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1 Food and Bioprocess Technology Volume 6 Issue 2 2013 (Doi 110.1007 - s11947-012-0800-2) Krishnaswamy, Kiruba Orsat, Valérie Gariépy, Yvan Thangavel - Optimization of Microwave-Assisted Extraction o
1 Food and Bioprocess Technology Volume 6 Issue 2 2013 (Doi 110.1007 - s11947-012-0800-2) Krishnaswamy, Kiruba Orsat, Valérie Gariépy, Yvan Thangavel - Optimization of Microwave-Assisted Extraction o
DOI 10.1007/s11947-012-0800-2
ORIGINAL PAPER
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- observed ones as follows: GAE r00.995, CAT r00.990,
chemicals that have antioxidant properties. The influence TAE r00.996, DPPH r00.996, and FRAP r00.996.
of independent variables such as microwave power (100,
150, and 200 W), extraction time (2, 4, and 6 min), and Keywords Gallic acid . Catechin . Tannic acid . Response
solvent concentration (30%, 45%, and 60% ethanol) and surface methodology . Antioxidant activity . FRAP . DPPH
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 Introduction
the microwave-assisted extraction (MAE) process was opti-
mized using a central composite design. The total phenols Antioxidants are substances that neutralize free radicals.
that were expressed as gallic acid equivalents (GAE), cate- Free radicals are damaging compounds in the human body
chin equivalents (CAT), and tannic acid equivalents (TAE) that alter cell membranes, causing cell damage and inflam-
were significantly influenced by the solvent concentration mation, promoting abnormal cell growth including various
and the time of extraction. A numerical optimization was kinds of cancer, and even causing cell death (Lee et al.
carried out to obtain the overall conditions for MAE of 2004). Free radicals are generated naturally in the body by
phenolic antioxidants from grape seed. The response varia- metabolic process and from exogenous factors like ultravi-
bles were maximized for 6 min of MAE of grape seed (GS) olet light, radiation, smoke, and air pollution (Kim et al.
with 32.6% ethanol at 121 W with a desirability function of 2006). The normal aerobic respiration in cells produces
0.947. The predicted extraction yields were 130.89, 21.6 natural antioxidants which are further supplemented by di-
1.59, and 15.91.32 mg GAE, CAT, and TAE, respectively etary sources like vitamins, carotenoids, flavonoids, etc.
per gram of GS. The predicted antioxidant activity per gram (Harman 1995). The effectiveness of the endogenous anti-
of dry weight GS was 80.9% for the inhibition of DPPH and oxidant system declines with aging. Hence it is required to
135 M ascorbic acid equivalents for FRAP test. The pre- incorporate antioxidative nutraceuticals in the prevention of
dicted response values were significantly correlated with the 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
K. Krishnaswamy : V. Orsat (*) : Y. Garipy carcinogenicity (Pokorn 1991).
Department of Bioresource Engineering, McGill University,
The present regulatory regimes on waste management and
21,111, Lakeshore Road,
Ste-Anne-de-Bellevue, QC H9X3V9, Canada disposal in the food industries has urged for an integrated
e-mail: valerie.orsat@mcgill.ca approach in context of recycling/reuse and waste recovery.
Recycling of fruit and vegetable waste is one of the most
K. Thangavel
important means of utilizing it in a number of innovative
Department of Food and Agricultural Process Engineering,
Tamil Nadu Agricultural University, ways yielding essentially new products meeting the require-
Coimbatore 641003, India ments in human, animal, and plant nutrition as well as in the
442 Food Bioprocess Technol (2013) 6:441455
pharmaceutical industry. Hence there is a growing interest in very important so that it can be utilized in the preparation of
exploiting agricultural waste materials to obtain phytochem- functional foods, dietary supplements, and in nutraceutical
icals and antioxidants which will provide satisfactory solu- and pharmaceutical products (Cacace and Mazza 2003).
