Vol.4, No.9B, 56-62 (2013) Agricultural Sciences
http://dx.doi.org/10.4236/as.2013.49B010
Copyright © 2013 SciRes. OPEN ACCESS
Polyphenol extraction from grape wastes: Solvent
and pH effect
Celia M. L ibrán, Luis Mayor, Esperanza M. Garcia-Castello*, Daniel Vidal-Brotons
Insti tuto Universitar io de Ingenier ía de Alimentos para el Desarrollo, Universitat Politécn ica de València, Camino de Vera s/n, 46022
Valencia, España; *Corresponding Author: egarcia1@iqn.upv.es
Received August 2013
AB STRACT
World wine industry transforms 10% - 25% of
raw grap es int o r esid ue s, m ainly represen ted b y
lees, grape marcs, seeds and stems. These by-
products are a rich source of polyphenols and
therefore they can be used to produce new
added value products. The aim of this work was
to determine the best process conditions (treat-
ment time, % of ethanol and pH of the solvent)
during solid-liquid extraction of pol yphenols from
grape marcs, by analyzing the phenolic content
of the extracts, namely: total polyphenol content,
flavanols, flavonols, phenolic acids and antho-
cyanins. Antioxidant activity of the extracts was
also deter mined. A n extraction time of two hours
was enough since longer times did not increase
process yields. Best extraction yields were ob-
tained for 75% etha nol s olutions. Bas ic pH l ed to
better yields in extracting media with low per-
centage of ethanol, whereas acid pH presented
better extraction yields in extracting media with
high percentage of ethanol. Among all the po-
lyphenols extracted, anthocyanins were the most
abundant representing over 40% of the total. In
general, the best process conditions were 2 h of
extraction in a 75% EtOH liquid mixture at pH = 2.
Keywords: Antiox idant s; B y-Products; Fruits;
Solvent Extraction; Wine
1. INTRODUCTION
In 2011, the world wine industry used 13,930,985 tons
of grapes for its transformation [1]. Among them, from
10% to 25% (w/w) changed into residues after grape
wine processing, being mainly represented by lees, grape
marcs, seeds, stems and stalks [2,3]. These wastes are of
difficult manageme nt due to t heir high biological oxygen
demand [4].
In recent years, scientists have realized of this envi-
ronmental problem and looked for solutions. Several stu-
dies marked these by-products as a rich source of poly-
phenols and therefore they could be used to produce new
added-value products [4-6]. Traditionally, these wastes
were used for animal feed but recently they have been
found as a low-cost source of antioxidants [7]. Some
authors [8] have summarized the health aspects derived
from the consumption of phenols from grape, mainly due
to their antioxidan t activity. Others s uggested its applica -
tion to food to extend their self-life and hence avoiding
the use of synthetic antioxidants such as butylated hy-
droxyanisole (BHA) or butylated hydroxytoluene (BHT)
which use is reg ulated by internatio nal agencies [9,10].
Most common groups of polyphenols found in grapes
are: anthocyanins, flavonols, flavanols and phenolic ac-
ids [11]. Their total content in grape and grape wastes
seemed to not vary among white and red varieties [12]
although the extraction procedure has a significant effect
on the q uantit y and quality of extracts [6,7,10] . Since t he
antioxidant power of grape extracts is in direct relation
with their total polyphenol content [5,12], the selection
of the best extraction conditions is of great importance,
because it could alter the characteristics of the final ex-
tract and then have an economic impact.
For these reasons, the aim of this study was to deter-
mine the best process conditions (treatment time, percen-
tage o f etha nol and p H of t he sol vent) dur ing so lid -liquid
extraction of polyphenols from grape marcs, by analyz-
ing the effect of these conditions on several extraction
yield s, name ly on to tal p henol ics, fla vono ids, flavano ids,
phenolic acids and anthocyanins and also on the antioxi-
dant power of the extracts.
2. MATERIALS A ND METHODS
2.1. Grape Marcs
Pressed marcs (from the vinification of Tempranillo
red grapes) were provided by the Enology Laboratory of
the Institute of Food Engineering for Development-Po-
lytechnic University of Valencia (Spain) and were stored
C. M. Librán et al. / Agricultural Sciences 4 (2013) 56-62
Copyright © 2013 SciRes. OPEN ACCESS
57
at 20˚C until their use. Homogeneous samples were
taken and thawed at room temperature previous to use
them in the experiments. They were dried at 25˚C in a
conditioning chamber (ACR-45/87, Dycometal, Spain)
up to moisture content of 16% - 18% (wet basis) (deter-
mined by dry weight in a vacuum oven (J.P Selecta, Va-
cioTem, Spain) at 70˚C till constant weight) and milled
to reach a final particle size between 0.5 and 2.5 mm [7,
10].
