Food Chemistry 207 (2016) 162169

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Influence of solvents on the composition of condensed tannins in grape

pomace seed extracts
Antonella Bosso , Massimo Guaita, Maurizio Petrozziello
Consiglio per la ricerca in agricoltura e lanalisi delleconomia agraria Centro di Ricerca per lEnologia, via P. Micca 35, 14100 Asti, Italy

a r t i c l e

i n f o

Article history:
Received 31 August 2015
Received in revised form 23 March 2016
Accepted 24 March 2016
Available online 25 March 2016
Keywords:
Grape pomace
Solvent extraction
Ultrasound assisted extraction
Condensed tannins
Phloroglucinolysis

a b s t r a c t
The extracting effectiveness of some solvents (water, ethanol, acetone, ethyl acetate), used as pure or in
binary and ternary mixtures, was studied for the extraction of seeds from the fermented pomace of
Nebbiolo cv. The aqueous mixtures of acetone provided the highest extraction yields in total polyphenols
and total flavonoids. Polyphenolic extracts were also obtained with aqueous mixtures of ethanol or
acetone at variable concentrations, and the condensed tannins were quantified with the phloroglucinolysis method. The aqueous mixtures of acetone were more effective than the corresponding aqueous
mixtures of ethanol.
The solvent influenced the extraction yield and the composition of the extracts: with the increase of the
yield, the mean degree of polymerization (mDP) of the condensed tannins increased. A significant
correlation was noticed between mDP and the molar percentages of (+)-catechin as terminal unit
(negative correlation), and of (
)-epicatechin and (
)-epicatechin-3O-gallate as extension units (positive
correlation).
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Polyphenols are an important quality factor for red wines, being
strictly related to sensory descriptors like astringency, bitterness
and structure. Moreover, various polyphenolic compounds, particularly resveratrol, quercetin, rutin, catechins and proanthocyanidins have been reported to have multiple biological activities,
including cardio-protective, anti-inflammatory, anti-carcinogenic,
antiviral and antibacterial properties attributed mainly to their
antioxidant and antiradical activity (Frankel, German, Kinsella,
Parks, & Kanner, 1993; King, Bomser, & Min, 2006; Santos-Buelga
& Scalbert, 2000; Teissedre, Frankel, Waterhouse, Peleg, &
German, 1996); they can also act as protective agents against
atherosclerosis and brain dysfunction (Gordon, 1996). Natural phenols have also been reported to have excellent properties as food
preservatives (Valenzuela, Nieto, Cassels, & Speisky, 1992) and
for many industrial applications (natural colorants for foods, or
in the production of paints, paper, and cosmetics).
Among the winemaking byproducts, the pomace is an important source of polyphenols. Generally, grape pomace accounts for
2030% of the initial weight of the grape (Rondeau, Gambier,
Jolibert, & Brosse, 2013), and it is characterized by a high residual
content of total polyphenols, depending on various factors such as
Corresponding author.
E-mail address: antonella.bosso@entecra.it (A. Bosso).
http://dx.doi.org/10.1016/j.foodchem.2016.03.084
0308-8146/ 2016 Elsevier Ltd. All rights reserved.

grape variety, winemaking technique, etc. (Makris, Boskou, &

Andrikopoulos, 2007).
Many works have dealt with the development of analytical
methods for the determination of the polyphenolic composition
of the grapes, with the aim of planning interventions from the
choice of the harvest date to the managing of the winemaking
and wine aging processes.
The analytical determination of grapes polyphenols is preceded
by the extraction of the polyphenolic fraction from the solid parts
of the berry (skins and seeds). Considering that the winemaking
byproducts (pomace), both fermented (red winemaking) and
unfermented (white winemaking), are an important source of
polyphenolic compounds, the extraction procedure represents a
fundamental step for the chemical characterization of grapes
polyphenols and for the industrial exploitation of the winemaking
byproducts.
To date no standard extraction method has been defined: the
most commonly used techniques for the isolation of phenolic
compounds from plant material are the extraction with solvents
(Baydar, Ozkan, & Sagdic, 2004; Bucic-Kojic, Planinic, Tomas,
Bilic, & Velic, 2007; Ignat, Volf, & Popa, 2011) and the extraction
with supercritical fluids, sometimes modified with co-solvents to
enhance their polarity (Bleve et al., 2008; Nahar & Sarker, 2005).
Regarding solid-liquid extraction, solvents like methanol, ethanol, acetone, diethyl ether, ethyl acetate (Bonilla, Mayen, Merida, &
Medina, 1999; Lafka, Sinanoglou, & Lazos, 2007), and their combi-

