J.

of Supercritical Fluids 43 (2007) 55–63

Subcritical (carbon dioxide + ethanol) extraction of polyphenols

from apple and peach pomaces, and determination of the
antioxidant activities of the extracts
İ. Hasbay Adil, H.İ. Çetin, M.E. Yener ∗ , A. Bayındırlı
Department of Food Engineering, Middle East Technical University, 06531 Ankara, Turkey
Received 20 January 2007; received in revised form 13 April 2007; accepted 30 April 2007

Abstract
The effects of pressure (20–60 MPa), temperature (40–60 ◦ C), ethanol concentration (14–20 wt.%) and extraction time (10–40 min) on subcritical
(CO2 + ethanol) extraction of polyphenols from apple and peach pomaces (moisture content ∼ = 14%, particle size = 0.638 mm) were determined. All
variables increased total phenolic contents (TPC) and antiradical efficiencies (AE) of the extracts significantly (p ≤ 0.05). The optimum pressure
and temperature were 54.6–57 MPa and 55.7–58.4 ◦ C for apple pomace, and 50.6–51 MPa and 50.9–52.3 ◦ C for peach pomace. The optimum
ethanol concentration and extraction time were 20% ethanol and 40 min for both pomaces. TPC of the extracts were 0.47 and 0.26 mg gallic acid
equiv./g sample and AE of the extracts were 3.30 and 1.5 mg DPPH• /mg sample for apple and peach pomaces, respectively. Low TPC and AE of the
extracts from peach pomace could be contributed to low concentrations of ferulic and p-coumeric acids, epicatechin, and presence of anthocyanins
in peach.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Subcritical carbon dioxide extraction; Co-solvent; Fruit; Pomace; Phenolic content; Antioxidant activity

1. Introduction organic solvents, long extraction time, limited solvent choice

Fresh fruits and vegetables, therefore their industrial by-
products are rich in antioxidants [1] such as ascorbic acid,
tocopherols, carotenoids, and polyphenols [2–5]. Polyphenols
in fruits and vegetables include mainly phenolic acids (hydrox-
ybenzoic and hydroxycinnamic acids), flavonoids (flavonols,
flavones, flavanones, isoflavones, flavanols, and anthocyanins)
[4]. Antioxidants are substances that are able to prevent or retard
the oxidation of lipids, proteins and DNA; and to protect the
compounds or tissues from damage caused by oxygen or free
radicals. Therefore, their health promoting effects reduce the
risk of various diseases [4].
Recovery of antioxidants from by-products of food process-
ing plants has gained importance [3,6] since the replacement
of synthetic antioxidants by natural ones have benefits due to
health implications and solubilities in food systems [7–9].
Solvent extraction offers good recovery of polyphenols how-
ever, has several drawbacks like the use of large amounts of
∗ Corresponding author. Tel.: +90 312 210 5630; fax: +90 312 210 2767.
E-mail address: eyener@metu.edu.tr (M.E. Yener).
for health assurance and possible degradation of target com-
pounds. There are many alternative methods that either eliminate
or reduce these drawbacks. These are microwave assisted extrac-
tion, pressurized liquid extraction, ultrasonic extraction, and
supercritical fluid extraction [5,10–12].
Supercritical fluid extraction is an alternative for the food
industry, especially using CO2 because it has a low critical tem-
perature (Tc = 31.1 ◦ C), it is safe, and non-toxic. Moreover, the
absence of light and air during extraction reduce the risk of
degradation reactions. However, since CO2 is non-polar it is
not a good solvent for polar polyphenols. Addition of organic
co-solvents like ethanol, methanol, acetone, increases the sol-
vating power of CO2 and the yield of extraction of polyphenols.
Ethanol is a permitted co-solvent in food industry. Consump-
tion of ethanol is not in large amounts as in conventional solvent
extraction, and it is easily eliminated from the extract by evap-
oration at room temperature.
When a co-solvent is added to CO2 , the critical temperature
of the resulting mixture is elevated [13] limiting the amount of
co-solvent which may be added if the extraction is to occur in
the supercritical region at 40–60 ◦ C. Addition of 5% ethanol to
CO2 increases the critical temperature of the mixture to 42.5 ◦ C