2.2. Reagents
Gallic acid, ethanol, methanol, hydrogen chloride and
sodium bisulfite were from Panreac. Sodium carbonate
was from Fluka. Caffeic acid, p-dimethylaminocinnamal-
dehyde (DMACA) and quercetin were from Sigma. Ca-
techi n, Trolox and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
were from Aldrich. Potassium hydroxide was from Ana-
laR.
2.3. Extraction Procedure
Solid-liquid extractions were carried out on an orbital
shaker (GFL Typ 3005 D-30938 Burgwedel, Germany) at
150 rpm and room temperature (20˚C - 23˚C), with 1/25
(w/v) ratio sample/solvent according to previous studies
[13,14]. After the extraction, liquid extracts were sepa-
rated from solids by centrifugation (3600 rpm for 10 min,
Selecta, Medifriger, BL-S, centrifuge), and then stored at
20˚C overnight up to their analysis.
2.4. Extraction Kinetics
Ethanol/ water mixtures at different ratios were used as
solvents with the necessary amounts of HCl or KOH to
regulate the liquid pH (always less than 1 mL). It was
necessary to correct the pH lecture by Eq.1, because the
equipment was calibrated with aqueous tampons.
0
pH pH
δ
= +
(1)
where pH is the corrected lecture and pH0 is the lecture
given by the pHmet er.
The values of δ were obtained from the literature,
ranging from 2.9 to 0 [15,16]. The liquid extracts were
analyzed for their total polyphenolic index (TPI) at dif-
ferent times during 8 h, for the determination of extrac-
tion kinetics.
2.5. Extractions at Fixed Time
A full factorial desig n [17] with five leve ls for ethano l
concentration (0%, 25%, 50%, 75% and 100%) and four
levels for pH (2, 5.3, 8.7 and 12) was used. Experiments
were d one in dup licate, giving a to tal of 40 r uns. Extra c-
tion time was fixed from the previous experiments de-
scribed in 2.4. The yields for each extracting condition
were determined by analyzing the concentration in the
extract of total polyphenols, flavonols, flavanols, phe-
nolic ac id s, ant ho cyani ns a nd ant io xid a nt ac ti vi t y. Al l t he
determinations were done by triplicate.
2.6. Chemical Analyses
2.6.1. Total Polyphenol Index and Total
Polyphenol Content
Total polyphenol index (TPI) was determined from the
Eq.2
280
*TPI An=
(2)
where A280 is the absorbance at 280 nm of the extract and
n is its dilution factor.
Total polyphenol content (TPC) was calculated from
the T PI , standar diz ed a gain st a gallic ac id c ur ve expressed
as mg gallic acid equivalent (GAE) per mL of extract [4].
Total polyphenol extraction yield was expressed as mg
GAE/g dry sample (3).
[ ]
GAE (mg)dry sample (g)
=GA (mg/mL)*Liquid (mL)dry mass (g)
(3)
2.6.2. Total Flavanols
Flavanols were determined after derivatization with
p-dimet hylami no-cinnamaldehyde (DMACA), since this
method has proved to have no interferences with antho-
cyanins. The followed method was adapted from refer-
ences [12,18]. Briefly, the extract was 1/10 (v/v) diluted
with MeOH, and then 1.5 mL of acidified DMACA solu-
tion were added to 0.3 mL of methanolic extract. The
mixture was allowed to react for 10 min at room temper-
ature, and the absorbance was read at 640 nm. Total fla-
vanol content was standardized against a catechin curve
expressed as mg of catechin equivalent (CE) per mL of
extract, and the flavanols extraction yield was expressed
as mg CE/g dry sample, using an equation similar to
Eq.3.
2.6.3. Total Flavonols and Phe noli c A c ids
Total flavonols and phenolic acids were determined
following the procedure described by references [7,19,
20]. Briefly, extracts were thor oughly mixed sequentially
with acidified ethanol and HCl 2%. Absorbances at 360
and 320 nm were measured for total flavonols and phe-
nolic acids, respectively. After the correspondent calibra-
tion curves, the results were expressed as mg of querce-
tin equivalent (QE) and mg of caffeic acid equivalent
(CAE) per mL of extra ct for t otal flavanols and phe nolic
acids, respectively. Both extraction yields were calcu-
lated using an eq uation similar to Eq.3.
