of the SP and 75.4 ± 33.1%
of the insoluble-PA were found remaining in the feces. This
was therefore much higher than the percentage of soluble-PA
reaming in feces (13.9 ± 2.4%). Soluble-PA had a significantly
higher absorption (digestibility, 86 ± 2%) than SP (38 ± 24%) and
insoluble-PA (25 ± 33%) (Fig. 2b). The total amount of TBA found in
feces is shown in Fig. 3 for the control group and the experimental
group fed with the grape by-product extract. The latter group had
significantly higher amount (P < 0.05) of TBA in feces.
Phenolic profile in plasma and rats liver
The concentrations of individual phenolic compounds found and
quantified in plasma and liver for the control and grape extract
groups are reported in Table 2. None of the four target com-
pounds were detected in plasma nor, in liver for rats fed with
the control diet. For the grape extract group, plasma and liver
concentrations of cat-(+) were significantly higher than those of
myr and quer (6164.1 ± 1590.6 ng mL−1 versus 25.2 ± 25.2 ng mL−1
and 36.2 ± 16.9 ng mL−1 respectively). Epic-(−) was not detected
in plasma, nor liver for any of the two groups. Although myr and
quer levels were in a comparable concentration between plasma
and liver, the concentration of cat-(+) was above 10-fold higher in
plasma than the liver, with a concentration of 701.2 ± 50.5 ng mL−1
protein.
Concentration of DOPAC and DOPPR in plasma, liver
and urine
DOPAC and DOPPR were not detected in plasma, liver and urine of
the control group. The oral administration of grape extract caused
the appearance of DOPAC in plasma, liver and urine in the animals

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 www.soci.org
Figure 3. Sum of total bile acids (TBA) found in feces at 24 h, after the
administration of a control diet and a diet enriched of grape extract
(5%), to male Wistar rats (approximately 200 g), over 12 days. *Statistically
significant (P < 0.05) difference. All analyses were performed in triplicate.
Data are expressed as the mean ± SD (seven animals per group). A post-hoc
study was performed using an unpaired Student’s t-test to compare the
control group and the group fed with grape extract.
fed with this diet. However, only DOPPR was detected in the urine
of grape extract group (Table 3).
Concentration of DOPAC and DOPPR in plasma, liver
and urine
DOPAC and DOPPR were not detected in plasma, liver and urine
of the control group The oral administration of grape extract
caused the appearance of DOPAC in plasma (80.5 ± 28.6 ng mL−1 ),
liver (1.0 ± 0.5 ng mL−1 ) and urine (9117.5 ± 2629.4 ng mL−1 ) in the
animals fed with this diet. However, only DOPPR was detected in
the urine of grape extract group (629.1 ± 245.8 ng mL−1 ).
Lipid concentration in plasma and liver
The content of lipid compound in plasma and liver is shown
in Table 4. The addition of grape extract in the diet caused a
significant decrease of the total cholesterol in plasma, compared
to the control group (P = 0.045). Total-chol/HDL ratio in plasma
was affected by the addition of grape extract (P < 0.05) in the
diet. Grape extract group showed a tendency to lower LDL-chol
levels and LDL/HDL ratio in plasma, with respect to the control
group (P = 0.099 and P = 0.068, respectively). HDL-chol and TG in
liver were not significantly affected (P > 0.1) by the presence of
grape extract in the diet. Nonetheless, total-chol and TG tended to
decrease and HDL-chol tended to increase in grape extract group,
respect to control group.
Statistical analysis: Pearson correlations
Table 5 shows the statistical correlations between the absorp-
tion of SP, soluble-PA, insoluble-PA, total phenols or TBA in feces
obtained from the group fed with grape extract, against several
parameters of the lipid fraction in plasma, or TBA in feces and
liver. For several compounds, a slight correlation was observed,
wileyonlinelibrary.com/jsfa © 2018 Society of Chemical Industry