A. Bosso et al. / Food Chemistry 207 (2016) 162169

nations have been widely used for the extraction of phenolics,

often as aqueous mixtures with different proportions of water
(Lafka et al., 2007).
The majority of the scientific studies on this subject have investigated the effect of the extraction conditions on the total polyphenols content of the extracts, while fewer of them were focused on
single classes of polyphenol compounds.
Downey and Hanlin (2010) compared the effectiveness of a
range of aqueous mixtures of acetone and ethanol for the extraction of condensed tannins from Shiraz grape skins, and they
observed that the extraction yields in tannins and the mDP of the
extracted proanthocyanidins changed with the type of solvent
(acetone or ethanol) or the percentage of water in the mixtures.
Among the polyphenolic compounds of the grapes, the condensed tannins (proanthocyanidins) are the most abundant in
the skins and seeds. The chemical/biological activity of the extracts
does not only depend on their total polyphenols content, but also
on specific subclasses of polyphenols: when the goal is the characterization of the polyphenolic extracts aimed at their practical use
(antiradical activity), Makris, Boskou, and Andrikopoulos (2007)
suggest to consider the total flavanols content (FTI) in addition to
the total polyphenols content.
The present work studied the influence of different extraction
modalities, particularly the type of solvent, on the composition in
condensed tannins of the extracts, working with Nebbiolo grapes
seeds obtained from fermented pomace. Firstly, two different
ternary solvent mixtures (acetone/ethanol/water and acetone/
ethanol/ethyl acetate) at different proportions were compared,
then the work focused on the use of a range of two binary aqueous
mixtures (acetone-water and ethanol-water). This work was aimed
at verifying the possibility to modulate the composition of the
extracting mixture in order to obtain a range of extracts different
from one another for the polyphenolic composition: once characterized for their chemical and biological properties, these extract
will find different possible applicative destinations.

163

ically processed to separate seeds from skins, and to obtain seeds

flour (threshing, sieving, grinding).
a) Extraction trials with ternary mixtures.
Two different ternary mixtures were compared for the
extraction of Nebbiolo seeds flour: acetone/ethanol/water and
acetone/ethanol/ethyl acetate. For each of the two mixtures, the
solvents were used in variable proportions, according to the
experimental scheme reported in Table 1.
The extraction protocol was as follows. 1 g seeds flour and 6 mL
solvent were placed in a 15 mL Corex tube, vortexed for 30 s, sonicated for 20 min (50 W, 48 kHz 10%), then again vortexed for
30 s. The sample was centrifuged at 18 C for 20 min at 2880g, then
the supernatant (5 mL) was separated from the solid residue, and
vacuum dried at 35 C with a Genevac evaporator (EZ-2, Genevac,
Ipswich, UK). The dry residue of the extract was re-dissolved in
2 mL of methanol for the subsequent analysis of polyphenols.
The content of total polyphenols and total flavonoids was determined by spectrophotometry (see below).
b) Extraction trials with aqueous mixtures of acetone and
ethanol.
The second trial concerned the extraction of polyphenols from
Nebbiolo seeds flour with aqueous solutions of acetone and ethanol at different title (0, 25, 50, 75 and 100% of acetone and ethanol,
respectively). The extraction ratio was 1:6 w/v flour/solvent
(1 g flour in 6 mL solvent), and extraction times in the sonicator
of 20 min and 40 min were compared. Each experiment was performed in duplicate. The obtained extracts were analyzed to determine the content of total polyphenols and total flavonoids by
spectrophotometry and condensed tannins by phloroglucinolysis
(see below).
2.3. Phenolic characterization of the extracts

2. Materials and methods

2.1. Reagents
The solvents used during the experiment were acetone and
ethyl acetate HPLC grade (Sigma Aldrich Co., St. Louis, MO, USA)
and ethanol 95% v/v (Beccaris Distilleries - Boglietto di Costigliole
d0 Asti, AT, Italy) for the extraction trials, methanol HPLC grade
(VWR Chemicals, Radnor, PA, USA) and acetonitrile HPLC grade
(Merck KGaA, Darmstadt, Germany) for the HPLC analysis of the
phloroglucinolysis reaction products. Other reagents were
phloroglucinol, L-ascorbic acid, sodium acetate, gallic acid monohydrate, Folin-Ciocalteu reagent, hydrochloric acid 37%, formic
acid 98%, phosphoric acid 85% (Sigma Aldrich Co., St. Louis, MO,
USA), sodium carbonate, acetic acid 100% (Merck KGaA, Darmstadt,
Germany). The standards used for the determination of the
response factors for the HPLC analysis of the phloroglucinolysis
reaction
products
were
(+)-catechin,
(
)-epicatechin,
(
)-epicatechin-3O-gallate (Sigma Aldrich Co., St. Louis, MO,
USA). Ultrapure water from a Milli-Q gradient A10 instrument
system (Millipore Corporation, Billerica, MA, USA) was used
throughout the experiment.
2.2. Extraction trials from grape seeds
During vintage 2012 the pomace of Nebbiolo cv. (kindly provided by Marchesi di Gresy winery, Barbaresco, CN - Italy) was sampled at the end of the alcoholic fermentation, after soft pressing
(0.5 bars); grape pomace was air dried (48 h, 35 C), then mechan-