2.6.4. Total Anthocyanins
Anthocyanins were measured through a chemical me-
(a)
C. M. Librán et al. / Agricultural Sciences 4 (2013) 56-62
Copyright © 2013 SciRes. OPEN A CCESS
58
thod based on their specific properties of bleaching by
SO2, and calculated by comparison with a standardized
anthocyanin solution according to reference [21]. The
anthocyanins extraction yield was expressed as mg an-
thocyanins/g dry sample, using equation similar t o Eq.3.
2.6.5. Antioxidant Activity
Antioxid ant ac tivit y was de termi ned by t he DP PH (2,2-
diphenyl-1-picrylhydrazyl) method described by refer-
ence [18]. Each extract was diluted 1/10 (v/v) with me-
thanol, and 3.8 mL of DPPH solution (60 μM in MeOH)
was added to 0.2 mL of methanolic sample. At t = 0 min
(A515(0)) and after 30 min (A515(30)) of reaction, absor-
bances were measured at 515 nm and the results were
expressed as percentage with Eq.4:
515515(0)515(30) 515(0)
%() *100AA AA∆= −
(4)
Afterwards, antioxidant activity was expressed as μM
of Trolox equivalent per mL of sample, by using a pre-
vious calibration curve. The yield was expressed as μmol
of Trolox equivalent/g dry sample, and was obtained
using and equation similar to Eq. 3.
2.7. Statistical Analysis
Each extraction, at the different conditions previously
explained, was assayed twice, and the obtained extracts
were chemically analyzed three times each. Therefore, a
descriptive analysis was performed, and all values were
averaged and given along with their confidence interval
(t student). Significant effect of ethanol concentrations
and pH were evaluated with an analyses of variance
(ANOVA, p < 0.05) and a Tukey test was carried out to
find differences among groups. Moreover, results were
fitted to a second order polynomic equation (Eq.5) that
considers lineal and quadratic effects as well as interac-
tion effects among the exp e rimental factors studied
(5)
where Y is the studied response and β0, βi, βii y βij are the
independent, lineal, quadratic and interaction coefficients,
respectively. Non-linear fit and goodness of fit (R2) were
performed through the STATGRAPHICS Centurion XVI
software (Statpoint Technologies Inc.).
3. RESULTS AND DISCUSSION
3.1. Extraction Kinetics
Figure 1 shows the evolution of TPI with time at dif-
ferent concentrations of ethanol in the extracting liquid
(0%, 50% and 100% of ethanol) and without fixing pH
(Figure 1(a)), with pH = 2 (Figu re 1(b)) and pH = 12
(Figure 1(c)).
(a)
(b)
(c)
Figure 1. Polyphenol extraction kinetics from wine wastes
samples extracted with different water/ethanol mixtures without
fixing pH (a), with pH = 2 (b) and with pH = 12 (c) expressed
as TPI (mean ± confidence interval). x, and : 0%, 50%
and100% of ethanol, respectively.
In general, at first stage, the TPI increased fast, fol-
lowed b y a slow incre ment and then remained practically
constant till the end of the process. This asymptotic be-
havior was found previously by other authors [10,22]. In
0
5
10
15
20
25
30
35
40
45
50
02468
TPI (A bs 280 n m)
Time (h)
0
5
10
15
20
25
30
35
40
45
50
0246 8
TPI (A bs 280 n m)
Time (h)
0
5
10
15
20
25
30
35
40
45
50
02468
TPI (A bs 280 n m)
Time (h)
C. M. Librán et al. / Agricultural Sciences 4 (2013) 56-62
Copyright © 2013 SciRes. OPEN ACCESS
59
Figure 1(b), can be observed that the TPI with 50%
EtOH was the highest (twofold the 100% EtOH and six-
fold the 0% EtOH) which means a positive effect on the
use of this organic solvent until certain concentration.
When the pH was fixed, the trend with percentage of
ethanol was the same but different behaviour was ob-
served at the different pH assayed. Hence, the acid pH
(Figure 1(b) ) increased the TPI for 0% and 50% EtOH,
but decreased it for 100% EtOH and the pH = 12 im-
proved the extraction for 0 and 100% EtOH and did not
affect the 50% EtOH.
Different authors marked as better extraction condi-
tions, concentrations of ethanol near to 50% finding de-
creases on TPI extraction yields with higher EtOH con-
centratio ns [6,10, 23]. The y sugge sted that e tha nol r educes
the dielectric constant of the solvent, thus increasing the
diffusion of the bioactive molecules with the solvent.
Ho wever, highly pur e organic solve nts, e .g. 10 0% EtOH,
could dehydrate the vegetable cells, making difficult the
diffusion of polyphenols from the plant material to the
extracting liquid.