R Olivero-David et al.
although a statistically significant effect was not obtained. Sig-
nificant negative correlations (P < 0.05) were observed between
soluble-PA absorption and total-chol, TG and LDL-chol (r = −0.976,
r = −0.903 and r = −0.849, respectively). However, the absorp-
tion of soluble-PA showed a tendency to negatively correlate
with LDL/HDL ratio (r = −0.803 and P = 0.054). The absorption of
TP negatively correlated (P < 0.05) with TBA/mg proteins in liver,
total-chol, TG and LDL-chol (r = −0.809, r = −0.886, r = −0.913 and
r = −0.886, respectively). The TBA in feces at 24 h negatively corre-
lated with the LDL/HDL ratio (r = −0.840 and P = 0.036).
DISCUSSION
The present study aimed to investigate the absorption, distribu-
tion and excretion of polyphenols from grape extract in terms of
their potential activity with respect to lowering the cholesterol
level. In our experiment, the diet containing grape extract was
generally well accepted by growing rats, as corroborated by sim-
ilar intake data from other studies.40,41 The group fed with grape
extract consumed high daily amounts of soluble-PA but lower lev-
els of SP and insoluble-PA as a result of the composition of the
extract. This group did not show a statistically significant difference
in the growth rate during the experimental period compared to
the control group. Nonetheless, 24 h after administering the grape
extract, high percentages of SP and insoluble-PA were found in
the feces compared to the other group. However, the amount of
soluble-PA and total polyphenols remaining in feces was moder-
ately low. The absorption of soluble-PA was 86%, whereas it was
much lower for insoluble-PA and SP. According to the literature, it
is estimated that 90–95% of dietary polyphenols are not absorbed
in the small intestine and pass intact to the colon, where an impor-
tant fraction is degraded by the gut microbiota.42,43 Proantho-
cyanins differ from other phenolic compounds because of their
polymeric nature, and the complexity of these molecules is likely
to cause difficult absorption in the small intestine and a lower
degradation degree by gut microbiota.44,45 Furthermore, accord-
ing to Touriño et al.,46 oligomeric and polymeric PAs after inges-
tion may suffer partial depolymerization, mainly as a result of
action of the gastric medium, into their constituent units, which
are then degraded further by the colonic microbiota into pheno-
lic acids. This could explain the differences of absorption values
between soluble-PA and insoluble-PA in the diet enriched with
grape extract in these studies. This increase in phenolic acids dur-
ing colonic fermentation could explain the high presence of SP
in feces (i.e. approximately 60%) 24 h after the consumption of
the grape extract-enriched diet. However, the total polyphenols
from grape extract were efficiently digested (i.e. absorption of
82 ± 2%). Also, it is important to consider that the amount of solu-
ble polyphenols and insoluble-PA ingested was much lower than
the soluble-PA. Therefore, despite the fact that the absorption of
soluble polyphenols and insoluble-PA are very low, the absolute
amounts ingested of these compounds are neglectable compared
to the soluble-PA.
According to previous studies, polyphenols reach their maxi-
mum plasma concentrations 1–2 h after ingestion and are rapidly
conjugated in the liver by the actions of sulphate and glucuronide
acid, with subsequent elimination in the form of urine or bile.47,48
Scalbert and Williamson 47 reported that the fast plasma decrease
of most flavonoids absorbed in the small intestine, as well as their
fast excretion, is the result of rapid conjugation of the aglycone to
sulfate and glucuronide groups. Accordingly, because of this rapid
elimination and the low concentrations found in the liver, plasma
J Sci Food Agric (2018)
Grape by-product in vivo bioavailability www.soci.org