tions to both food processing industries and the society. In this Microwave-assisted extraction (MAE) has been reported
context, winery waste could be an excellent source for obtain- as a novel method for extraction of bioactive compounds. It
ing natural antioxidants from grape pomace (Bail et al. 2008). utilizes microwave energy to cause molecular movements
Grape (Vitis vinifera L.) is one of the worlds most and rotation of liquids with permanent or induced dipoles
important fruit crop with a global production of around 67 (Sun et al. 2007). When a biological material with favorable
million tons in 2009 (FAOSTAT 2011) and about 80% of the dielectric properties (plant material along with solvent for
total production is used for wine making. The grape pomace extraction) is placed in a microwave field, the molecules try
obtained, as winery by-product, constitutes 1020% of the to align with the oscillating electromagnetic field either by
weight of grapes (Garcia-Marino et al. 2006). It was distortion or distribution of electron cloud within the mole-
reported that the grape seed present in the pomace weighs cule or by physical rotation of the molecular dipoles which
3852% on dry matter basis (Maier et al. 2009). Grape seed leads to rapid heating of solvent and sample matrix (Dandekar
consists of 35% fiber, 29% polyphenols, 15% lipids, 3% and Gaikar 2002). Thus MAE is advantageous over conven-
ash, and 7% water (Mayer et al. 2008). Grape seeds are rich tional extraction techniques with improved efficiency, reduced
sources of monomeric phenolic compounds (Fig. 1) such as extraction time, rapid and volumetric heating of the absorbing
(+)-catechins, ()-epicatechin, (+)-gallocatechins, ()-epi- medium, low solvent consumption, higher selectivity of target
gallocatechin, and their dimeric, trimeric, and tetrameric molecules, and high potential for automation (Pallaroni et al.
proanthocyanidins (Baoshan Sun and Spranger 2005; Freitas 2002, Thostenson and Chou 1999, Wang et al. 2010). In
et al. 1998). conventional techniques such as heating, boiling, or refluxing,
Grape seed extract has been a matter of intense inves- there is a loss of flavonoids due to ionization, hydrolysis, and
tigations with respect to its potential beneficial effects on oxidation on prolonged extraction time. MAE is the simplest,
human health as it possesses a broad spectrum of biological, fastest, and most economical technique for extraction. The
pharmacological, and chemoprotective properties against enhancement of product recovery by microwave is generally
free radicals and oxidative stress. Recent reports indicate a attributed to its heating effect, which occurs due to the dipole
wide range of biological activities, e.g., antioxidant proper- rotation of the solvent in the microwave field. This causes the
ties and radioprotective effects (Castillo et al. 2000), anti- solvent temperature to rise, which then increases the solubility
bacterial activity (Jayaprakasha et al. 2003), prevention of of the compound of interest such as flavonoids (Wang et al.
cataract (Yamakoshi et al. 2002), antihyperglycemic effects 2010). Microwave-assisted extraction has been used to extract
(Pinent et al. 2004), anti-inflammatory effects (Terra et al. many different bioactive compounds, such as 1,8 cineole,
2007), enhancement of postprandial lipemia (Del Bas et al. menthone, and menthol from peppermint and 1,8 cineole
2005), improvement of insulin sensitivity, and prevention of and camphor from rosemary leaves (Chen and Spiro 1994),
hypertriglyceridemia and cardiovascular disease (Al-Awwadi essential oil from Lippia sidoides (Craveiro et al. 1989),
et al. 2005), prevention of in vitro low density lipoprotein alkaloid from lupin seeds (Lupinus mutabilis) (Ganzler et al.
oxidation (Meyer et al. 1997), modulation of the expression of 1990), secoisolariciresinol diglucoside from flaxseeds (Nemes
antioxidant enzyme systems (Puiggrs et al. 2005), and pro- and Orsat 2010), pectin from apple pomace (Wang et al.
tective effects against oxidative damage in mouse brain cells 2007), phenolic antioxidants from peanut skin (Ballard et al.