Total polyphenols and total flavonoids were determined by

spectrophotometry (Di Stefano, Cravero, & Gentilini, 1989):
for total flavonoids, the extract was diluted 50-fold with
hydrochloric ethanol (ethanol:H2O:HCl 70:30:1) and the absorbance at 280 nm was measured. The results were expressed as
(+)-catechin equivalents per 1 g dry weight of seeds flour.
for total polyphenols, 1 mL extract was diluted 20-fold with
water, and 1 mL of diluted sample was added to 1 mL of
Folin-Ciocalteu reagent, basified with 4 mL of sodium carbonate, and filled up to 20 mL. After 90 min, the absorbance at
750 nm was measured against a blank. The results were
expressed as gallic acid equivalents (GAE) per 1 g dry weight
of seeds flour.
2.4. Characterization of condensed tannins by phloroglucinolysis
Phloroglucinolysis is the acid-catalyzed cleavage of the condensed tannins (proanthocyanidins) in the presence of excess
phloroglucinol as nucleophile molecule (Kennedy & Jones, 2001).
The method consists of a sample preparation step, followed by
the phloroglucinolysis reaction and the HPLC analysis of the reaction products. The operating protocol (Ducasse et al., 2010, with
some modifications) was as follows. The extracts obtained during
the extraction trials (5 mL) were evaporated to dryness under vacuum at 35 C. The dry residue was dissolved in 2.5 mL of 2% aqueous acetic acid (using ultrasound, when necessary) and 1.5 mL was
applied on a C-18 solid phase extraction cartridge (Sep-Pak tC18
1 g, Waters, Milford, MA, USA). Sugars and organic acids were

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A. Bosso et al. / Food Chemistry 207 (2016) 162169

Table 1
Experimental design for the study of the ternary mixtures. The percentage of the individual solvent is displayed and the results of the trials as concentrations in total polyphenols
and total flavonoids (mg/g d.w.) are reported.

Run Ordera

Acetone

Ethanol

Water

Total flavonoids

Total
polyphenols (GAE)

Acetone

Ethanol

Ethyl
acetate

Total
flavonoids

Total polyphenols
(GAE)

14
15
9
10
7
1
2
4
5
3
6
8
13
11
12

100
0
0
50
50
0
0
50
50
66
16.5
16.5
33
33
33

0
100
0
50
0
50
50
0
50
16.5
66
16.5
33
33
33

0
0
100
0
50
50
50
50
0
16.5
16.5
66
33
33
33

0.50
1.84
0.72
2.64
12.60
4.92
4.90
12.07
2.85
7.67
6.38
7.74
9.80
9.71
10.15

0.92
2.14
0.91
2.94
8.78
4.18
4.35
8.32
2.86
6.05
4.68
5.75
7.64
7.61
7.83

100
0
0
50
50
0
0
50
50
66
16.5
16.5
33
33
33

0
100
0
50
0
50
50
0
50
16.5
66
16.5
33
33
33

0
0
100
0
50
50
50
50
0
16.5
16.5
66
33
33
33

0.50
1.84
0.27
2.64
0.37
1.56
1.42
0.34
2.85
1.31
2.29
0.98
2.13
2.06
2.21

0.92
2.14
0.58
2.94
0.61
1.49
1.45
0.50
2.86
1.48
2.43
1.29
2.37
2.18
2.43

Run Order is the order of execution of the extraction tests.

eluted with 5 mL of 2% aqueous acetic acid, the phenolic compounds were recovered with 8 mL of methanol. The eluent volume
was evaporated to dryness under vacuum, re-dissolved in 400 lL
of the reactive solution (250 mg phloroglucinol + 50 mg ascorbic
acid in 5 mL methanol/HCl 98/2 v/v) and heated for 25 min at
50 C (water bath). The reaction was stopped with 400 lL of aqueous 200 mM sodium acetate, and the sample was filtered with a
0.45 lm polypropylene syringe filter (Whatman Inc., NJ - USA)
directly into the HPLC vial. Samples were analyzed with an Agilent
1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA) with a
reversed-phase Atlantis T3 dC18 column (5 lm packing,
250  2.1 mm i.d.; Waters, Milford, MA, USA) protected by a guard
column of the same material (10  2.1 mm i.d.; Waters, Milford,
MA). The mobile phase was a gradient of acetonitrile/water/formic
acid (80/18/2 v/v/v; solvent B) in water/formic acid (98/2 v/v;
solvent A), at a flow rate of 0.30 mL/min at 30 C. Proportions of
solvent B were as follows: 05 min with 0% B; 520 min, 06.5%;
2031 min with 6.5%; 3135 min, 6.510%; 3565 min, 1020%;
6570 min with 20%; 7075 min, 20100%; and 7580 min,
1000%. The injection volume for all samples was 10 lL.
The monomers (terminal subunits and extension subunitphloroglucinol adducts) were detected by a DAD detector (Agilent
G1315D), identified by comparison with the retention times and
quantified from peak areas at 280 nm with the Chemstation software (Agilent Technologies, Palo Alto, CA, USA) using external calibration with known concentrations of flavan-3-ol monomers from
Sigma (Saint Louis, MO).
The total condensed tannins content, their mean degree of
polymerization (mDP), and the percentage of each constitutive
unit were determined. The molar concentration of the subunits
was calculated from their molar absorptivities, as reported by
Kennedy and Jones (2001), and expressed as molar percentage.
To calculate the apparent mean degree of polymerization
(mDP), the sum of the terminal and extension subunits
(in moles) was divided by the sum of the terminal subunits
(in moles). The total condensed tannins, expressed in mg/g of
dried weight of seeds flour, were determined as the sum of
the quantified subunits.
For the quantification of the different subunits in units of
weight, the response factors of (+)-catechin, (
)-epicatechin and
(
)-epicatechin-3O-gallate were calculated by direct HPLC
injection of the standards. The response factors of the extension
subunits were then calculated using the response factor of
(+)-catechin and considering the respective values of the molar
extinction coefficients (Kennedy & Jones, 2001).

