Food Bioprocess Technol (2013) 6:441455
DOI 10.1007/s11947-012-0800-2
ORIGINAL PAPER
Optimization of Microwave-Assisted Extraction of Phenolic
Antioxidants from Grape Seeds (Vitis vinifera)
Kiruba Krishnaswamy & Valrie Orsat & Yvan Garipy &
K. Thangavel
Received: 27 June 2011 / Accepted: 1 February 2012 / Published online: 26 February 2012
# Springer Science+Business Media, LLC 2012
Abstract Grape seeds (Vitis vinifera) are rich in phyto-
chemicals that have antioxidant properties. The influence
of independent variables such as microwave power (100,
150, and 200 W), extraction time (2, 4, and 6 min), and
solvent concentration (30%, 45%, and 60% ethanol) and
their interactions on total phenols and the antioxidant activ-
ity (1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric ion
reducing antioxidant power (FRAP)) were determined; and
the microwave-assisted extraction (MAE) process was opti-
mized using a central composite design. The total phenols
that were expressed as gallic acid equivalents (GAE), cate-
chin equivalents (CAT), and tannic acid equivalents (TAE)
were significantly influenced by the solvent concentration
and the time of extraction. A numerical optimization was
carried out to obtain the overall conditions for MAE of
phenolic antioxidants from grape seed. The response varia-
bles were maximized for 6 min of MAE of grape seed (GS)
with 32.6% ethanol at 121 W with a desirability function of
0.947. The predicted extraction yields were 130.89, 21.6
1.59, and 15.91.32 mg GAE, CAT, and TAE, respectively
per gram of GS. The predicted antioxidant activity per gram
of dry weight GS was 80.9% for the inhibition of DPPH and
135 M ascorbic acid equivalents for FRAP test. The pre-
dicted response values were significantly correlated with the
K. Krishnaswamy : V. Orsat (*) : Y. Garipy
Department of Bioresource Engineering, McGill University,
21,111, Lakeshore Road,
Ste-Anne-de-Bellevue, QC H9X3V9, Canada
e-mail: valerie.orsat@mcgill.ca
K. Thangavel
Department of Food and Agricultural Process Engineering,
Tamil Nadu Agricultural University,
Coimbatore 641003, India
observed ones as follows: GAE r00.995, CAT r00.990,
TAE r00.996, DPPH r00.996, and FRAP r00.996.
Keywords Gallic acid . Catechin . Tannic acid . Response
surface methodology . Antioxidant activity . FRAP . DPPH
Introduction
Antioxidants are substances that neutralize free radicals.
Free radicals are damaging compounds in the human body
that alter cell membranes, causing cell damage and inflam-
mation, promoting abnormal cell growth including various
kinds of cancer, and even causing cell death (Lee et al.
2004). Free radicals are generated naturally in the body by
metabolic process and from exogenous factors like ultravi-
olet light, radiation, smoke, and air pollution (Kim et al.
2006). The normal aerobic respiration in cells produces
natural antioxidants which are further supplemented by di-
etary sources like vitamins, carotenoids, flavonoids, etc.
(Harman 1995). The effectiveness of the endogenous anti-
oxidant system declines with aging. Hence it is required to
incorporate antioxidative nutraceuticals in the prevention of
diseases. The health-related issues with the growing popu-
lation has led to the search of naturally occurring antioxi-
dants in food to replace synthetic antioxidants, which are
being restricted, due to their adverse side effects, such as
carcinogenicity (Pokorn 1991).
The present regulatory regimes on waste management and
disposal in the food industries has urged for an integrated
approach in context of recycling/reuse and waste recovery.
Recycling of fruit and vegetable waste is one of the most
important means of utilizing it in a number of innovative
ways yielding essentially new products meeting the require-
ments in human, animal, and plant nutrition as well as in the
442
pharmaceutical industry. Hence there is a growing interest in
exploiting agricultural waste materials to obtain phytochem-
icals and antioxidants which will provide satisfactory solu-
tions to both food processing industries and the society. In this
context, winery waste could be an excellent source for obtain-
ing natural antioxidants from grape pomace (Bail et al. 2008).
Grape (Vitis vinifera L.) is one of the worlds most
important fruit crop with a global production of around 67
million tons in 2009 (FAOSTAT 2011) and about 80% of the
total production is used for wine making. The grape pomace
obtained, as winery by-product, constitutes 1020% of the
weight of grapes (Garcia-Marino et al. 2006). It was
reported that the grape seed present in the pomace weighs
3852% on dry matter basis (Maier et al. 2009). Grape seed
consists of 35% fiber, 29% polyphenols, 15% lipids, 3%
ash, and 7% water (Mayer et al. 2008). Grape seeds are rich
sources of monomeric phenolic compounds (Fig. 1) such as
(+)-catechins, ()-epicatechin, (+)-gallocatechins, ()-epi-
gallocatechin, and their dimeric, trimeric, and tetrameric
proanthocyanidins (Baoshan Sun and Spranger 2005; Freitas
et al. 1998).
Grape seed extract has been a matter of intense inves-
tigations with respect to its potential beneficial effects on
human health as it possesses a broad spectrum of biological,
pharmacological, and chemoprotective properties against
free radicals and oxidative stress. Recent reports indicate a
wide range of biological activities, e.g., antioxidant proper-
ties and radioprotective effects (Castillo et al. 2000), anti-
bacterial activity (Jayaprakasha et al. 2003), prevention of
cataract (Yamakoshi et al. 2002), antihyperglycemic effects
(Pinent et al. 2004), anti-inflammatory effects (Terra et al.
2007), enhancement of postprandial lipemia (Del Bas et al.
2005), improvement of insulin sensitivity, and prevention of
hypertriglyceridemia and cardiovascular disease (Al-Awwadi
et al. 2005), prevention of in vitro low density lipoprotein
oxidation (Meyer et al. 1997), modulation of the expression of
antioxidant enzyme systems (Puiggrs et al. 2005), and pro-
tective effects against oxidative damage in mouse brain cells
(Guo et al. 2007). Thus effective extraction and concentration
of these phenolic bioactive compounds from the grape seed is
Fig. 1 Monomeric units of
proanthocyanidins present in
grape seeds (Baoshan Sun
and Spranger 2005)
Food Bioprocess Technol (2013) 6:441455
very important so that it can be utilized in the preparation of
functional foods, dietary supplements, and in nutraceutical
and pharmaceutical products (Cacace and Mazza 2003).
Microwave-assisted extraction (MAE) has been reported
as a novel method for extraction of bioactive compounds. It
utilizes microwave energy to cause molecular movements
and rotation of liquids with permanent or induced dipoles
(Sun et al. 2007). When a biological material with favorable