(Guo et al. 2007). Thus effective extraction and concentration 2009), glycyrrhizic acid from licorice roots (Pan et al. 2000),
of these phenolic bioactive compounds from the grape seed is curcuminoids from Curcuma longa (Dandekar and Gaikar
2002), Zearalenone from wheat and corn (Pallaroni et al. 15-s interval for 15 s to avoid heating of the sample (Gokturk
2002), anthocyanin from red raspberries (Sun et al. 2007), Baydar et al. 2007). The grape seed powder (10 g) was
tea polyphenols and tea caffeine from green tea leaves (Pan et defatted with 30 ml of hexane in an Erlenmeyer flask at
al. 2003), etc. 60 C for 6 h. The hexane containing the lipid portion was
The optimization of microwave-assisted extraction of poly- filtered, and the grape seed powder was kept in the fume hood
phenolic antioxidants from grape seeds was conducted in the to allow the residual hexane to evaporate.
present study with the objective of recovering maximum
antioxidants. Response surface methodology (RSM) was used Microwave Extraction of Phenolic Antioxidants
to evaluate the effect of multiple factors like microwave
power, solvent concentration, extraction time, and their inter- Dry defatted grape seed powder (0.50.05 g) was placed in
actions and was estimated by least square regression. From the a 250-ml quartz vessel. Fifty milliliters of different ethanol
literature it was found that Hong et al. (2001) used microwave concentrations (30%, 45%, 60%; v/v) was used for extrac-
power of 150300 W and time from 20 to 200 s to extract tion. The vessel was introduced into the microwave cavity
phenolic compounds from grape seed. Mayer et al. (2008) and fitted with a condenser. A focused-type, open-vessel
used microwave power of 150 W and 200 s for extraction of microwave system (Star System 2, CEM Matthews, USA)
proanthocyanidins from grape seed. Thus 100300 W and 1 operating at 800 W maximum power with a frequency of
5 min were taken as the minimum and maximum values for 2,450 MHz was used. The microwave power applied
the screening design. A response surface study was initially was intermittent with power on for 30 s/min. The
conducted using a two-level, three-factorial design for screen- extracts were centrifuged for 20 min and filtered with
ing the effects of the factors, ethanol concentration of 50% and 0.22-m filters (Whatman Puradisc 13-mm nylon syringe
90%, microwave power levels of 100 and 300 W, and extrac- filters) for the determination of total phenols, color, and anti-
tion time of 1 and 5 min on the extraction yields of total oxidant activity.
phenols, the antioxidant activity, and the color of the extracts.
Then the design was augmented to central composite design Experimental Design
by the addition of center and axial points.
Face-centered central composite design (CCD) was used to
determine the optimal conditions of microwave-assisted ex-
Materials and Methods traction of total phenols (TP), antioxidants activity (ferric
ion reducing antioxidant power (FRAP) and DPPH), and
Materials and Reagents color from grape seeds. Central composite design is highly
efficient and provides sufficient information on the effect of
V. vinifera (var. Bangalore blue), a variety of grapes, were process variables for resourceful optimization with reduced
procured from the local markets of Coimbatore, Tamil number of total experimental runs. The three independent
Nadu, India. This variety of grapes is widely grown in the factors studied were the microwave power, X1 (100, 150,
southern states of India like Karnataka and Tamil Nadu. The and 200 W), extraction time, X2 (2, 4, and 6 min), and
grapes were processed for the production of juice in the Post ethanol concentration, X3 (30%, 45%, and 60% ethanol),
Harvest Technology Centre, Tamil Nadu Agricultural Uni- which were coded at three levels as shown in Table 1.
versity, Coimbatore. The residual pomace was collected and The CCD included eight factorial points, six axial points,
the seeds were manually removed from the pomace. The and three center points, which totals 17 experimental runs,
seeds were cleaned, lyophilized in a batch-type freeze dryer and was used to fit the second-order polynomial model
(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 Table 1 Independent variables in CCRD for optimization of grape
seed extract
tannic acid, sodium carbonate, 1,1-diphenyl-2-picrylhydrazyl
(DPPH), and 2,4,6-tripyridyl-s-triazine were of analytical and Independent variables Coded levels
HPLC grade obtained from Sigma Chemicals Co (St. Louis,
1 0 1
MO, US).