Table 2. Concentration of (+)-catechin, (−)-epicatechin, myricetin and quercetin in plasma (ng mL−1 ) and liver (ng mg−1 protein), after administra-
tion of a control diet compared to a diets enriched with grape extract, in male Wistar rats at the end of day 12

Plasma (ng mL−1 ) Liver (ng mg−1 protein)

Cat-(+) Epic-(−) Myr Quer Cat-(+) Epic-(−) Myr Quer

Control ND ND ND ND ND ND ND ND
Grape extract 6164.1 ± 1590.6 ND 25.2 ± 22.8 36.2 ± 16.9 701.2 ± 50.5 ND 18.8 ± 15.3 23.1 ± 12.3

All analyses were performed in triplicate. Data are expressed as the mean ± SD (seven animals per group). Cat-(+), (+)-Catechin; Epic-(−),
(−)-Epicatechin; Myr, myricetin; Quer, quercetin; ND, not detected. The limit of detection for all compounds was 1 ng mL−1 .

Table 3. Concentration of 3,4-hydroxyphenylacetic and 3,4-hydroxyphenylpropionic acids in plasma (ng mL−1 ), liver (ng mg−1 protein) and urine
(ng mL−1 ) of male Wistar rats, after administration of a control diet versus a grape extract-enriched diet

Plasma (ng mL−1 ) Liver (ng mg−1 protein) Urine (ng/total urine 24 h)
DOPAC DOPPR DOPAC DOPPR DOPAC DOPPR

Control ND ND ND ND ND ND
Grape extract 80.5 ± 28.6 ND 1.0 ± 0.5 ND 9117.5 ± 2629.4 629.1 ± 245.8

All analyses were performed in triplicate. Data are expressed as the mean ± SD (seven animals per group). DOPAC, 3,4-Hydroxyphenylacetic acid;
DOPPR, 3,4-Hydroxyphenylpropionic acid; ND, not detected.