The pH effect has not been extensively studied before
this work. Reference [ 6] a ssayed its effect on the stability
of extr acts. They fo und that p H 3 and 5 mainta in the a n-
tioxidant power instead of pH 7 and 9 which showed
reductions of this property of the extracts.
All this results showed that indistinctly the pH or the
EtOH concentration in the extracting medium, at 2 hours
of extraction the TPI yield was at least 90% of the max-
imu m attai ned dur ing the kinetics experiments. Therefore,
this time was used for the next extractions.
3.2. Total Polyphenol Yields
Extraction of total polyphenols (Figure 2), according
to analysis of variance, was significantly affected (p <
0.05) by the ethanol concentration and the pH. Results
ranged from 4.58 to 28.06 mg GAE/g dry sample, de-
pending on the extraction conditions (0% EtOH, pH = 2
and 0% EtOH and pH = 12, respectively) and similar
results were achieved by reference [10] with 50% EtOH
extracting solutions.
It is also observed an increase in the polyphenol ex-
traction yield with basic pH for aqueous extractions (0%
and 25% EtOH) and this tendency changed at higher
EtOH concentration, where acid pH had the better ex-
traction yields. However, the highest TPCs were ob ta ined
with 75% EtOH at all the assayed pH, with exception of
pH = 12.
3.3. Flavonol, Flavanol, Phenolic Acid and
A nthocyanin Extraction Y ields
Figure 3 summarizes the results for the different phe-
nolic compounds identified in the liquid extracts. As ob-
Figure 2. Total polyphenol content yield (mg of GAE/g dry
sample, mean ± CI) at 2 hours and 25˚C with different pH and
ethanol concentrations in the extracting media. , , and
: pH = 2, pH = 5.33, pH = 8.66 and pH = 12 , respectively; a,
b, c and d, represent significant differences among groups (p <
0.05)
served with the total polyphenols, the results were sig-
nificantly affected (p > 0.05) by both ethanol concentra-
tion and pH of the extracting medium.
The extraction yields of flavonols, flavanols, phenolic
acids and anthocyanins ranged from 0.03 - 4.98 mg QE,
0.09 - 1.83 mg CE, 0.39 - 5.02 mg CAE and 0.85 - 9.83
mg anthocyanins per g of dry sample, respectively. Among
the total polyphenols extracted, more than 40% where
from the anthocyanin group. Reference [4] studies on
polyphenol extraction with water/ethanol mixtures got
similar range of values for flavonols and phenolic acids,
altho ugh sli ghtly lo wer, and very lo wer for anthocyanins
(almost tenfold le ss).
In general, all the compounds showed higher extrac-
tion yields with higher concentrations of ethanol until
75%. This behaviour could be attributed to the change on
polyphenol solubility, den sity or dielec tric consta nt of the
extracting liquid due to the presence of ethanol [20].
Phenolic acids and flavonols extraction (Figure s 3(a)
and (c), respectively) showed similar values. Aqueous
solutions (0% and 25% EtOH) get better yields when
increasing pH, but higher concentration of ethanol, changed
this trend and better yields were achieved with acid pH.
Also , flava nols a nd antho cya nin extr actio ns (Figures 3(b)
and (d), respectively) showed similar behaviour although
this change was found at 25% EtO H.
3.4. Antioxidant Activity
Figure 4 illustrates the antioxidant activity of the liq-
uid extracts. The analysis of variance found a significant
effect (p < 0.05) of pH and ethanol concentration for all
the extractio n co nd itions.
0
5
10
15
20
25
30
35
0
25
50
75
100
mg GAE/g dry sample
% Ethanol
a
a
b
a
a
c
aa
b
bb
a
c
bb
c
a
a
a
b
C. M. Librán et al. / Agricultural Sciences 4 (2013) 56-62
Copyright © 2013 SciRes. OPEN A CCESS
60
Figure 3. Extraction yields at 2 hours and 25˚C at different pH and ethanol % in the extracting media: (a) flavonols; (b) flavanols; (c)
phenolic acids and (d) anthocyanins (mean ± confidence interval). White, light grey, dark grey and black: pH = 2, pH = 5.33, pH =
8.66 and pH = 12, respectively; a, b c and d represent significant d ifferences among groups ( p < 0.05).
Figure 4. Antioxidant activity at 2 hours and 25˚C with differ-
ent pH and ethanol % in the extracting media. , , and
: pH = 2, pH = 5.33, pH = 8.66 and pH = 12, resp ectively; a,
b, c and d, represent significant differences among groups (p <
0.05).