dielectric properties (plant material along with solvent for
extraction) is placed in a microwave field, the molecules try
to align with the oscillating electromagnetic field either by
distortion or distribution of electron cloud within the mole-
cule or by physical rotation of the molecular dipoles which
leads to rapid heating of solvent and sample matrix (Dandekar
and Gaikar 2002). Thus MAE is advantageous over conven-
tional extraction techniques with improved efficiency, reduced
extraction time, rapid and volumetric heating of the absorbing
medium, low solvent consumption, higher selectivity of target
molecules, and high potential for automation (Pallaroni et al.
2002, Thostenson and Chou 1999, Wang et al. 2010). In
conventional techniques such as heating, boiling, or refluxing,
there is a loss of flavonoids due to ionization, hydrolysis, and
oxidation on prolonged extraction time. MAE is the simplest,
fastest, and most economical technique for extraction. The
enhancement of product recovery by microwave is generally
attributed to its heating effect, which occurs due to the dipole
rotation of the solvent in the microwave field. This causes the
solvent temperature to rise, which then increases the solubility
of the compound of interest such as flavonoids (Wang et al.
2010). Microwave-assisted extraction has been used to extract
many different bioactive compounds, such as 1,8 cineole,
menthone, and menthol from peppermint and 1,8 cineole
and camphor from rosemary leaves (Chen and Spiro 1994),
essential oil from Lippia sidoides (Craveiro et al. 1989),
alkaloid from lupin seeds (Lupinus mutabilis) (Ganzler et al.
1990), secoisolariciresinol diglucoside from flaxseeds (Nemes
and Orsat 2010), pectin from apple pomace (Wang et al.
2007), phenolic antioxidants from peanut skin (Ballard et al.
2009), glycyrrhizic acid from licorice roots (Pan et al. 2000),
curcuminoids from Curcuma longa (Dandekar and Gaikar
Food Bioprocess Technol (2013) 6:441455
2002), Zearalenone from wheat and corn (Pallaroni et al.
2002), anthocyanin from red raspberries (Sun et al. 2007),
tea polyphenols and tea caffeine from green tea leaves (Pan et
al. 2003), etc.
The optimization of microwave-assisted extraction of poly-
phenolic antioxidants from grape seeds was conducted in the
present study with the objective of recovering maximum
antioxidants. Response surface methodology (RSM) was used
to evaluate the effect of multiple factors like microwave
power, solvent concentration, extraction time, and their inter-
actions and was estimated by least square regression. From the
literature it was found that Hong et al. (2001) used microwave
power of 150300 W and time from 20 to 200 s to extract
phenolic compounds from grape seed. Mayer et al. (2008)
used microwave power of 150 W and 200 s for extraction of
proanthocyanidins from grape seed. Thus 100300 W and 1
5 min were taken as the minimum and maximum values for
the screening design. A response surface study was initially
conducted using a two-level, three-factorial design for screen-
ing the effects of the factors, ethanol concentration of 50% and
90%, microwave power levels of 100 and 300 W, and extrac-
tion time of 1 and 5 min on the extraction yields of total
phenols, the antioxidant activity, and the color of the extracts.
Then the design was augmented to central composite design
by the addition of center and axial points.
Materials and Methods
Materials and Reagents
V. vinifera (var. Bangalore blue), a variety of grapes, were
procured from the local markets of Coimbatore, Tamil
Nadu, India. This variety of grapes is widely grown in the
southern states of India like Karnataka and Tamil Nadu. The
grapes were processed for the production of juice in the Post
Harvest Technology Centre, Tamil Nadu Agricultural Uni-
versity, Coimbatore. The residual pomace was collected and
the seeds were manually removed from the pomace. The
seeds were cleaned, lyophilized in a batch-type freeze dryer
(Ilshin, version 1.0, Model FD5505) until constant weight
was obtained. They were packed in polyethylene bags and
stored at 18 C until further analysis. All chemicals used
such as FolinCiocalteau reagent, gallic acid, catechin and
tannic acid, sodium carbonate, 1,1-diphenyl-2-picrylhydrazyl
(DPPH), and 2,4,6-tripyridyl-s-triazine were of analytical and
HPLC grade obtained from Sigma Chemicals Co (St. Louis,
MO, US).
Sample Preparation
Dried grape seeds were milled in a coffee grinder (Proctor
Silex, Model E167C) for 2 min, the process was stopped at
443
15-s interval for 15 s to avoid heating of the sample (Gokturk
Baydar et al. 2007). The grape seed powder (10 g) was
defatted with 30 ml of hexane in an Erlenmeyer flask at
60 C for 6 h. The hexane containing the lipid portion was
filtered, and the grape seed powder was kept in the fume hood
to allow the residual hexane to evaporate.
Microwave Extraction of Phenolic Antioxidants
Dry defatted grape seed powder (0.50.05 g) was placed in
a 250-ml quartz vessel. Fifty milliliters of different ethanol
concentrations (30%, 45%, 60%; v/v) was used for extrac-
tion. The vessel was introduced into the microwave cavity
and fitted with a condenser. A focused-type, open-vessel
microwave system (Star System 2, CEM Matthews, USA)
operating at 800 W maximum power with a frequency of
2,450 MHz was used. The microwave power applied
was intermittent with power on for 30 s/min. The
extracts were centrifuged for 20 min and filtered with
0.22-m filters (Whatman Puradisc 13-mm nylon syringe
filters) for the determination of total phenols, color, and anti-
oxidant activity.
Experimental Design
Face-centered central composite design (CCD) was used to
determine the optimal conditions of microwave-assisted ex-
traction of total phenols (TP), antioxidants activity (ferric
ion reducing antioxidant power (FRAP) and DPPH), and
color from grape seeds. Central composite design is highly
efficient and provides sufficient information on the effect of
process variables for resourceful optimization with reduced
number of total experimental runs. The three independent
factors studied were the microwave power, X1 (100, 150,
and 200 W), extraction time, X2 (2, 4, and 6 min), and
ethanol concentration, X3 (30%, 45%, and 60% ethanol),
which were coded at three levels as shown in Table 1.
The CCD included eight factorial points, six axial points,
and three center points, which totals 17 experimental runs,
and was used to fit the second-order polynomial model
Table 1 Independent variables in CCRD for optimization of grape
seed extract
Independent variables Coded levels
1 0 1
Microwave power (W) X1 100 150 200
Extraction time (min) X2 2 4 6
Ethanol concentration (% v/v) X3 30 45 60
Power (100 W014%, 150 W020%, 200 W027% of maximum power)
444 Food Bioprocess Technol (2013) 6:441455