90 min after ingestion, whereas the maximum concentration of

Table 4. Lipid profile and total bile acids in plasma and liver, after
administration of a control diet compared to a diets enriched with (−)-epic was found at 60 min. However, 150 min after intake, the
grape extract in male Wistar rats concentration was lowered by approximately 15%, probably as a
result of the binding of plasma proteins with quer.
Control Grape extract According to the results of the present study, the plasma concen-
tration of cat-(+) was much higher compared to myr and quer in
Plasma
rats fed with a diet enriched in grape extract. However, although
Total-chol (mg dL−1 ) 71.33 ± 12.89* 60.98 ± 3.61
the rats fed with grape extract consumed similar amounts of
HDL-chol (mg dL−1 ) 23.28 ± 4.76 24.30 ± 2.97
epic-(−) compared to myr and quer, the first compound was not
TG (mg dL−1 ) 112.33 ± 38.72 100.90 ± 55.27
detected in liver, nor in plasma. This result suggests that the excre-
LDL-chol (mg dL−1 ) 44.21 ± 11.82 35.93 ± 7.88
tion of epic-(−) is faster than that of myr and quer. The half-life for
Total-chol/HDL ratio 3.14 ± 0.65* 2.54 ± 0.32
quer has been reported to be much higher than for cat-(+) and
LDL/HDL ratio 1.91 ± 0.37 1.51 ± 0.44
epic-(−).49 The slow excretion of quer can be therefore attributed
Liver
not only to its high affinity for plasma albumin,50,51 but also to
Total-chol (𝜇g mg−1 protein) 7.45 ± 0.92 6.22 ± 1.22
phase II metabolism occurring later on.52
HDL-chol (𝜇g mg−1 protein) 0.82 ± 0.25 1.18 ± 0.47
DOPAC and DOPPR were analyzed to measure the products
TG (𝜇g mg−1 protein) 51.76 ± 5.84 45.76 ± 5.27
of polyphenol digestion. Although there are several other com-
TBA (mmol mg−1 protein) 29.56 ± 2.38 31.45 ± 1.57
pounds that can be potentially biosynthesized from polyphenols
All analyses were performed in triplicate. Data are expressed as over the digestion, these two compounds are the most abundant
the mean ± SD (of seven animals per group). Asterisks indicate a and have been used as the target compounds in several stud-
statistically significant difference based on an unpaired Student’s
ies in previous years.46,47 Our data showed the presence of low
t-test (P < 0.05). HDL-chol, HDL-cholesterol; LDL-Chol; TBA, Total Bile
Acids; TG, triglycerides; Total-chol, total-cholesterol; LDL, low density DOPAC concentrations in plasma and liver as a result of the oral
lipoprotein; HDL, high density lipoprotein. administration of grape phenolic extracts, whereas a different
behaviour was observed for DOPPR. By contrast, the level of
DOPAC in urine was significantly higher than DOPPR concentra-
and urine, which makes any analysis difficult, (+)-cat and (−)-epic tion (i.e. 9117.5 ± 2629.4 and 629.1 ± 245.8 ng/total, respectively).
have been previously analyzed after enzymatic hydrolysis. As expected, DOPAC and DOPPR were not detected in plasma, liver
For this reason, the samples of plasma, liver and urine were selec- and urine of control group because of an absence of polyphenols
tively incubated with 𝛽-glucuronidase and sulfatase for determina- in the diet of the control group. Previous studies reported that
tion of total amounts of cat-(+), epic-(−), myr and quer. Lee et al.34 polyphenols that are not absorbed in the small intestine can be
reported that the use of glucuronidase and sulfatase allows the hydrolyzed by the colonic microbiota to give less complex ben-
analysis of free and conjugated forms of each phenolic compound. zoic acid derivatives.43–45 The hydrolysis depends of the chemical
In addition, the use of this procedure allows an assessment of structure of flavonoids. The flavonol degradation results in the
whether the polyphenols are derived from sulfate and glucuronide biosynthesis of DOPAC, whereas the degradation of flavones,
conjugates.34 flavanones and flavanols mainly gives DOPPR.43–48 Metabolites
From a trial carried out in the present study involving the such as DOPAC and DOPPR or other absorbed polyphenols are
administration of 1 g of grape extract in Wistar rats, we found that conjugated in the liver by methylation, sulphation or glucuronida-
the maximum concentration of (+)-cat and quercetin occurred at tion. However, they were not targeted in our experiment, and

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 www.soci.org R Olivero-David et al.