2.5. Statistical analysis

a) Extraction trials with ternary mixtures.
A Modified Simplex Centroid design (Mixture design) (Table 1)
was built with the 3 solvents, respectively acetone/ethanol/water
and acetone/ethanol/ethyl acetate, as independent factors. The
data matrices were then processed with the MODDE software (Version 10.0) to obtain the Contour Plots.
b) Extraction trials with aqueous mixtures of acetone and
ethanol.
Data were processed with two-factor ANOVA and Tukeys test
to study the effect of the solvent concentration and the extraction
time, separately for the two solvents (acetone and ethanol). The
Correlation analysis was also applied to study the relationships
between mDP and the concentration of condensed tannins and
the percentage weight of each subunit. The Correlation analysis
was applied to the complete data set, and separately for the ethanol and acetone extracts. SPSS for Windows, version 15.0 (SPSS
Inc., Chicago Il USA, 2004) was used.
3. Results
3.1. Extraction trials with ternary mixtures
Fig. 1 shows the trend of the total flavonoids content of the
grape seeds extracts when varying the composition of the ternary

Fig. 1. Contour Plot of the total flavonoids content of the grape seeds extracts as a
function of the composition of the ternary mixture acetone/ethanol/water.

165

A. Bosso et al. / Food Chemistry 207 (2016) 162169

acetone extracts was approximately 1.5 times higher than in the

ethanol extracts. Working with fermented pomace of different cultivars Ky, Lorrain, Kolbas, Crozier, and Teissedre (2014) found total
polyphenols contents (GAE) in the seeds that were 26 times
higher than the average flavonoids content we observed for the
Nebbiolo cultivar. In a later work, comparing different fermented
pomace from some Italian cultivars (data not published), we measured in the seeds of Albarossa cv. an average polyphenols content
that was more than double the values reported here for Nebbiolo
cv., while for other cultivars it was similar or lower. Therefore, a
high variability exists in the polyphenols content of the extracts
and this variability is probably related to the cultivar of origin
and to the extraction degree achieved during the winemaking
process.
Fig. 2. Contour Plot of the total flavonoids content of the grape seeds extracts as a
function of the composition of the ternary mixture acetone/ethanol/ethyl acetate.

3.2.2. Concentration of total condensed tannins

The concentration of total condensed tannins (proanthocyanidins) in the seeds extracts was determined as the sum of the extension and terminal subunits expressed as mg/g d.w. of seeds flour.
Fig. 3 reports the concentration trend of the total condensed tannins in the extracts obtained with ethanol and acetone at different
concentrations in water, separately for extraction times of 20 min
and 40 min.
The type of solvent and its proportion in water influenced the
yield in polyphenols extracted from grape seeds. Acetone was
more effective than ethanol, with more than double the extractive
effectiveness at 75% v/v in water. These results perfectly agree with
the trend reported by Downey and Hanlin (2010) for skins extracts.
The extraction efficiency increased with the increase of the solvents concentration in water up to 50%, then for acetone it

mixture acetone/ethanol/water. The maximum extraction (more

than 15 mg/g d.w.) was achieved with the binary mixture acetone/water at 50% v/v. The lowest extraction yields were obtained
with 100% acetone or 100% water. A similar trend was observed for
the total polyphenols content (data not shown).
As regards the mixture acetone/ethanol/ethyl acetate (Fig. 2),
the maximum extraction of total flavonoids (about 2.8 mg/g d.w.)
was achieved with the binary mixture ethanol/acetone with percentages of ethanol varying between 50% and 70%. A similar trend
was observed for the total polyphenols content (data not shown).
At optimal extraction conditions for both tests, the extractive
effectiveness was about 5 times higher for the acetone/water mixture than for the ethanol/acetone mixture.

3.2. Extraction trials with aqueous mixtures of acetone and ethanol

Condensed tannins (mg/g)

The second experiment regarded the comparison between the

extractive capacity of aqueous mixtures of acetone and ethanol
at different percentages, working with the same matrix (Nebbiolo
seeds from fermented pomace). The polyphenolic extracts were
analyzed to determine the content of total flavonoids by spectrophotometry and to characterize the condensed tannins (proanthocyanidins) with the phloroglucinolysis method.
3.2.1. Concentration of total flavonoids
The total flavonoids concentration in the seeds extracts (Table 2)
varied with the concentration of the solvent in water from
0.77 mg/g d.w. (pure water) to 5.38 mg/g d.w. for ethanol (75% of
solvent) and 7.98 mg/g dw for acetone (50% solvent): the maximum yields were obtained with aqueous mixtures at 75% ethanol
or 50% acetone, and the total flavonoids concentration in the

7
6
5

Ethanol 20'

Ethanol 40'

Acetone 20'
Acetone 40'

2
1
0
0

25

50

75

100

% solvent

Fig. 3. Concentration trend of the total condensed tannins in the extracts obtained
with ethanol and acetone at different concentrations in water.