Microwave power (W) X1 100 150 200
Sample Preparation Extraction time (min) X2 2 4 6
Ethanol concentration (% v/v) X3 30 45 60
Dried grape seeds were milled in a coffee grinder (Proctor
Silex, Model E167C) for 2 min, the process was stopped at 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 The DPPH assay was used to determine the free radical
scavenging activity of grape seed extract. The method by
A colorimetric method using FolinCiocalteau assay was Nair et al. (2007) was adopted with some modifications
used to determine the total phenols content in the grape seed (Singh et al. 2011). An aliquot of 50 l ethanolic extract
extract. The method used by Jayaprakasha et al. (2003), of grape seed having 1 mg of GS/ml of ethanol was added to
Jayaprakasha and Jaganmohan Rao (2000), and Singh et 1.5 ml of DPPH (3.94 mg/100 ml ethanol). The discolor-
al. (2011) was used with slight modifications. One milliliter ation of purple is due to the paring of the free electron with
of the grape seed extract having 1 mg of grape seed/ml of the free radical scavenger present in DPPH. The color
ethanol was mixed with 7.5 ml of double deionized water, change was measured with a spectrophotometer (Ultrospec
followed by the addition of 0.5 ml of FolinCiocalteau 2100 pro, Biochrom Ltd., Cambridge, England) at 517-nm
reagent, and 1 ml of 7.5% sodium carbonate solution. The absorbance after 20 min. The percentage of free radicals
mixture was incubated at room temperature in the dark for scavenged by DPPH radical was calculated using the fol-
the development of color for 30 min. The absorbance was lowing equation:
measured at 765 nm using a spectrophotometer (Ultrospec
2100 pro, Biochrom Ltd., Cambridge, England). The total DPPH% inhibition
phenols concentration was expressed in terms of gallic acid
Abs control 517nm Abs sample 517nm
equivalents (GAE), catechin equivalents (CAT), and tannic 100
Abs control 517nm
acid equivalents (TAE) based on the standard curves given
2
in Table 2 which were plotted for 30%, 45%, and 60% ethanol
concentrations in respective equivalents (Y0concentration, where, Abs control 0 absorption of blank sample and Abs
x0absorbance at 765 nm). sample 0 absorption of grape seed extract.
Reducing Power for each sample. From the obtained L*, a*, and b* values, the
total change in color of the extract (E) was calculated using
The FRAP assay takes advantage of electron transfer reactions the following equation:
and is used to measure the total antioxidant activity of natural q
compounds. Herein a ferric salt, Fe(III)(TPTZ)2Cl3 (TPTZ) E L0 L2 a0 a2 b0 b2 4
2,4,6-tripyridyl-s-triazine is used as an oxidant. Antioxidants
are used as reductants in a redox-linked colorimetric method. Where L0, a0, and b0 denotes the value of pure ethanol at 30%,
The method used by Benzie and Strain (1996) and Deighton et 45%, and 60%, v/v used as standard reference to compare the
al. (2000) was used in this experiment with some modifica- color change of the ethanolic extract.
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 Statistical Analysis
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 Statistical analysis was performed using the JMP 8 software
FRAP reagent, vortexed for 60 s, and allowed to stand for (SAS Institute Inc., Cary, NC, USA). The data were ana-
6 min at room temperature. The control consisted of 1.5 ml lyzed by analysis of variance (ANOVA) and the adequacy of
FRAP reagent to which were added 50 l distilled water. The the response surface model was determined by evaluating
color change was read with a spectrophotometer at 593 nm. the lack of fit and coefficient of determination (R2). The
Ascorbic acid, which is an antioxidant, was used as a standard statistical significance of the model and its variables was
solution to determine the reactivity of FRAP solutions using determined at 5% probability level (p<0.05). The optimal
the standard curve shown in Eq. 3 (R2 00.997). conditions for MAE of grape seed phenolic antioxidants
were obtained based on modeling and desirability function
Absorbance of grape seed extract 593nm that could be visually explained in terms of three-dimensional
response surface plots and contour plots.