similar to that shown in Eq. 1 (Haaland 1989, Abdeshahian Determination of Antioxidant Activity
et al. 2010).
Several methods are used to evaluate antioxidant activities
of natural compounds in foods or biological systems with
X
3 X
3 3 X
X
Y b0 b i xi b ii x2i b ij xi xj 1 varying results. Depending upon the reactions involved,
i1 i1 i<j1 these assays can roughly be classified into two types: assays
based on hydrogen atom transfer reactions and assays based
where Y is the predicted response, 0 is the regression on electron transfer (ET). The ET-based assays measure the
coefficient for the intercept (a constant), i is the coefficient capacity of an antioxidant in the reduction of an oxidant,
for the linear effect, ii is the coefficient for the quadratic which changes color when reduced. The degree of color
effect, ij is the coefficient for the interaction effect, of change is correlated with the samples antioxidant concen-
variables i and j, Xi, and Xj are independent variables. The trations. The ET-based assays include FRAP and DPPH. The
JMP software Version 8 (SAS Institute Inc., Cary, NC, antioxidant activity of the microwave-assisted extracts of
USA) was used for the statistical analysis and to generate grape seed was determined by both FRAP and DPPH.
the response surface plots for the quadratic models.
Antiradical Scavenging Activity

Analysis of Total Phenolic Compounds
A colorimetric method using FolinCiocalteau assay was
used to determine the total phenols content in the grape seed
extract. The method used by Jayaprakasha et al. (2003),
Jayaprakasha and Jaganmohan Rao (2000), and Singh et
al. (2011) was used with slight modifications. One milliliter
of the grape seed extract having 1 mg of grape seed/ml of
ethanol was mixed with 7.5 ml of double deionized water,
followed by the addition of 0.5 ml of FolinCiocalteau
reagent, and 1 ml of 7.5% sodium carbonate solution. The
mixture was incubated at room temperature in the dark for
the development of color for 30 min. The absorbance was
measured at 765 nm using a spectrophotometer (Ultrospec
2100 pro, Biochrom Ltd., Cambridge, England). The total
phenols concentration was expressed in terms of gallic acid
equivalents (GAE), catechin equivalents (CAT), and tannic
acid equivalents (TAE) based on the standard curves given
in Table 2 which were plotted for 30%, 45%, and 60% ethanol
concentrations in respective equivalents (Y0concentration,
x0absorbance at 765 nm).
The DPPH assay was used to determine the free radical
scavenging activity of grape seed extract. The method by
Nair et al. (2007) was adopted with some modifications
(Singh et al. 2011). An aliquot of 50 l ethanolic extract
of grape seed having 1 mg of GS/ml of ethanol was added to
1.5 ml of DPPH (3.94 mg/100 ml ethanol). The discolor-
ation of purple is due to the paring of the free electron with
the free radical scavenger present in DPPH. The color
change was measured with a spectrophotometer (Ultrospec
2100 pro, Biochrom Ltd., Cambridge, England) at 517-nm
absorbance after 20 min. The percentage of free radicals
scavenged by DPPH radical was calculated using the fol-
lowing equation:
DPPH% inhibition
 
Abs control 517nm  Abs sample 517nm
 100
Abs control 517nm
2
where, Abs control 0 absorption of blank sample and Abs
sample 0 absorption of grape seed extract.