Table 2
Total flavonoids content and composition of the condensed tannins. Different letters along the column discriminate the extracts significantly different from one another (p < 0.05,
Tukeys test), separately for ethanol and acetone aqueous mixtures.
Solvent

Total flavonoids
(mg/g d.w.)

mDP

% gallates

Extension units (molar%)

EGC

Terminal units (molar%)

EC

ECG

EC

ECG

31.6
19.8
13.3
15.5
14.4

c
b
a
ab
a

34.0 c
24.4 b
9.3 a
8.7 a
7.5 a

1.3
4.3
4.7
4.3
3.9

a
bc
c
bc
b

31.6
13.6
10.3
10.0
24.2

d
b
a
a
c

34.0 b
9.8 a
9.4 a
9.4 a
10.4 a

1.3
4.6
4.2
4.3
4.8

a
c
c
bc
c

Ethanol

0
25
50
75
100

0.77
2.47
5.28
5.38
2.25

a
c
d
e
b

1.5
2.1
3.7
3.6
3.9

a
b
c
c
c

1.9 a
9.0 b
13.9 c
12.7 bc
13.1 c

0.0
2.4
1.0
0.4
0.0

a
d
c
b
a

18.0
20.3
22.1
25.7
26.0

a
b
c
d
d

14.5
24.2
40.5
37.0
39.0

a
b
d
c
c

0.6
4.8
9.2
8.4
9.2

a
b
c
c
c

Acetone

0
25
50
75
100

0.77
2.18
7.98
7.64
1.29

a
c
e
d
b

1.5
3.6
4.2
4.2
2.6

a
c
c
c
b

1.9 a
13.3 c
15.4 cd
16.2 d
11.5 b

0.0
0.5
1.2
1.0
0.0

a
b
d
c
a

18.1
21.6
17.9
18.0
23.8

a
b
a
a
c

14.5
41.2
45.8
45.4
30.2

a
c
d
d
b

0.6 a
8.7 c
11.3 d
11.9 d
6.6 b

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A. Bosso et al. / Food Chemistry 207 (2016) 162169

remained quite stable up to 75% aqueous solutions, while for ethanol it steadily decreased from 50 to 100% in water. A dramatic
decrease of the extraction efficiency was observed for the pure solvents (100%), particularly in the case of 100% acetone that showed
less extractive effectiveness than 100% ethanol and 100% water. On
the contrary, in the case of skins Downey and Hanlin (2010)
observed that 100% acetone, though showing a decrease of effectiveness, remained twice as effective than 100% ethanol and
100% water.
The maximum average concentration of condensed tannins was
8.3 mg/g for 50% acetone and 4.3 mg/g for 50% ethanol. The concentrations of condensed tannins and total flavonoids were of the
same order of magnitude, but the related concentration trends,
when varying the concentration of the solvent in water, were similar but not overlapping. With ethanol, a modest but significant
increase in the extraction yield of total flavonoids was observed
when passing from 50% to 75% ethanol, while in the same interval
the content of condensed tannins decreased. With acetone, the
trend was reversed: from 50% to 75% acetone the content of
condensed tannins increased, while the concentration of total flavonoids decreased. Moreover, in the extracts obtained with 100%
acetone the decrease in extracted flavonoids was lower than the
decrease in condensed tannins. The determination of the total
flavonoids content, being based on the measurement of the absorbance at 280 nm, normally includes other non-flavonoid phenolic
molecules and non-phenolic molecules whose extraction is differently affected by the same solvents.
Regarding the extraction time, with the ethanol solutions no
significant changes in concentration were observed by increasing
the duration of the extraction from 20 min to 40 min, whereas a
statistically significant increase of the extraction yield (F = 7),
though modest and with no practical relevance, was observed with
the acetone solutions. Bucic-Kojic et al. (2007) reported that
8090% of the total polyphenols extracted within 200 min was
extracted during the first 40 min of extraction, independently from
the temperature and the particle size of the vegetal matrix. In our
experiment, the use of ultrasound allowed the extraction process
to be accelerated further, while in previous reported work
(Bucic-Kojic et al., 2007) only periodical stirring of the mass was
carried out at 15 min intervals.
3.2.3. mDP of the condensed tannins
The average polymer length of the condensed tannins in the
extracts varied with the solvent and its concentration in water
(Table 2).
The mean polymerization degree (mDP) varied from 1.5 to 4.2
units. The condensed tannins extracted in pure water had the lowest mDP. Low mDP values were also obtained with 25% ethanol and
100% acetone (2.1 and 2.5 units, respectively).
In the case of ethanol, the mDP of the extracted tannins strongly
increased by increasing the solvent concentration up to 50%, then it
remained stable, while in the case of acetone the mDP increased up
to 50%, it remained stable in the interval 5075%, then it dramatically decreased to a minimum value at 100% acetone.
At a given concentration of the solvent in the aqueous mixture,
the mDP values of the acetone extracts were higher than the corresponding values of the ethanol extracts. These results confirm
those reported by Downey and Hanlin (2010) for skins extracts,
but in our study the differences were quantitatively lower, though
still statistically significant. This could be due to the lower mDP of
the seeds tannins (Souquet, Cheynier, Brossaud, & Moutounet,
1996), that allowed less variability than for the skins tannins.
3.2.4. Degree of galloylation (%) of the condensed tannins
Differences were observed in the galloylation degree of the
extracts (Table 2), that was maximum when both solvents were