Table 5. Statistical correlations between the digestibility coefficient (DC) of the phenol fractions or total bile acids in feces at 24 h, and several
parameters in plasma, liver and feces of male Wistar rats fed a control diet or a diets enriched with phenols from grape extract, over 12 days (except
TBA concentation in feces)

TBA/mg Total-chol HDL-chol LDL-chol LDL/HDL Total-chol/

TBA feces protein liver plasma plasma TG plasma plasma ratio HDL ratio

DC soluble phenols −0.035 0.948 0.393 0.440 −0.155 0.769 0.626 0.184 −0.422 0.404 0.474 0.342 0.054 0.919 −0.709 0.115
DC soluble-PA −0.601 0.207 0.733 0.097 −0.976*** 0.001 −0.395 0.438 −0.903* 0.014 −0.849* 0.033 −0.803 0.054 −0.005 0.992
DC insoluble-PA 0.230 0.661 0.728 0.101 −0.106 0.841 0.633 0.177 −0.083 0.876 0.020 0.970 −0.375 0.464 −0.766 0.076
DC total phenols −0.510 0.301 −0.809 0.051 −0.886* 0.019 −0.150 0.776 −0.913* 0.011 −0.886* 0.019 −0.718 0.108 −0.226 0.667
TBA feces at 24 h 1 −0.171 0.746 0.630 0.180 0.633 0.117 0.702 0.120 −0.743 0.091 −0.840* 0.036 −0.3850 0.451

Pearson correlation coefficient (r values are reported in bold, whereas the statistically significant differences are presented in italics). Statistically
significant difference: *P < 0.05; ***P < 0.001. DC, digestibility coefficient; HDL-chol, HDL-cholesterol; insoluble-PA, insoluble proanthocyanidins;
LDL-Chol, LDL-cholesterol; TBA, soluble-PA, soluble proanthocyanidins; total bile acids, TG, triglycerides; total-chol, total cholesterol.

further studies are needed to quantify the obtained products.
They can be excreted by urine or bile, or directly pass the entero-
cytes and return to the small intestine to pass again through the
colon.47 This could explain why low concentrations of plasmatic
and liver DOPAC were found in the grape extract-group only.
By contrast, the amount of DOPAC in the urine after 24 h was
significantly higher than DOPPR levels (i.e. 9117.5 ± 2629.4 and
629.1 ± 245.8 ng, respectively). The low absorption of SP could be
also explained by the formation of DOPAC and DOPPR metabolites
in the colon, which cause an increase of SP.
Several animal models have been previously reported in the
literature with respect to assessing the effect of cholesterol on
atherogenesis and cardiovascular diseases, although no sin-
gle model is considered as ideal for extrapolating results for
humans.53 Therefore, rats are commonly used in experimental
models to investigate the cholesterol metabolism, including
several previous studies that have used rats in the evaluation
of cholesterolemia.41,54,55 A decrease of cardiovascular risk and
lipemia associated with the administration of polyphenols derived
from various foods have been reported in cellular, animal and
humans model.15,16,21,22 The absolute cholesterol concentration,
as well as HDL, LDL and TG values, in both groups of rats used
in the present study were comparable with those reported in
other studies.40,56 In general terms, the lipid profile was similar in
the control and grape extract group. Nonetheless, the total-chol
concentration was moderately lower and LDL-chol tended to
decrease in plasma as a result of the administration of grape
extract. Differences in lipid levels between the two groups were
attributed to the presence of polyphenol in the diets, which may
affect lipemia.33 Furthermore, the total-chol/HDL-chol ratio, which
is used as a marker of hypercholesterolemia,57 was statistically sig-
nificantly lower in rats fed with polyphenols, suggesting that the
grape extract-enriched diet moderately increases the proportion
of HDL-chol in the lipoprotein. Yubero et al.22 found that the levels
of LDL-chol and total-chol were lower in humans who ingested
grape extract at a dose of 700 mg day−1 at breakfast compared
to a group consuming 700 mg day−1 of maltodextrin as a placebo
over 8 weeks. The findings of the present study are in agreement
with these results and indicate that grape extract consumption
can be effective with respect to lowering both cholesterolemia
and the cardiovascular risk.
To identify which parameters mostly explained the effect of
grape extract consumption on lipemia, a statistical analysis
was performed to correlate plasma lipid composition and the
absorption data. Although the consumption of grape extract
produced a moderate effect in total-chol and total-chol/HDL-chol
ratio, polyphenol absorption showed a negative correlation with
lipemia. According to the correlations observed, an increase of
soluble-PA and total phenol absorption after the consumption
of grape extract was correlated with lipemia, as well as with the
levels of total-chol, LDL-chol, TG and LDL/HDL ratio in plasma.
However, the main effect was attributed to the soluble-PA because
these provide their higher absorption. This relationship between
the soluble-PA absorption and the various parameters related
to lipemia indicates the potential effect that the consumption
of grape extract can exert on the reduction and prevention of
hyperlipidemia. Bile acid content in feces can be used to check the
interference of some diet components or certain drugs with the
enterohepatic cycle of bile salts (BS). This interference promotes
an increase of hepatic synthesis of BS such as from circulating
cholesterol.58 In the present study, grape extract consumption
produced an increase in the excretion of the TBA in feces over
24 h. A greater presence of TBA in feces was generally related to a
decrease in LDL/HDL ratio. The results indicate that this phenolic
extract promotes TBA excretion, because binding inhibits TBA
reabsorption. To compensate for the lower TBA level, the liver
increases plasmatic cholesterol uptake to biosynthesize BS. This
higher excretion in extract-fed rats can increase the catchment of
plasmatic cholesterol for the endogenous synthesis of BS and this
explain the lower cholesterol levels in the grape extract group.
However, similar results for TBA were obtained in liver of rats of
both groups. Nonetheless, liver TBA levels showed a decreasing
trend when total phenol absorption increased. This effect could be
explained by BS being incorporated into the enterohepatic cycle
to compensate for the bile acids excreted in feces, as mediated by
the presence of polyphenols.32,58 Ngamukote et al.59 reported that
cat-(+), epic-(−) and gallic acid from grape seed extracts showed
important binding capacities with bile acids. This suggests the
possibility that the binding of polyphenols with bile acids might
be related to the increase of faecal excretion of bile acids and may
promote the decrease of plasma cholesterol. However, further
studies are needed to confirm this hypothesis.
In addition, further studies are needed to fully characterize all
of the metabolites arising from polyphenol degradation during
digestion, as well as to clarify the pharmacokinetics of this extract
and all of the mechanisms acting on lipid metabolism.
CONCLUSIONS
The present study has reported on the use of a grape pheno-
lic extract with respect to assessing its potential in vivo effects
on lipemia in male Wistar rats. We have used a food industry