The extracts from 75% EtOH had the highest antioxi-
dant activity (12.95 - 15.63 µM Trolox/g dry sample)
according to the highest polyphenol extraction (Figu re
2). However little concordance was found in other ex-
tracts: 0% EtOH and pH 5.33 and 8.66 showed good
antioxidant conditions (14.10 and 13.45 µM Trolox/g dry
sample, respectively), although their concentration of po-
lyphe nols were not the highe st. Previo us works i ndicat ed
the degree of correlation between antioxidant activity
and polyphenol contents depends not only on the total
polyphenol content, but also on the composition of ex-
tracts [4].
Reference [6] recommended pH lower than 5 to pre-
serve the antioxidant activity during storage with 60%
EtOH. In general, this fact was in accordance with our
results, with exception of 100% EtOH which increased
their antioxidant activity at higher p H .
3.5. Response Surface A nalysis
Table 1 summarizes the coefficients of the response
surface equations and the goodness of fit with the para-
meter R2. The values of R2 were not too high but were
similar than those obtained by previous authors on the
extraction of polyphenols from different vegetables [20,
24].
Among these results, the most adequate were for an-
thocyanins (82.39%), phenolic acids (79.59%) and fla-
vonols (76.77%).
0
2
4
6
8
10
12
0
25
50
75
100
mg Q E/ g dry sample
%Ethanol
a
d
bb
d
a
cc
bba
a,b
b
a
b
c
(a)
0
2
4
6
8
10
12
0
25
50
75
100
mg CAE/g dry sample
% Ethanol
ca
d
db
bb
b
bca
a
b
aa
a
(c)
0
2
4
6
8
10
12
0
25
50
75
100
mg CE/g dry sample
%Ethanol
acb
c
aa
cd
a,b
d
b
b
(b)
0
2
4
6
8
10
12
0
25
50
75
100
mg ant hocyani ns/g dry sample
%Ethanol
a,b
b, c
a
b
ab
c
c
d
c
b
a
b
a
bb
c
b
a
b
(d)
0
2
4
6
8
10
12
14
16
18
0
25
50
75
100
µM Trolox eq/g dry sample
%Ethanol
b
c
c
c
a
b
a
b, c
c
a
b
c
b
b
bb
b
a
a,b
a
C. M. Librán et al. / Agricultural Sciences 4 (2013) 56-62
Copyright © 2013 SciRes. OPEN ACCESS
61
Table 1. Coefficients o f the response su rface equations.
Response ( mg/grdry sample) Coefficients* R2 (%)
β0 β1 β2 β11 β22 β12
Total polyphenols 5.9180 0.7745 0 .4323 0.1268 0.00 33 0.0162 55.26
Flavonols 0.3447 0.1502 0.0904 0.0232 0.0005 0.0031 76.77
Flava nols 0.6 202 0.1168 0.0376 0.0094 0.0003 0.0012 64.98
Phenolic acids 0.3768 0.1381 0 .1117 0.0270 0.0007 0.0038 79.59
Anthocyanins 0.8954 0.1604 0.2262 0 .0139 0.0018 0.0039 82.39
Antioxidant activity 5.8228 1.8654 0.1608 0.1586 0.0022 0.0061 58.20
*Subindexes: 0 = independent term; 1 = pH, lineal term; 2 = % ethanol, lineal term; 11 = pH, quadratic term; 22 = ethanol, quadra t ic te rm; 12 = pH*temperature,
interac tion ter m .
Response surface plots (not sho wn) exhibited the trends
previously commented in this work. In general, extrac-
tion yields increased with higher concentrations of etha-
nol until 75% and, basic pH improved the extraction of
aqueous samples (0% and 25% EtOH) while acid pH was
better for ethanol concentrated samples.
4. CONCLUSI O NS
This study reflects the importance of controlling the
studied extraction conditions (time, pH, % ethanol) to
obtain an extra ct with the hi gh est polyphenol content and
with an a dequate antioxidant activity.
An extraction time of two hours was enough since
longer time did not increase process yields. Best extrac-
tion yields were obtained for 75% ethanol solutions. Ba-
sic pH led to better yields in extracting media with low
ethanol percentage, whereas acid pH presented better
extraction yields in extracting media with high ethanol
percentage. Among all the polyphenols extracted, antho-
cyanins were the most abundant representing over 40%
of the total. In general, the best process conditions were 2
h of extraction in a 75% EtOH liquid mixture at pH = 2.
5. ACKNOWLEDG E ME NTS
The authors wish to acknowledge to the Enology Laboratory of the
Institute of Food Engineering for Development-Polytechnic University
of Valencia (Spain) for providing the grape marcs used in this work,
and FOMESA for the financial support. Author Luis Mayor acknowl-
edges JCI2009-04923 grant to MINECO (Spain).
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