employed at 5075% in water. Acetone extracted a greater galloylated fraction than ethanol: the concentration of the galloylated
fraction in the extracts, at the maximum extraction yield for both
solvents (50% v/v), was about 2.2 times higher for acetone than
for ethanol. On the contrary, the galloylated fraction was almost
absent in the 100% aqueous extracts.
3.2.5. Composition in monomer subunits
The composition of condensed tannins in monomer subunits is
reported in Table 2. Seven different monomer subunits were
identified: (+)-catechin, (
)-epicatechin and (
)-epicatechin-3Ogallate, as both terminal and extension subunits, plus (
)-epigallocatechin as extension subunit. In some extracts the presence of
(
)-epigallocatechin was detected in trace amounts: the presence
of this subunit, which is usually found only in the skins tannins,
was probably due to the adsorption on grape seeds of a small
amount of condensed tannins from the skins during the fermentative maceration.
The extraction with 100% water, besides determining low
extraction yields, had a significant influence on the profile of the
flavanol subunits that form the condensed tannins. Statistically
significant differences for all parameters were noticed between
the trials extracted with 100% water and those obtained with aqueous solutions of ethanol or acetone. In particular, in the 100% water
extracts the terminal subunits prevailed over the extension subunits (low mDP); the terminal subunits were almost exclusively
represented by (+)-catechin and (
)-epicatechin, roughly in the
same proportion, and the percentage of galloylated forms was very
low. Among the extension subunits, (+)-catechin slightly exceeded
(
)-epicatechin, and (
)-epicatechin-3O-gallate was present at
very low concentrations.
As regards the ethanol extracts, the percentage values of the
terminal and extension subunits were similar at 0% and 25% solvent. At 50% ethanol the percentage of the terminal subunits
decreased, due to the increase of mDP, and then it remained
unchanged with higher concentrations of ethanol. The terminal
subunits profiles were similar at 0% and 25% solvent, then changed:
(+)-catechin became the dominant unit followed by (
)-epicatechin and (
)-epicatechin-3O-gallate; for the extracts with 50%,
75% and 100% ethanol the terminal subunits profile remained
unchanged. Slight differences were found between the different
mixtures for the content of (
)-epicatechin-3-O-gallate (3.94.7%).
In the case of the acetone mixtures, the percentage weight of
the terminal subunits and their profiles remained unchanged at
25%, 50% and 75% solvent. The percentages of (+)-catechin and
(
)-epicatechin were similar, and lower than in the ethanol
extracts (with a lower mDP). Greater differences were observed
with 100% acetone, when the extraction yield dramatically
decreased.
As regards the extension subunits, an increase was generally
observed in the total percentage value of the subunits, particularly
due to (
)-epicatechin. With ethanol, the extracts at 50%, 75% and
100% solvent had the same extension subunits profile, characterized by the predominance of (
)-epicatechin (3740%), followed
by (+)-catechin (2226%) and (
)-epicatechin-3O-gallate (89%).
With acetone, the extension subunits profile remained stable in
the range 2575% solvent: (
)- epicatechin was the prevailing
subunit (41.245.8%), followed by (+)-catechin (18.022.0%) and
(
)-epicatechin-3O-gallate (8.711.9%).
Contrary to our experimental observations, Downey and Hanlin
(2010) working with Syrah skins noticed more constancy in the
extension subunits profile between ethanol and acetone extracts.
Apparently, in our case the extension and terminal subunits profiles remained unchanged for each extraction modality in the intervals with unvaried mDP (2575% for acetone and 50100% for
ethanol) (Table 2): any variation of solvent concentration within

A. Bosso et al. / Food Chemistry 207 (2016) 162169

Table 3
Correlation coefficients, calculated in the complex and distinctly by type of solvent,
between mDP and the total content of condensed tannins and the percentage values
of each subunit.
mDP
Acetone

Ethanol

Total

Extension units

EGC
C
EC
ECG

0.84

0.19
0.99
0.98

0.22
0.81
0.95
0.95

0.02
0.17
0.96
0.95

Terminal units

C
EC
ECG

0.99

0.84
0.70

0.90

0.96
0.70

0.93

0.86
0.56

% gallates
Condensed tannins (mg/g d.w.)