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Grape by-product in vivo bioavailability
by-product from the grape production industry, with the grape
by-product extract added as a feed, which makes our research
of interest for further practical applications in the food indus-
try and pharmaceutical sector. The absorption and excretion of
soluble polyphenols, as well as soluble and insoluble proantho-
cyanins, from grape extract before and after their ingestion was
shown, as well as their bioavailability and excretion, with respect to
understanding the fate of specific phenolic compounds and some
phenolic metabolites. The diet containing grape extract gener-
ally showed acceptable levels of absorption for individual pheno-
lic compounds and mainly soluble proanthocyanidins. The results
suggest the convenience of including grape extract to obtain a
partial blockage of the reabsorption of bile salts, which promotes
the decrease of plasmatic cholesterol levels through the activation
of bile salt hepatic synthesis.
Future works should investigate the full range of metabolites
produced from the phenolic compounds (e.g. glucuronic, sulphate
and other derivatives), which can be carried out using liquid
chromatography (LC-MS) for their full characterization. LC-MS can
be also used to quantify the full range of metabolites produced
during the digestion of this grape by-product extract.
In addition, further research should investigate the effect of
grape extract-enriched diet on lipid metabolism in other ani-
mal models, including human subjects with hypercholesterolemia.
Further research is also required to understand the optimization
of acceptable doses related to human intake, depending on the
target subject and the use (e.g. whether it is intended as a supple-
ment or to be used by the food industry as an ingredient for the
formulation of functional foods).
ACKNOWLEDGEMENTS
The present study was supported by INNPRONTA project from
Centro para el Desarrollo Tecnológico Industrial (CDTI). Ministe-
rio de Economía, Industria y Competitividad, (Spain) CDTI Min-
istry of Economy and Competitiveness (Spain). We thank Bodegas
Matarromera SL Valbuena de Duero (Spain) for providing the grape
extract. The authors declare that they have no competing financial
interests.
REFERENCES
1 Ali K, Maltese F, Choi YH and Verpoorte R, Metabolic constituents of
grapevine and grape-derived products. Phytochem Rev 9:357–378
(2010).
2 Xia EQ, Deng GF, Guo YJ and Li HB, Biological activities of polyphenols
from grapes. Int J Mol Sci 11:622–646 (2010).
3 He F, Mu L, Yan GL, Liang NN, Pan QH, Wang J et al., Biosynthesis
of anthocyanins and their regulation in colored grapes. Molecules
15:9057–9091 (2010).
4 Ananga A, Georgiev V and Tsolova V, Manipulation and engineering
of metabolic and biosynthetic pathway of plant polyphenols. Curr
Pharm Des 19:6186–6206 (2013).
5 Nassiri-Asl M and Hosseinzadeh H, Review of the pharmacological
effects of Vitis vinifera (grape) and its bioactive constituents: an
update. Phytother Res 30:1392–1403 (2016).
6 Crozier A, Jaganath IB and Clifford MN, Dietary phenolics: chemistry
bioavailability and effects on health. Nat Prod Rep 26:1001–1043
(2009).
7 Giovinazzo G and Grieco F, Functional properties of grape and wine
polyphenols. Plant Food Hum Nutr 70:454–462 (2015).
8 Stalmach A, Mullen W, Steiling H, Williamson G, Lean ME and Crozier
A, Absorption metabolism and excretion of green tea flavan-3-ols in
humans with an ileostomy. Mol Nutr Food Res 54:323–334 (2010).
9 Sánchez-Patán F, Monagas M, Moreno-Arribas MV and Bartolomé B,
Determination of microbial phenolic acids in human faeces by
UPLC-ESI-TQ MS. J Agric Food Chem 59:2241–2247 (2011).
J Sci Food Agric (2018) © 2018 Society of Chemical Industry
www.soci.org
10 Van Acker SA, Tromp MN, Haenen GR, Van der Vijgh WJF and Bast A,
Flavonoids as scavengers of nitric oxide radical. Biochem Biophys Res
Commun 214:755–759 (1995).
11 Thomas P, Wang YJ, Zhong JH, Kosaraju S, O’Callaghan NJ, Zhou
XF et al., Grape seed polyphenols and curcumin reduce genomic
instability events in a transgenic mouse model for Alzheimer’s
disease. Mutat Res 661:25–34 (2009).
12 Sakurai T, Kitadate K, Nishioka H, Fujii H, Kizaki T, Kondoh Y et al.,
Oligomerized grape seed polyphenols attenuate inflammatory
changes due to antioxidative properties in coculture of adipocytes
and macrophages. J Nutr Biochem 21:47–54 (2010).
13 Manach C, Mazur A and Scalbert A, Polyphenols and prevention of
cardiovascular diseases. Curr Opin Lipidol 16:77–84 (2005).
14 Lecour S and T Lamont K, Natural polyphenols and cardioprotection.