Table 2 Standard curve for dif-

ferent solvent concentrations in Sample no. Standard curve Equation R2
terms of standard equivalents
1 Gallic acid standard curve, 30% EtOH Y 81:75x  0:040 0.997
2 Gallic acid standard curve, 45% EtOH Y 91:13x  0:052 0.999
3 Gallic acid standard curve, 60% EtOH Y 92:58x  0:019 0.999
4 Catechin standard curve, 30% EtOH Y 50:39x 0:036 0.999
5 Catechin standard curve, 45% EtOH Y 51:57x 0:045 0.991
6 Catechin standard curve, 60% EtOH Y 49:17x 0:091 0.991
7 Tannic acid standard curve, 30% EtOH Y 69:18x 0:052 0.997
x concentration (in milligrams/ 8 Tannic acid standard curve, 45% EtOH Y 69:87x 0:018 0.999
milliliters), Y absorbance at 9 Tannic acid standard curve, 30% EtOH Y 87:66x 0:002 0.993
765 nm
Food Bioprocess Technol (2013) 6:441455
Reducing Power
The FRAP assay takes advantage of electron transfer reactions
and is used to measure the total antioxidant activity of natural
compounds. Herein a ferric salt, Fe(III)(TPTZ)2Cl3 (TPTZ)
2,4,6-tripyridyl-s-triazine is used as an oxidant. Antioxidants
are used as reductants in a redox-linked colorimetric method.
The method used by Benzie and Strain (1996) and Deighton et
al. (2000) was used in this experiment with some modifica-
tions. Acetic acid buffer of 250 ml, 25 ml of TPTZ (2,4,6-
tripyridyl-s-triazine) and 25 ml of ferric chloride solution was
added together at 10:1:1 ratio, to make the FRAP reagent.
Grape seed ethanolic extract (50 l) was added to 1.5 ml of
FRAP reagent, vortexed for 60 s, and allowed to stand for
6 min at room temperature. The control consisted of 1.5 ml
FRAP reagent to which were added 50 l distilled water. The
color change was read with a spectrophotometer at 593 nm.
Ascorbic acid, which is an antioxidant, was used as a standard
solution to determine the reactivity of FRAP solutions using
the standard curve shown in Eq. 3 (R2 00.997).
Absorbance of grape seed extract 593nm
244:7  Conc: of grape seed extract  0:014 3
Determination of Extract Color
The color of the extract was measured using a colorimeter
(Konica Minolta, Japan), equipped with 1 cm measuring
head. The colorimeter works on the principle of focussing
the light and measuring the energy reflected from the sample
across the entire visible spectrum. The color meter relies on
standard observer curves that define the quantity of red,
green, and blue colors. The primary light requires matching
a series of colors across the visible spectrum and the math-
ematical model used to describe these colors as L*a*b*
color space. The colorimeter provides reading in terms of
L*, a*, and b*, where luminance (L) forms the vertical axis,
which indicates whiteness to darkness. The chromatic portion
of the solids is defined by: a (+) redness, a () greenness, b (+)
yellowness, and b () blueness.
The colorimeter was calibrated using the standard white
calibration plate provided by the manufacturer before the
start of the measurements. Buci Koji et al. (2009) and
Wrolstad et al. (2005) similarly used the L, a, and b system
to determine the color changes of grape seed extract and
anthocyanin pigments. One milliliter of the microwave-
assisted grape seed extracts was placed in small vials of
1 cm in diameter to match the orifice of the colorimeter. This
setup was covered with a black cover in order to prevent the
scattering of light. L* (light/dark), a* (a+, redness/green a)
and b* (b+, yellowness/blueness b) values were recorded
445
for each sample. From the obtained L*, a*, and b* values, the
total change in color of the extract (E) was calculated using
the following equation:
q
E L0  L2 a0  a2 b0  b2 4
Where L0, a0, and b0 denotes the value of pure ethanol at 30%,
45%, and 60%, v/v used as standard reference to compare the
color change of the ethanolic extract.
Statistical Analysis
Statistical analysis was performed using the JMP 8 software
(SAS Institute Inc., Cary, NC, USA). The data were ana-
lyzed by analysis of variance (ANOVA) and the adequacy of
the response surface model was determined by evaluating
the lack of fit and coefficient of determination (R2). The
statistical significance of the model and its variables was
determined at 5% probability level (p<0.05). The optimal
conditions for MAE of grape seed phenolic antioxidants
were obtained based on modeling and desirability function
that could be visually explained in terms of three-dimensional
response surface plots and contour plots.
Results and Discussion
Fitting the Model
A two-level, three-factorial design was used for screening the
factors. The studied factors and their levels were: the micro-
wave power at 100 and 300 W, the extraction time of 1 and
5 min, and the concentration of ethanol 50% and 90%, v/v.
The results obtained indicated that the yield of total phenolics
can be maximized by increasing the extraction time and
decreasing the solvent concentration. At increased microwave
power level of 300 W in the screening experiment, rapid
heating of the extraction medium was seen. Due to high
cavitation, bubbling of the substance occurred. This led to
the entry of the extraction medium into the condenser. Based
on the observation from the screening design, the microwave
level was fixed between 100 and 200 W in the CCD. Hence,
for further experiments, it was decided to modify the experi-
mental domain as follows: microwave powers between 100
and 200 W, extraction time of 2 and 6 min, and concentration
of ethanol of 30% and 60%. The different combinations of the
independent variables and their corresponding response in
terms of total phenols are presented in Table 3.
Total Phenols
Response surface regression models were fitted to the ex-
perimental data. The ANOVA of the quadratic regression
446 Food Bioprocess Technol (2013) 6:441455

Table 3 Face-centered central composite design (CCD) with observed response for total phenols from microwave-assisted extraction of grape
seeds

Design MWP, Time, Sol. conc. Total phenols (mg/g)

points Watt Min % v/v
GAE CAT TAE

Actual Predicted STD Actual Predicted STD Actual Predicted STD

error error error

R1 200 2 30 10.373 10.470 0.165 16.829 16.977 0.292 12.258 12.324 0.230
R2 100 6 30 12.269 12.345 0.165 19.905 20.034 0.292 14.498 14.580 0.230
R3 200 6 30 13.235 13.289 0.165 21.473 21.592 0.292 15.640 15.777 0.230
R4 100 2 30 11.351 6.423 1.238 18.416 9.760 2.182 13.414 7.067 1.720
R5 100 2 60 9.138 9.084 0.165 17.206 17.087 0.292 9.651 9.514 0.230
R6 200 2 60 9.689 9.613 0.165 18.243 18.114 0.292 10.233 10.151 0.230
R7 100 6 60 10.510 10.413 0.165 19.788 19.640 0.292 11.100 11.034 0.230
R8 200 6 60 15.241 7.839 1.238 28.696 15.008 2.182 16.096 7.610 1.720
R9 100 4 45 9.920 9.996 0.145 17.530 17.669 0.255 12.938 13.059 0.201
R10 200 4 45 10.809 10.733 0.145 19.100 18.961 0.255 14.098 13.977 0.201
R11 150 2 45 9.580 9.613 0.145 16.928 17.027 0.255 12.495 12.647 0.201
R12 150 6 45 11.720 11.687 0.145 20.710 20.611 0.255 15.286 15.134 0.201
R13 150 4 30 11.755 11.527 0.145 19.071 18.675 0.255 13.891 13.606 0.201
R14 150 4 60 9.905 10.133 0.145 18.650 19.046 0.255 10.461 10.746 0.201
R15 150 4 45 10.995 10.955 0.104 19.430 19.359 0.183 14.341 14.289 0.144
R16 150 4 45 10.886 10.955 0.104 19.236 19.359 0.183 14.198 14.289 0.144
R17 150 4 45 10.984 10.955 0.104 19.411 19.359 0.183 14.327 14.289 0.144