0.95
0.94

0.91
0.65

0.91
0.76

these intervals, therefore, did not cause qualitative differences in

the condensed tannins composition.
Table 3 shows the correlation coefficients, calculated in the
complex and distinctly by type of solvent, between mDP and the
total content in condensed tannins and the percentage value of
each subunit. The correlation coefficients were higher with acetone
than with ethanol, and for the totality of the data the coefficients
values resulted intermediate (Table 3).
A positive correlation was observed between the total content
in condensed tannins and their mDP: the correlation was stronger
for the acetone extracts (r = 0.94) than for the ethanol extracts
(r = 0.65). Generally, with the increase of mDP a significant
increase of the percentage values of (
)-epicatechin and (
)epicatechin-3-O-gallate as extension units was observed; on the
other side, a significant decrease of the (+)-catechin and (
)-epicatechin content as terminal units was observed.
4. Discussion
Many works in the literature have studied the factors that influence the solid-liquid extraction process of polyphenols from grapes
skins and seeds, both fermented and unfermented. Bucic-Kojic
et al. (2007) observed that the studied factors (temperature and
duration, particles size and duration, temperature and solid/liquid
ratio) were independent of one another, while Lafka et al. (2007)
observed that the effect of the solvent type was independent of
the extraction duration and of the solid/liquid ratio. No significant
interactions between the various factors have been observed, that
could cause the inversion of the extraction yield of a solvent with
respect to another when varying the extraction conditions (duration, solid/liquid ratio). When operating with factors independent
of one another the experimental plan of the trials can be simplified,
and the various factors can be studied independently without running the risk of undermining the general validity of the results,
thus maintaining the possibility to extend the obtained results to
other situations; moreover, the results can be compared to other
works that studied the same factors, albeit under different
conditions.
The operating conditions of the extraction processes were
chosen according to the data reported in the literature. As regards
the duration of the contact between flour and solvent, the existing
literature reports extraction times varying from a few tens of minutes (Bonilla et al., 1999), to a few hours (Mildner-Szkudlarz,
Zawirska-Wojtasiak, & Goslinski, 2010), up to 24 h for extractions
with periodic re-suspensions of the solid phase (Lafka et al.,
2007; Spigno, Tramello, & De Faveri, 2007). Carrera, RuizRodriguez, Palma, and Barroso (2012) observed that the use of
ultrasounds, extraction conditions and yields being equal, reduced
10-fold (from 60 min to 6 min) the duration of the extraction. This

167

explains why in our case, with the use of ultrasound, no significant

increase in the extraction of polyphenols was observed when varying the duration of the extraction from 20 to 40 min, and why
20 min can therefore be considered sufficient for the performance
of the process.
Regarding the proportion between the weight of solid phase
(flour) and the volume of solvent, the ratio 1:6 was chosen. In
the literature this ratio ranges from 1:3 to 1:12 (Lafka et al.,
2007); other authors used the ratios 1:4 (Mildner-Szkudlarz
et al., 2010; Spigno et al., 2007) and 1:51:5.5 (Makris, Bokou, &
Andrikopoulos, 2006; Nawaz, Shi, Mittal, & Kakuda, 2006).
In the present work two different ternary mixtures were studied (ethanol/acetone/ethyl acetate and ethanol/acetone/water);
the combination of ethyl acetate and water (Bonilla et al., 1999)
was not considered because we chose not to use solvents
immiscible with each other. During the second experiment only
two solvents were employed: acetone, because it gave the best
extraction yields as aqueous solution, and ethanol, the most widely
used solvent and the preferred one when the extracts are prepared
for food applications. The use of ethanol is particularly interesting
for the sustainability of the extraction process because also ethanol, like grape pomace, is a winemaking byproduct (from the distillation of pomace) (Makris et al., 2006).
The study focused on the effect of the type and concentration of
solvent on the extraction of the most abundant polyphenolic fraction present in grape seeds and skins: the condensed tannins. Pure
solvents (100% solvent) proved to be less effective for the extraction of tannins than their aqueous mixtures, and the same was
observed for 100% water. These data are in agreement with various
authors (Alonso, Bouzeix, & Revilla, 1991; Pinelo, Rubilar, Jerez,
Sineiro, & Nunez, 2005; Yilmaz & Toledo, 2006).
The highest extractions yields were obtained with aqueous
solutions at 50% ethanol and 5075% acetone. Working with aqueous solutions of ethanol at concentrations ranging from 28.5 to
85.5%, Makris et al. (2006) found that the highest extractions yields
from different kinds of byproducts (skins, seeds and stems from
white grapes) were obtained with aqueous solutions at 57% ethanol; in the case of stems, a further increase in extraction yields
was achieved by acidifying the medium (0.1% HCl).
Spigno et al. (2007) observed an increase in the extraction of
total polyphenols (as GAE) from fermented grape marc of Barbera
cv. by increasing the percentage of water in ethanol from 10% to
30%, then from this point up to additions of 60% (40% ethanol)
the extraction yields in polyphenols remained unchanged, and only
an increase in total extract was observed. Yilmaz and Toledo
(2006) reported that the extraction yield in polyphenols (as GAE)
from seeds flour increased with the percentage of water in the
ethanol mixture from 0% to 30%, then it remained constant
between 30% and 50%, and it dropped at higher concentrations of
water.
Mildner-Szkudlarz et al. (2010) observed an increase of 22% in
the extraction yield of phenols from fermented grape marc with
aqueous solutions at 70% ethanol instead of 90% ethanol. In the
work by Downey and Hanlin (2010) the amount of total tannins
extracted from Shiraz grapes skins increased with the proportions
of ethanol in water, until a maximum concentration was reached at
50% ethanol; the concentrations of extracted tannins decreased
with mixtures containing more than 50% ethanol in water. The
same Authors observed a maximum concentration of extracted
tannins with aqueous mixtures of acetone at concentrations
between 50% and 70%.
According to Downey and Hanlin (2010), we observed that acetone was twice as effective than ethanol at the same concentration
in water. However, when ethanol was used as pure (100% ethanol)
it was more effective than 100% acetone for the extraction of
polyphenols from grape seeds. Our results agree with those