Mini Rev Med Chem 11:1191–1199 (2011).
15 Wu JM and Hsieh TC, Resveratrol: a cardioprotective substance. Ann N
Y Acad Sci 1215:16–21 (2011).
16 Wu JM, Hsieh TC and Wang Z, Review Article Cardioprotection by
resveratrol: a review of effects/targets in cultured cells and animal
tissues. Am J Cardiovasc Dis 1:38–47 (2011).
17 Yang CS, Ju J, Lu G, Xiao H, Hao X, Sang S et al., Cancer prevention by
tea and tea polyphenols. Asia Pac J Clin Nutr 17:245–248 (2008).
18 Khan N and Mukhtar H, Tea and Health: Studies in Humans. Curr Pharm
Des 19:6141–6147 (2013).
19 Sabu MC, Smitha K and Kuttan R, Anti-diabetic activity of green tea
polyphenols and their role in reducing oxidative stress in experimen-
tal diabetes. J Ethnopharmacol 83:109–116 (2002).
20 Del Rio D, Calani L, Cordero C, Salvatore S, Pellegrini N and Brighenti F,
Bioavailability and catabolism of green tea flavan-3-ols in humans.
Nutrition 26:1110–1116 (2010).
21 Pérez-Jiménez J, Serrano J, Tabernero M, Arranz S, Díaz-Rubio ME,
García-Diz L et al., Effects of grape antioxidant dietary fiber in car-
diovascular disease risk factors. Nutrition 24:646–653 (2008).
22 Yubero N, Sanz-Buenhombre M, Guadarrama A, Villanueva S, Carrión
JM, Larrarte E et al., LDL cholesterol-lowering effects of grape extract
used as a dietary supplement on healthy volunteers. Int J Food Sci
Nutr 64:400–406 (2013).
23 Woerdeman J, Del Rio D, Calani L, Eringa EC, Smulders Y and Serné
EH, Red wine polyphenols do not improve obesity associated
insulin-resistance: a randomized controlled trial. Diabetes Obes
Metab 20:206–210 (2018).
24 Martínez GA, Rebecchi S, Decorti D, Domingos JM, Natolino A, Del Rio D
et al., Towards multi-purpose biorefinery platforms for the valorisa-
tion of red grape pomace: production of polyphenols volatile fatty
acids polyhydroxyalkanoates and biogas. Green Chem 18:261–270
(2016).
25 Sanz-Buenhombre M, Villanueva S, Moro C, Tomás-Cobos L, Viadel B
and Guadarrama A, Bioavailability and the mechanism of action of a
grape extract rich in polyphenols in cholesterol homeostasis. J Funct
Foods 21:178–185 (2016).
26 Moro C, Procedimiento de extracción de polifenoles a partir de orujo de
uva procedente de destilación. Spanish Patent ES 2319032 B1 (2010).
27 USP, 731 Loss on Drying. USP 34 United States Pharmacopeia. The
United States Pharmacopeial Convention Inc., Rockville, MD (2011).
28 USP, 851 Spectrophotometry and Light-scattering. USP 34 United States
Pharmacopeia. The United States Pharmacopeial Convention Inc.,
Rockville, MD (2011).
29 Reeves PG, Nielsen FH and Fahey GC, AIN-93 purified diets for labora-
tory rodents: final report of the American Institute of Nutrition ad
hoc writing committee on the reformulation of the AIN-76A rodent
diet. J Nutr 123:1939–1951 (1993).
30 Singleton VL, Orthofer R and Lamuela-Raventós RM, Analysis of total
phenols and other oxidation substrates and antioxidants by means
of Folin-Ciocalteu reagent. Methods Enzymol 299:152–178 (1999).
31 Reed JD, McDowell RT, Van Soest PJ and Horvath PR, Condensed
tannins: a factor limiting the use of cassava forage. J Sci Food Agric
33:213–220 (1982).
32 Ruiz-Roso B, Quintela JC, de la Fuente E, Haya J and Pérez-Olleros
L, Insoluble carob fiber rich in polyphenols lowers total and LDL
cholesterol in hypercholesterolemic subjects. Plant Food Hum Nutr
65:50–56 (2010).
33 Reed JD, Effects of proanthocyanidins on digestion of fiber in forages.
J Range Manag 54:466–473 (2001).
34 Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S et al., Analysis of plasma
and urinary tea polyphenols in human subjects. Cancer Epidemiol
Biomarkers Prev 54:393–399 (1995).
wileyonlinelibrary.com/jsfa
 www.soci.org
35 Suárez Valles B, Santamaria Victorero J, Mangas Alonso JJ and Blanco
Gomis D, High-performance liquid chromatography of the neutral
phenolic compounds of low molecular weight in apple juice. J Agric
Food Chem 42:2732–2736 (1994).
36 Wang Y, Fice DS and Yeung PK, A simple high-performance liquid
chromatography assay for simultaneous determination of plasma
norepinephrine epinephrine dopamine and 3 4-dihydroxyphenyl
acetic acid. J Pharm Biomed Anal 21:519–525 (1999).
37 Iuvone PM, Boatright JH and Bloom MM, Dopamine mediates the
light-evoked suppression of serotonin N-acetyltransferase activity in
retina. Brain Res 418:314–324 (1987).
38 Bradford MM, A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:248–254 (1976).
39 Van der Meer R, De Vries HT and Glatz JFC, t-Butanol extraction of
feces: a rapid procedure for enzymic determination of fecal bile
acids, in Cholesterol Metabolism in Health and Disease: Studies in The