244:7 Conc: of grape seed extract 0:014 3
Table 3 Face-centered central composite design (CCD) with observed response for total phenols from microwave-assisted extraction of grape
seeds
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
is a reasonable agreement with the adjusted R2 of 0.9738 Similarly, a regression analysis was carried out to quan-
in the model. The lack of fit was not significant (p> tify the total phenols in terms of catechin equivalents. The
0.05), suggesting that the model was well fitted and could response predictions of total phenols in terms of catechin
be used to predict the total phenols from microwave-assisted equivalents was significant in the given model with p0
grape seed extract. 0.0009 (Eq. 6). The R2 value for the model was 0.9806 with
The model indicated that the linear effects of extraction time a reasonable agreement with the adjusted R2 of 0.9457, and
and solvent concentration had the greatest significance on total the lack of fit was not significant 0.0678 (p>0.05) suggest-
phenols of the extract. Values of (p) less than 0.05 indicate the ing that the model could be used to predict the response. It
significant terms in the model, based on which the linear terms was found that microwave power, time and the interaction of
such as microwave power, time of extraction, solvent concen- MWP and solvent concentration, time and solvent concen-
tration, bilinear terms such as MWP Sol. Conc., Time Sol. tration, and MWP2 were significant factors in the model.
Conc., and quadratic terms of MWP2, were found to be sig- The predicted response for total phenols in terms of catechin
nificant. The predictive, second-order, polynomial model for equivalents, in term of coded factors level, is given in the
total phenols content of microwave-assisted grape seed Eq. 6 neglecting some insignificant terms:
extracts, in terms of coded factors levels, is shown in Eq. 5.
TPCATmg=g dw GS 19:359 0:646MWP
TPGAEmg=g dw GS 10:955 0:368MWP
1:791Time
1:03705Time
0:185Sol:Conc:
0:697Sol:Conc:
1:414MWP Time
0:775MWP Time
1:547MWP Sol:Conc:
0:879MWP Sol:Conc:
1:930Time Sol:Conc:
1:148Time Sol:Conc:
1:044 MWP2
0:590 MWP2
0:54 Time2
0:305 Time2
0:498Sol:Conc:2
0:125Sol:Conc:
2
5 6
The Pearsons correlation coefficient for actual vs. pre- The predictive response of total phenols in terms of tannic
dicted value is r00.995 (Fig. 2). Hence this model can be acid equivalents by regression analysis was found to be signif-
used to predict total phenolics under different experimental icant (p value<0.0001). The R2 value of the model was found
conditions during microwave-assisted extraction. 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
13.5 equation in terms of coded factors levels for total phenols is:
13
12.5 TPTAEmg=g dw GS 14:288 0:458MWP
GAE Actual
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 Food Bioprocess Technol (2013) 6:441455
The predicted vs. actual plot obtained for total phenols in compared to the microwaveless control method. Pan et al.
terms of catechin equivalents and tannic acid equivalents (2000) studied the MAE of glycyrrhizic acid from licorice
were similar to that obtained for gallic acid equivalents root with extraction times of 45 min, ethanol concentra-
(Fig. 2) with Pearsons correlation coefficient (r) for GAE tions of 5060% (v/v).
r 00.995, CAT r00.990, TAE r00.996. Thus the given Similar surface plots were obtained for total phenols in
models can be used to predict the total phenols of terms of tannic acid equivalents and catechin equivalents.