168

A. Bosso et al. / Food Chemistry 207 (2016) 162169

reported by Lafka et al. (2007) regarding fermented grape marc.

Differently, Downey and Hanlin (2010) observed with Shiraz skins
that 100% acetone was twice as effective than 100% ethanol. The
observed differences in the results may depend either on the characteristics of the raw material used (skins in the case of Downey &
Hanlin, seeds in our case) or also on the different degradation
grade of the skins (fresh skins in the case of Downey & Hanlin,
fermented skins in the case of Lafka et al.).
Considering the three organic solvents used as pure, the extraction yields in polyphenols increased with the increase of the
dielectric constant (e): 6.02 for ethyl acetate, 20.7 for acetone,
24.3 for ethanol. The same principle is valid for methanol, not used
in this work, which has a higher e (32.7): under the same conditions, methanol is more extracting than ethanol (Lafka et al.,
2007). Differently, the yields decreased with water, which is the
most polar solvent (e = 80.1). The binary mixtures ethanol/water
have e values intermediate between those of the pure solvents
and proportional to their relative concentrations: for solutions of
ethanol in water ranging from 25% to 50% and to 75% the e values
vary from 66 to 52 and 36.
The polarity of a solvent depends on its dielectric constant,
which affects the capacity of the solvent to solubilize molecules
with different degree of polarity. The polyphenolic compounds
contained in grape seeds and in whole pomace typically consist
of molecules belonging to classes different from one another for
the degree of polarity. On the basis of the results obtained for
ethanol/water mixtures, the higher extraction yields in condensed
tannins were obtained for e values ranging between 50 to 60.
Regarding the qualitative aspects, we observed that the increase
of the extraction yields in condensed tannins was associated with
an increase of their mean degree of polymerization (mDP), in particular when acetone was used: in other words, the extraction proceeds from the lower mDP tannins to the more polymerized ones.
With the variation of mDP, some variations in the percentage
weight of some subunits were observed, in particular when acetone was used: among the extension units, the percentage weight
of (
)-epicatechin and (
)-epicatechin-3O-gallate increased, and
among the terminal units the percentage weight of (+)-catechin
and (
)-epicatechin decreased.
Our data differ from those reported by Downey and Hanlin
(2010), which observed for the skins extracts the influence of the
solvent concentration on the mDP of tannins, but not on the subunits profile. However, we noticed that within the solvent concentration range which corresponds to the highest extraction yields
the changes in mDP and subunits profile were modest, both for
acetone and ethanol, and that the differences between the subunits
profiles of the extracts obtained with the two solvents are possibly
related to the differences in average mDP. In the ethanol extracts
(lower mDP), indeed, (+)-catechin was more abundant both as
extension unit and as terminal unit, while (
)-epicatechin and
(
)-epicatechin-3O-gallate were less abundant as extension units,
compared to the acetone extracts.

5. Conclusions
The study focused on the extraction of polyphenols from grape
seeds deriving from the fermented pomace of Nebbiolo cv., with
the use of some solvents used as pure or as binary or ternary
mixtures.
As regards the use of aqueous solutions of ethanol and acetone,
the variation of their composition (type of solvent and its percentage in water) caused not only variations in the extraction yields of
polyphenols, but also qualitative modifications of the composition
of the extracts: with higher yields their mDP increased and their
monomer profile changed. In particular, with the increase of

mDP the degree of galloylation as well as the percentage of

(
)-epicatechin and (
)-epicatechin-3-O-gallate as extension units
significantly increased, while the percentage of (+)-catechin as
terminal unit decreased. These results suggest the possibility to
obtain polyphenolic extracts qualitatively different by varying
the composition of the extraction mixture.
In future perspective, limited to the applications in the wine
field, the knowledge of selective extraction procedures of specific
classes of polyphenolic compounds will be useful to produce oenological grape tannins different from one another for their composition and, therefore, for their chemical properties (antioxidant,
antioxidase, antiradical effect, clarifying action, etc.) and sensory
characteristics.
Acknowledgements
The work was funded by Fondazione Cassa di Risparmio of Asti
(Italy) in the context of the SALUVA projects.
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