GAE gallic acid equivalents, CAT catechin equivalents, TAE tannic acid equivalents

model for total phenols expressed in gallic acid equivalents terms of gallic acid equivalents was significant with p val-
is given in Table 4. The model obtained for total phenols in ue00.0002, with R2 value of 0.9906. It was found that there

Table 4 ANOVA for total phenols by MAE of grape seeds

Total phenols (mg gallic acid equivalents g1 dw)

Source SS df MS F value p value

Model 17.087250 9 1.89858 58.9808 0.0002a
Lack of fit 0.15374735 3 0.051249 14.2319 0.0664
Error 0.160949 5 0.03219
Term Estimate Std error t ratio F value p value
Intercept 10.955 0.103586 105.76 <0.0001a
MWP (100, 200) 0.36855 0.069487 5.30 28.1308 0.0032a
Time (2, 6) 1.03705 0.069487 14.92 222.7352 <0.0001a
Sol conc. (30, 60) 0.69735 0.069487 10.04 100.7143 0.0002a
MWP time 0.77575 0.315043 2.46 6.0632 0.0571
MWP sol conc. 0.8795 0.315043 2.79 7.7935 0.0384a
Time sol conc. 1.14825 0.315043 3.64 13.2841 0.0148a
MWP MWP 0.5905 0.163783 3.61 12.9988 0.0155a
Time time 0.305 0.163783 1.86 3.4679 0.1216
Sol conc. sol conc. 0.125 0.163783 0.76 0.5825 0.4798
R2 0.990669
Adjusted R2 0.973872
a
Significant
Food Bioprocess Technol (2013) 6:441455 447

is a reasonable agreement with the adjusted R2 of 0.9738
in the model. The lack of fit was not significant (p>
0.05), suggesting that the model was well fitted and could
be used to predict the total phenols from microwave-assisted
grape seed extract.
The model indicated that the linear effects of extraction time
and solvent concentration had the greatest significance on total
phenols of the extract. Values of (p) less than 0.05 indicate the
significant terms in the model, based on which the linear terms
such as microwave power, time of extraction, solvent concen-
tration, bilinear terms such as MWP Sol. Conc., Time Sol.
Conc., and quadratic terms of MWP2, were found to be sig-
nificant. The predictive, second-order, polynomial model for
total phenols content of microwave-assisted grape seed
extracts, in terms of coded factors levels, is shown in Eq. 5.
TPGAEmg=g dw GS 10:955 0:368MWP
1:03705Time
 0:697Sol:Conc:
 0:775MWP  Time
 0:879MWP  Sol:Conc:
 1:148Time  Sol:Conc:
 
 0:590 MWP2
 
 0:305 Time2
 0:125Sol:Conc:
2
5 6
The Pearsons correlation coefficient for actual vs. pre-
dicted value is r00.995 (Fig. 2). Hence this model can be
used to predict total phenolics under different experimental
conditions during microwave-assisted extraction.
13.5
13
12.5
GAE Actual
Similarly, a regression analysis was carried out to quan-
tify the total phenols in terms of catechin equivalents. The
response predictions of total phenols in terms of catechin
equivalents was significant in the given model with p0
0.0009 (Eq. 6). The R2 value for the model was 0.9806 with
a reasonable agreement with the adjusted R2 of 0.9457, and
the lack of fit was not significant 0.0678 (p>0.05) suggest-
ing that the model could be used to predict the response. It
was found that microwave power, time and the interaction of
MWP and solvent concentration, time and solvent concen-
tration, and MWP2 were significant factors in the model.
The predicted response for total phenols in terms of catechin
equivalents, in term of coded factors level, is given in the
Eq. 6 neglecting some insignificant terms:
TPCATmg=g dw GS 19:359 0:646MWP
1:791Time
0:185Sol:Conc:
 1:414MWP  Time
 1:547MWP  Sol:Conc:
 1:930Time  Sol:Conc:
 
 1:044 MWP2
 
 0:54 Time2
 0:498Sol:Conc:2
The predictive response of total phenols in terms of tannic
acid equivalents by regression analysis was found to be signif-
icant (p value<0.0001). The R2 value of the model was found
to be 0.9939 with a reasonable agreement with the adjusted R2
of 0.9829 and insignificant lack of fit (p>0.05). The final
equation in terms of coded factors levels for total phenols is:
TPTAEmg=g dw GS 14:288 0:458MWP