Netherlands, ed. by Beynen AC, Geelen MJH, Katan MB and Schouten
JA. Ponsen & Looijen, Wageningen, pp. 113–119 (1985).
40 Pérez-Olleros L, García Cuevas M, Ruiz-Roso B and Requejo A, Compar-
ative study of natural carob fibre and psyllium husk in rats. Influence
on some aspects of nutritional utilisation and lipidaemia. J Sci Food
Agric 79:173–178 (1999).
41 Olivero-David R, Schultz-Moreira A, Vázquez-Velasco M,
González-Torres L, Bastida S, Benedí J et al., Effects of Nori-and
Wakame-enriched meats with or without supplementary choles-
terol on arylesterase activity lipaemia and lipoproteinaemia in
growing Wistar rats. Br J Nutr 106:1476–1486 (2011).
42 Gu L, Kelm MA, Hammerstone JF, Beecher G, Holden J, Haytowitz D
et al., Concentrations of proanthocyanidins in common foods and
estimations of normal consumption. J Nutr 134:613–617 (2004).
43 Monagas M, Urpi-Sarda M, Sánchez-Patán F, Llorach R, Garrido I,
Gómez-Cordovés C et al., Insights into the metabolism and microbial
biotransformation of dietary flavan-3-ols and the bioactivity of their
metabolites. Food Funct 1:233–253 (2010).
44 Baba S, Osakabe N, Natsume M and Terao J, Absorption and urinary
excretion of procyanidin B2 [epicatechin-(4𝛽-8)-epicatechin] in rats.
Free Radic Biol Med 33:142–148 (2002).
45 Donovan JL, Lee A, Manach C, Rios L, Morand C, Scalbert A et al., Pro-
cyanidins are not bioavailable in rats fed a single meal containing a
grapeseed extract or the procyanidin dimer B 3. Br J Nutr 87:299–306
(2002).
46 Touriño S, Pérez-Jiménez J, Mateos-Martín ML, Fuguet E, Vinardell MP,
Cascante M et al., Metabolites in contact with the rat digestive tract
after ingestion of a phenolic-rich dietary fiber matrix. J Agric Food
Chem 59:5955–5963 (2011).
wileyonlinelibrary.com/jsfa © 2018 Society of Chemical Industry
View publication stats
R Olivero-David et al.
47 Scalbert A and Williamson G, Dietary intake and bioavailability of
polyphenols. J Nutr 130:2073S–2085S (2000).
48 Manach C, Scalbert A, Morand C, Rémésy C and Jiménez L, Polyphenols:
food sources and bioavailability. Am J Clin Nutr 79:727–747 (2004).
49 Hollman PC, Van Trijp JM, Buysman MN, Mengelers MJ, de Vries JH
and Katan MB, Relative bioavailability of the antioxidant flavonoid
quercetin from various foods in man. FEBS Lett 418:152–156 (1997).
50 Manach C, Morand C, Texier O and Favier ML, Quercetin metabolites
in plasma of rats fed diets containing rutin or quercetin. J Nutr
125:1911–1922 (1995).
51 Dangles O, Dufour C, Manach C, Morand C and Remesy C, Binding
of flavonoids to plasma proteins. Methods Enzymol 335:319–333
(2000).
52 O’Leary KA, Day AJ, Needs PW, Mellon FA, O’Brien NM and Williamson
G, Metabolism of quercetin-7-and quercetin-3-glucuronides by an in
vitro hepatic model: the role of human 𝛽-glucuronidase sulfotrans-
ferase catechol-O-methyltransferase and multi-resistant protein 2
(MRP2) in flavonoid metabolism. Biochem Pharmacol 65:479–491
(2003).
53 Jacques H, Effects of dietary fish protein on plasma cholesterol and
lipoproteins in animal models and in humans, in Dietary Proteins,
Cholesterol Metabolism and Atherosclerosis, ed. by Sugano M and
Beynen AC. Karger, Basel, pp. 59–70 (1990).
54 Schultz Moreira AR, Olivero-David R, Vázquez-Velasco M,
González-Torres L, Benedí J, Bastida S et al., Protective effects of sea
spaghetti-enriched restructured pork against dietary cholesterol:
effects on arylesterase and lipoprotein profile and composition of
growing rats. J Med Food 17:921–928 (2014).
55 González-Torres L, Vázquez-Velasco M, Olivero-David R, Bastida S,
Benedí J, González RR et al., Glucomannan and glucomannan plus
spirulina added to pork significantly block dietary cholesterol effects
on lipoproteinemia arylesterase activity and CYP7A1 expression in
Zucker fa/fa rats. J Physiol Biochem 71:773–784 (2015).
56 Pérez-Olleros L, Garcia-Cuevas M and Ruiz-Roso B, Influence of pulp
and natural carob fiber on some aspects of nutritional utilization and
cholesterolemia in rats. Food Sci Technol Int 5:425–430 (1999).
57 Hetta M, Hassan S, Abdel-Tawab S, Bastawy M and Mahmoud B,
Hypolipidaemic effect of Acanthophora spicifera (red alga) and
Cystoseira trinode (Brown alga) on albino rats. Iran J Sci Technol
33:287–297 (2009).
58 Gálvez J, Rodríguez D and Camueso D, Fibra dietética, en , in Tratado de
Nutrición. Bases Fisiológicas y Bioquímicas de la Nutrición, ed. by Gil A.
Editorial Médica Panamericana, Madrid, pp. 233–256 (2017).
59 Ngamukote S, Mäkynen K, Thilawech T and Adisakwattana S,
Cholesterol-lowering activity of the major polyphenols in grape
seed. Molecules 16:5054–5061 (2011).
J Sci Food Agric (2018)