microwave-assisted extraction of grape seeds in milligrams The optimum extraction conditions for maximum total phe-
equivalents per gram of dry weight (dw) grape seed, nols from grape seed was found at 170 W, 6 min, and 30%
respectively. ethanol concentration yielding 13.50.48 mg GAE/g dw
GS with a desirability of 0.982. The microwave power of
Effect of Process Variables on Total Phenols 168 W, 6 min, and 30% ethanol concentration yielded more
total phenols in terms of catechin equivalents at 220.86 mg
The relationship between independent and dependent vari- CAT/g dw GS with the desirability of 0.997. The predicted
able can be well illustrated with three-dimensional response optimum extraction conditions in terms of tannic acid equiv-
surfaces. Figure 3 gives the interaction of MWP and time at alents were 160 W, 6 min, and 33.7% ethanol concentration
a fixed solvent concentration of 45% ethanol concentration. with 0.992 desirability giving 16.10.56 mg TAE/g dw GS.
It was found that there was an increase in total phenols with Ghafoor et al. (2009) studied the extraction of phenols
an increase in time of extraction. By keeping time constant and antioxidant from grape seed by ultrasound-assisted ex-
with constant solvent concentration and increasing the traction. The surface plots obtained were similar to the
MWP, there was a gradual increase in the total phenols. present study with 53.15% ethanol concentration v/v,
The maximum total phenol from the extract was obtained 56.03 C, and 29.03 min of extraction time yielding maxi-
with increased extraction time. It revealed that by decreasing mum total phenols of 5.44 mg GAE/100 ml. In the present
the solvent concentration from 60% to 30% and by increas- study with microwave-assisted extraction, it was possible to
ing the MWP from 100 to 170 W, it increased the total obtain a high amount of total phenols in less time (6 min)
phenols. Wang et al. 2010 reported that 70% ethanol con- with reduced 3034% (v/v) of ethanol. The amount of total
centration, microwave power 255 W, and extraction time phenols obtained by Li et al. (2008) was in the range of 7.5
6.5 min gave maximum flavonoids from Radix puerariae. to 44 mg GAE/100 g dry matter. Mandic et al. (2008)
MAE of secoisolariciresinol diglucoside from flaxseed by reported 3.59 to 11.7 g GAE/kg of dry grape seed using
Nemes and Orsat (2009) reported MWP of 135 W, sodium ethanolic solvent for extraction. Jayaprakasha et al. (2001)
hydroxide concentration of 0.5 M, and 3 min extraction time reported a range of 18.2 to 20.8 CAT/100 g grape seed extract
which gave 10% increase in the extraction yield when 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
Antiradical Activity
Table 5 Observed responses for the antioxidant activity and color value of the microwave-assisted extraction of grape seed in the experimental design
DPPH FRAP
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)
Antioxidant activity
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 453
cyanidin-based anthocyanin pigments; it was found that efficient and economical. Thus the maximum desirability in
methoxylation and hydroxylation of the B ring had predict- terms of total phenols was obtained for catechin (d00.997)
able effect on color. Katalinic et al. (2004) compared the which was the basic monomeric unit present in grape seed
reducing power (FRAP) and antioxidant effectiveness of red phenols. In terms of antioxidant activity, the desirability func-
wines with white wines. It was found that average FRAP of tion for both DPPH and FRAP was almost same. Katalinic et
red wines was almost tenfold higher than the average FRAP al. (2004) studied the antioxidant effectiveness of grape wines
of white wines, which was mainly due to the presence of in comparison with (+)-catechin and demonstrated significant
anthocyanin pigments and catechins that could significantly antioxidant capacity with FRAP assay. FRAP is a very rapid
participate in the antioxidant power of red wine. This indi- and simple method and is very convenient for screening large
cates that grape seed antioxidants make significant contri- number of samples (Katalinic et al. 2004). Thus a multiple
bution to the color of the extract. Thus chromatic parameters response optimization was done for total phenols in terms of
can be a helpful means for comparison and quick assess- catechin, and FRAP resulted 21.61.51 mg CAT/g dw GS
ment of phenolic content in grape seed extract. Further and 135 M of ascorbic acid equivalents/g dw GS with a
studies are required in this area to understand the concepts maximum desirability of 0.991 at 122 W, 6 min, and 33.7%
of coloring compounds and antioxidant activity. ethanol concentration.
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