12
1:243Time
11.5
11  1:429Sol:Conc:
10.5  1:015MWP  Time
10
 1:155MWP  Sol:Conc:
9.5
9  1:498Time  Sol:Conc:
9 9.5 10 10.5 11 11.5 12 12.5 13 13.5  
 0:770 MWP2
GAE Predicted P=0.0002  
 0:398 Time2
RSq=0.99 RMSE=0.1794
 2:112Sol:Conc:2
Fig. 2 Predicted (in milligrams GAE/g) vs. actual (in milligrams
GAE/g) for total phenols in MAE of grape seed 7
448
The predicted vs. actual plot obtained for total phenols in
terms of catechin equivalents and tannic acid equivalents
were similar to that obtained for gallic acid equivalents
(Fig. 2) with Pearsons correlation coefficient (r) for GAE
r 00.995, CAT r00.990, TAE r00.996. Thus the given
models can be used to predict the total phenols of
microwave-assisted extraction of grape seeds in milligrams
equivalents per gram of dry weight (dw) grape seed,
respectively.
Effect of Process Variables on Total Phenols
The relationship between independent and dependent vari-
able can be well illustrated with three-dimensional response
surfaces. Figure 3 gives the interaction of MWP and time at
a fixed solvent concentration of 45% ethanol concentration.
It was found that there was an increase in total phenols with
an increase in time of extraction. By keeping time constant
with constant solvent concentration and increasing the
MWP, there was a gradual increase in the total phenols.
The maximum total phenol from the extract was obtained
with increased extraction time. It revealed that by decreasing
the solvent concentration from 60% to 30% and by increas-
ing the MWP from 100 to 170 W, it increased the total
phenols. Wang et al. 2010 reported that 70% ethanol con-
centration, microwave power 255 W, and extraction time
6.5 min gave maximum flavonoids from Radix puerariae.
MAE of secoisolariciresinol diglucoside from flaxseed by
Nemes and Orsat (2009) reported MWP of 135 W, sodium
hydroxide concentration of 0.5 M, and 3 min extraction time
which gave 10% increase in the extraction yield when
Fig. 3 Response surface plot for total phenols in terms of GAE
Food Bioprocess Technol (2013) 6:441455
compared to the microwaveless control method. Pan et al.
(2000) studied the MAE of glycyrrhizic acid from licorice
root with extraction times of 45 min, ethanol concentra-
tions of 5060% (v/v).
Similar surface plots were obtained for total phenols in
terms of tannic acid equivalents and catechin equivalents.
The optimum extraction conditions for maximum total phe-
nols from grape seed was found at 170 W, 6 min, and 30%
ethanol concentration yielding 13.50.48 mg GAE/g dw
GS with a desirability of 0.982. The microwave power of
168 W, 6 min, and 30% ethanol concentration yielded more
total phenols in terms of catechin equivalents at 220.86 mg
CAT/g dw GS with the desirability of 0.997. The predicted
optimum extraction conditions in terms of tannic acid equiv-
alents were 160 W, 6 min, and 33.7% ethanol concentration
with 0.992 desirability giving 16.10.56 mg TAE/g dw GS.
Ghafoor et al. (2009) studied the extraction of phenols
and antioxidant from grape seed by ultrasound-assisted ex-
traction. The surface plots obtained were similar to the
present study with 53.15% ethanol concentration v/v,
56.03 C, and 29.03 min of extraction time yielding maxi-
mum total phenols of 5.44 mg GAE/100 ml. In the present
study with microwave-assisted extraction, it was possible to
obtain a high amount of total phenols in less time (6 min)
with reduced 3034% (v/v) of ethanol. The amount of total
phenols obtained by Li et al. (2008) was in the range of 7.5
to 44 mg GAE/100 g dry matter. Mandic et al. (2008)
reported 3.59 to 11.7 g GAE/kg of dry grape seed using
ethanolic solvent for extraction. Jayaprakasha et al. (2001)
reported a range of 18.2 to 20.8 CAT/100 g grape seed extract
for different ratios of ethanol water mixture. Since different
grape variety, solvents combinations, and methods of extrac-
tion were used, the comparison of the results can only be
informative. The variation in the results depends upon factors
such as concentration of phenolic compounds in different
grape cultivars, and it is also influenced by viticulture practi-
ces and environmental factors such as maturity stage, area of
production, and seasonal conditions (Gmez-Alonso et al.
(2007), Cheynier and Rigaud (1986), Mazza and Francis
(1995), Koundouras et al. (2006)).
Antioxidant Activity
The antioxidant activity in terms of DPPH and FRAP for
different combinations of the independent variables are pre-
sented in Table 5.
Antiradical Activity
The radical scavenging activity of the grape seed extract was
determined by the DPPH method. To increase the fitness of
the model, log transformation of the response was done.
Transformation of the variables is said to enhance the
Food Bioprocess Technol (2013) 6:441455 449

Table 5 Observed responses for the antioxidant activity and color value of the microwave-assisted extraction of grape seed in the experimental design

Antioxidant activity Color value

DPPH FRAP

Actual Predicted STD error Actual Predicted STD error L* a* b* E

R1 67.113 67.173 0.012 115.803 115.883 0.010 31.110 1.327 2.167 2.421
R2 73.065 85.081 0.025 124.210 141.193 0.021 29.630 1.087 2.940 3.996
R3 77.232 76.922 0.011 130.095 129.644 0.010 30.350 0.940 2.663 3.233
R4 56.994 56.736 0.011 101.512 101.091 0.010 35.417 0.813 1.453 2.555
R5 79.464 80.007 0.012 133.247 134.008 0.010 30.840 0.393 3.860 3.352
R6 80.357 80.581 0.012 134.508 134.807 0.010 27.977 0.643 4.640 5.821
R7 79.762 79.913 0.011 133.668 133.872 0.010 28.180 0.530 4.860 5.813
R8 61.012 61.461 0.012 107.186 107.871 0.010 28.217 0.437 3.683 5.075
R9 79.464 79.135 0.010 133.247 132.839 0.008 32.387 0.433 3.083 2.627
R10 76.042 75.514 0.009 128.414 127.669 0.008 32.187 0.863 3.437 3.075
R11 73.661 73.225 0.009 125.051 124.495 0.008 32.690 0.393 3.233 2.715
R12 78.720 78.313 0.010 132.197 131.613 0.008 33.600 0.400 2.377 1.924
R13 71.131 71.680 0.010 121.478 122.323 0.008 34.240 0.633 2.520 2.155
R14 77.530 76.086 0.009 130.515 128.467 0.008 28.350 0.573 4.550 5.479
R15 76.190 77.056 0.005 128.624 129.810 0.005 28.217 0.350 4.047 5.956
R16 77.083 77.056 0.005 129.885 129.810 0.005 28.113 0.376 4.060 6.048
R17 76.190 77.056 0.005 128.624 129.810 0.005 28.583 0.420 4.457 5.928

E total change in color of the extract, DPPH (percent inhibition), FRAP (in micromoles of ascorbic acid eq/g dw)

interpretability of the results (Zumbo and Harwell 1999). A Reducing Power

regression model was used to fit the DPPH data, and the
ANOVA results are presented in Table 6. It was found that
linear terms such as microwave power, time of extraction,
and solvent concentration and quadratic terms indicating
interactions such as MWP time, MWP Sol. Conc., Time
Sol. Conc., and Sol. Conc.2 were highly significant factors
(p<0.05). The model was significant with p value<0.0001
and a nonsignificant lack of fit 0.1825 (p>0.05). The R2
value of the model was 0.9937 and the adjusted R2 was
0.9843. Thus this model can be used to predict the radical
scavenging activity for the microwave-assisted extraction of
grape seed extract. The predictive model in terms of coded
factors levels for DPPH is given in Eq. 8.
FRAP assay was used to determine the reducing power of
the antioxidants present in microwave-assisted grape seed
extracts. Similar to DPPH, the best fit to the FRAP data was
obtained with log transformation. The model p value
<0.0001 implies the model is significant. The lack of fit
was not significant, 0.1831 (p>0.05). The R2 value of the
model is 0.9935 and the adjusted R2 of 0.9838. It was found
that the linear and the quadratic terms which were signifi-
cant in DPPH model were similar for the FRAP model. The
values obtained for antioxidant activity from DPPH and
FRAP showed significant correlation (r01). The predicted
response for FRAP in terms of coded values for factors
levels is given in Eq. 9.

Log DPPH % inhibition Log FRAP M of Ascorbic acid eq=g dw

4:3445  0:0234MWP 0:0335Time 4:8660  0:0198MWP 0:0277Time

0:0298Sol:Conc:  0:0674MWP  Time 0:0245Sol:Conc:  0:0554MWP  Time

 0:0404MWP  Sol:Conc:  0:0326MWP  Sol:Conc:  0:0837Time

   
 0:1015Time  Sol:Conc: 0:0032 MWP 2
 Sol:Conc: 0:0032 MWP2
   
 0:0174 Time2  0:0424Sol:Conc:2 8  0:0140 Time2  0:0349Sol:Conc:2 9
 450

Table 6 ANOVA for antioxidant activity of MAE of grape seed

Antioxidant activity

DPPH (% inhibition) FRAP (M of ascorbic acid eq)/g dw

Source SS df MS F value p value SS df MS F value p value

Model 0.15008640 9 0.016676 105.9275 <0.0001a 0.10116425 9 0.011240 102.2450 <0.0001a
Lack-of-fit 0.00085406 4 0.000214 4.7175 0.1825 0.00059617 4 0.000149 4.6977 0.1831
Error 0.00094459 6 0.000157 0.00065962 6 0.000110
Term Estimate Std Error t ratio F value p value Estimate Std Error t ratio F value p value
Intercept 4.3445263 0.005425 800.79 <0.0001a 4.8660683 0.004534 1073.3 <0.0001a
MWP (100, 200) 0.023418 0.004839 4.84 23.4238 0.0029a 0.019847 0.004043 4.91 24.0930 0.0027a
Time (2, 6) 0.0335854 0.004839 6.94 48.1792 0.0004a 0.0277993 0.004043 6.88 47.2688 0.0005a
Sol conc (30, 60) 0.02983 0.004839 6.16 38.0072 0.0008a 0.0245048 0.004043 6.06 36.7291 0.0009a
MWP time 0.067422 0.005627 11.98 143.5712 <0.0001a 0.055474 0.004702 11.80 139.1828 <0.0001a
MWP sol conc. 0.04043 0.005627 7.19 51.6272 0.0004a 0.032653 0.004702 6.94 48.2241 0.0004a
Time sol conc. 0.101595 0.005627 18.06 325.9954 <0.0001a 0.083779 0.004702 17.82 317.4527 <0.0001a
MWP MWP 0.0032098 0.007786 0.41 0.1699 0.6945 0.0032216 0.006506 0.50 0.2452 0.6381
Time time 0.0174 0.007786 2.23 4.9940 0.0668 0.014003 0.006506 2.15 4.6318 0.0749
Sol conc. sol conc. 0.042491 0.007786 5.46 29.7822 0.0016a 0.0349 0.006506 5.36 28.7710 0.0017a
R2 0.993746 0.993522
Adjusted R2 0.984364 0.983805
a
Significant
Food Bioprocess Technol (2013) 6:441455
Food Bioprocess Technol (2013) 6:441455 451

Effect of Process Variables on Antioxidant Activity

It was found that by increasing the time of extraction with

decrease in solvent concentration and MWP, the antioxidant
activity of the extract increased in terms of DPPH and
FRAP. Figures 4 and 5 represent the response surface plot
for antioxidant activity in terms of DPPH percent inhibition
and FRAP. The Pearsons correlation coefficient for DPPH
and FRAP was 0.996. It was found that microwave power,
time of extraction and solvent concentration and the inter-
action of microwave power with time and solvent concen-
tration, and interactions of time and solvent concentration
played a significant role in the antioxidant activity of DPPH
and FRAP. The diffusion of the antioxidants from the solids
matrix to the solvent is enhanced when the time of extrac-
tion increases. Grape seeds are natural reservoir of low
molecular weight catechins (monomers, dimmers, trimers,
and oligomers) and flavonoids with orthodiphenolic struc- Fig. 5 Response surface plot for antioxidant activity in FRAP
ture in the B ring (Katalinic et al. 2004). Spranger et al.
(2008) reported that the radical scavenging activity of grape
seed by DPPH is positively related to their degree of poly- the results obtained by different methods are not always
merization, i.e., polymer > oligomer > monomer (catechin). comparable (Spranger et al. 2008). But similar results were
As grape seed has high proanthocyanidin content, it has obtained by Ramchandani et al. (2010) for grape seed (Ban-
higher antioxidant activity when compared to vitamin C galore blue variety) with a percent inhibition range from
and vitamin E (Spranger et al. 2008, Ramchandani et al. 50% to 80%, proving that this variety of grape seed has a
2010). Since the structural features of phenolic compounds high potential antioxidant activity and could efficiently be
are reported responsible for the antioxidant activity, mea- extracted by microwave-assisted extraction. Pan et al. (2008)
surement of total phenols may be related to their antioxidant studied the antioxidant activity of microwave-assisted extract
activity (Katalinic et al. 2004). The methods for assessing of longan peel and found that antioxidant of microwave-
antioxidant activity vary considerably which depends upon assisted extract of Longan peel was superior to Soxhlet extract
the type of radical generated from a given compound; hence, of Longan peel. This suggests that microwave-assisted

Fig. 4 Response surface plot for antioxidant activity in DPPH percent Fig. 6 Response surface plot for total change in color of extract (E
inhibition or DE)
452 Food Bioprocess Technol (2013) 6:441455

extraction of antioxidants could be an effective method with increase in the solvent concentration and extraction time
respect to time and less solvent wastage. The highly signifi- there was an increase in the color of the extract. Canals et
cant correlation coefficient was obtained for FRAP and DPPH al. (2005) stated that when the ethanol concentration is high,
data (r01). This indicates that both FRAP and DPPH can be the extraction of proanthocyanidins from skin and seed
successfully used to obtain the antioxidant activity of the significantly increases. The color of the extract with 45%
grape seed extract. and 60% ethanol concentration had more intense color than
30% EtOH concentration. The values obtained in this study
Effect of Process Variables on Color were similar to the values obtained by Buci Koji et al.
(2009) who studied the influence of solvent and temperature
Color denotes visual appearance of the product, whereas on extraction of phenolic compounds and color of the
pigments or colorants are the chemical compounds that extracts from grape seed.
impart the observed color (Wrolstad et al. 2005).The color The color of the extract is directly related to the presence
measurements are presented in Table 5. It was found that of anthocyanin color pigments and may be due to the
solvent concentration played a significant role in the color of interactions of non-pigmented compounds like catechin
the extract as shown in Fig. 6 which represents the response and epicatechin (Buci Koji et al. 2009). Stintzing et al.
surface graph for total change in color. It was found that (2002) studied the color and antioxidant properties of

13
0.89356
13.05879

12
GAE

11
10
9
21
1.593564
21.60461

20
CAT

19
18
17

15
1.316428
15.96802
TAE

13
11

9
30.633, 140.26577.6113, 84.4823

80
80.97392
DPPH

75
70
65
135
135.3632

130
FRAP

125
120
115
0 0.25 0.75 1
Desirability
0.947546

100

120

140
160

180

200

30
35
40
45
50
55
60

0.25

0.5

0.75
2

121.5393 6 32.631016
mwp time sol con Desirability

Fig. 7 Overall optimum conditions of microwave-assisted extraction of phenolic antioxidants from grape seed
Food Bioprocess Technol (2013) 6:441455
cyanidin-based anthocyanin pigments; it was found that
methoxylation and hydroxylation of the B ring had predict-
able effect on color. Katalinic et al. (2004) compared the
reducing power (FRAP) and antioxidant effectiveness of red
wines with white wines. It was found that average FRAP of
red wines was almost tenfold higher than the average FRAP
of white wines, which was mainly due to the presence of
anthocyanin pigments and catechins that could significantly
participate in the antioxidant power of red wine. This indi-
cates that grape seed antioxidants make significant contri-
bution to the color of the extract. Thus chromatic parameters
can be a helpful means for comparison and quick assess-
ment of phenolic content in grape seed extract. Further
studies are required in this area to understand the concepts
of coloring compounds and antioxidant activity.
Optimum Conditions for Maximum Extraction from Grape
Seeds
It is relatively simple to determine the exact optimum con-
ditions for single response using RSM (Bezerra et al. 2008,
Lundstedt et al. 1998). The predicted optimal conditions for
obtaining maximum total phenols were: 170 W, 6 min, and
30% ethanol concentration for 13.50.48 mg GAE/g dw GS
with a desirability of 0.982; 168 W, 6 min, and 30% EtOH
for 220.86 mg CAT/g dw GS with a desirability of 0.997;
and 160 W, 6 min, and 33.7% ethanol for 16.10.56 mg
TAE/g dw GS with a desirability of 0.992. The maximum
antioxidant activity was found at 100 W, 6 min, and 39.4%
ethanol concentration for DPPH providing 86.5% DPPH
inhibition with desirability of 0.997, and 100 W, 6 min,
and 39.2% ethanol concentration for FRAP giving 143
(M of ascorbic acid eq)/g dw GS with a desirability of
0.998.
When various responses have to be considered at the
same time, it is necessary to find optimal compromises
between the total number of response taken into account.
Thus Derringer function or desirability function (d) is the
most important and most currently used multicriteria method-
ology in optimization procedures (Bezerra et al. 2008). In
order to maximize the response of the dependent variables, a
numerical optimization was carried by statistical means which
provided the overall optimum conditions of extraction of
antioxidants from grape seed at 121 W, 6 min, and 32.6%
ethanol concentration with desirability of 0.947 (Fig. 7). The
numerical optimization provided the maximum predicted
total phenols in terms of various equivalents as 130.89 mg
GAE/g dw grape seed (GS), 21.61.59 mg CAT/g dw GS,
and 15.91.32 mg TAE/g dw GS, and the values for antiox-
idant activity were 80.9% DPPH inhibition and 135 M of
ascorbic acid equivalents/g dw GS for FRAP.
The application of desirability function in optimization of
multiple responses in extraction procedures makes it more
453
efficient and economical. Thus the maximum desirability in
terms of total phenols was obtained for catechin (d00.997)
which was the basic monomeric unit present in grape seed
phenols. In terms of antioxidant activity, the desirability func-
tion for both DPPH and FRAP was almost same. Katalinic et
al. (2004) studied the antioxidant effectiveness of grape wines
in comparison with (+)-catechin and demonstrated significant
antioxidant capacity with FRAP assay. FRAP is a very rapid
and simple method and is very convenient for screening large
number of samples (Katalinic et al. 2004). Thus a multiple
response optimization was done for total phenols in terms of
catechin, and FRAP resulted 21.61.51 mg CAT/g dw GS
and 135 M of ascorbic acid equivalents/g dw GS with a
maximum desirability of 0.991 at 122 W, 6 min, and 33.7%
ethanol concentration.
Conclusion
Thus the principles of multiple response optimization using
desirability function can be applied to the development of
extraction procedures that demand a search for optimal con-
ditions for a set of response simultaneously. In our study, we
were able to extract maximum phenolic antioxidant with lower
solvent consumption and extraction time by microwave-
assisted extraction when compared with results reported in
the literature. With the increasing consumer interest in func-
tional foods, food companies are looking to incorporate new
bioactive ingredients to improve health. Thus grape seed which
is considered as a winery waste can be used as a high value
functional food ingredient as substituted or alternative antiox-
idant to the synthetic antioxidants. This will in turn increase the
economic value of the grape seed obtained from grape pomace.
Acknowledgments We thank the Dept. of Foreign Affairs and Inter-
national Trade, Canada for providing the Canadian Commonwealth
Scholarship, 2010. Thanks to Simona Nemes and Ashutosh Singh,
Dept. of Bioresource Engineering, McGill University for their technical
expertise and support.
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