0896-8446/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2007.04.012
56 İ.H. Adil et al. / J. of Supercritical Fluids 43 (2007) 55–63
Table 1
Critical temperature and pressure of CO2 –ethanol mixtures (calculated by using
SF-Solver Software, ISCO Inc., Lincoln, NE, USA)
Ethanol concentration (wt.%) Tc (◦ C)
0 31.1
5 42.5
10 53.7
14 62.8
17 69.5
20 76.1
100 243.3
(Table 1) The extraction performed at 20 MPa (this could be any
pressure above the Pc of the mixture), 50 ◦ C with 5% ethanol as a
co-solvent is a supercritical (CO2 + ethanol) extraction whereas,
the extraction performed at 20 MPa, 40 ◦ C with 5% ethanol as
a co-solvent is a subcritical (or near critical) (CO2 + ethanol)
extraction. Therefore, addition of high concentrations of ethanol
(14–20%) requires the extraction to be at subcritical conditions
at 40–60 ◦ C (Table 1).
Supercritical or subcritical CO2 extraction with ethanol
and/or methanol as a co-solvent has been applied to extract
polyphenols from grape seeds [14,15], from wine industry by-
products [16], from pistachio hulls [17], and from olive leaves
[18]. Hydroxycinnamic acids (p-coumaric acid, caffeic acid and
ferulic acid [19]), and coumeric acid isomers (o-, m-, p-coumeric
acids [20]), are slightly soluble in supercritical CO2 withouth
addition of a co-solvent. However, addition of 5–30% ethanol
is required to increase the solubility of quercetin [21], cate-
chin [22], epicatechin [23] and resveratrol [24] in supercritical
CO2 .
Apple has the highest total phenolic content (296.3 ± 6.4 mg
gallic acid equiv./100 g of fresh fruit) [25]. Peels of apple
have higher phenolic content than the peeled fruit [26]. Apple
is among the fruits with high antioxidant activity, as well
(97.6 ± 4.6 ␮mol of vitamin C equiv./g of fresh fruit) [25].
Poyphenols in apple include hydroxycinnamic acids such as
caffeic [26,27], p-coumeric [26], ferulic [26] and chlorogenic
[12,28] acids, flovonols such as quercetin glycosides (quercetin-
3-arabinose, quercetin-3-xyloside, quercetin-3-galactosidase,
quercetin-3-glucoside, quercetin-3-rhamnoside) [12,27–29] and
flavanols such as (+)catechin [29], (−)epicatechin [12,27–29],
procyanidin B2 [12,28]. Among these quercetin (34.7%),
epicatechin (19.9%), and procyanidin B2 (19%) contribute
significantly to the total antioxidant activity of apples
[27].
Peach has an intermediate total phenolic content
(84.6 ± 0.7 mg gallic acid equiv./100 g of fresh fruit) with
an antioxidant activity of 49.5 ± 2.8 ␮mol of vitamin C equiv./g
of fresh fruit [25]. Similar to apple, phenolic content is higher
in the peels of peach [26]. Polyphenols in peach include
hydroxycinnamic acids such as caffeic [26,30,31], p-coumeric
[26], ferulic [26] and chlorogenic [30–32] acids, flovonols such
as quercetin glycosides (quercetin-3-galactoside, quercetin-3-
glucoside, quercetin-3-rutinoside) [31,32], flavanols such as
catechin [31,32], epicatechin [32] and procyanidin B1 [32],
and anthocyanins such as cyanidin-3-glucoside and cyanidin-
3-rutinoside [32,33]. Contrary to apple, catechin content in
peach is higher than the contents of epicatechin and quercetin
glycosides [26,32].
Pc (MPa) Based on the solubility data of hydroxycinnamic acids
7.38 [19,20], quercetin [21], catechin [22], epicatechin [23] in super-
7.32 critical CO2 there is a potential for recovery of these polyphenols
7.27 from apple and peach pomaces by subcritical (CO2 + ethanol)
7.22 extraction. Therefore, the aim of this study was to investigate
7.19
7.15
the subcritical (CO2 + ethanol) extraction of polyphenols from
6.13 apple and peach pomaces and, to determine the optimum extrac-
tion conditions (pressure, temperature, ethanol concentration in
CO2 and extraction time) considering the total phenolic contents
and antioxidant activities of the extracts using response surface
methodology (RSM).
2. Materials and methods
2.1. Materials
The pomaces from ripe apple (mixture of Starking and
Amasya varieties) and ripe peach (Hale Haven variety)
were obtained from the fruit juice production pilot plant of
Ankara University, Department of Food Engineering. Unlike
to apple juice, peach juice production involves a heat treatment
(85–90 ◦ C) before pressing. The pomaces remaining after press-
ing consist of pressed skins and pulp residue. The pomaces
were freeze-dried at −5 ◦ C and 0.47 kPa (Model FD8, Heto
Lab. Equipment, Allerød, Denmark). Moisture content of the
freeze-dried pomaces were determined by placing about 2 g of
pomace in previously dried and weighed containers and keep-
ing at 100 ◦ C until constant weight. Moisture contents of apple
and peach pomaces were found as 13.82 ± 1.07% (n = 5) and
14.75 ± 0.92% (n = 5), respectively.
The dried samples were ground using kitchen-type grinder
(Moulinex, France), sieved and fractionated according to parti-
cle size by certified sieves (Endecotts Ltd., London, England).
Sieving was performed by a shaker (Octagon 200, Endecotts
Ltd., London, England). The particles that passed through the
sieve with an opening size of 0.850 and retained on that
of 0.425 mm were used. Therefore, the average particle size
was 0.638 mm by sieve analysis. CO2 used in the extrac-
tions was 99.9% pure (Bos, İstanbul, Turkey) and ethanol
(99.8%, Riedel Inc., Steinheim, Germany) was added as a co-
solvent.
2.2. Subcritical (CO2 + ethanol) extraction
Extractions were done by Supercritical Fluid Extraction Sys-
tem (SFX System 5100, ISCO Inc., Lincoln, NE, USA), which
consists of an extractor (SFX 3560) and two syringe pumps
(Model 100DX) that enables co-solvent addition. A 1 g of sam-
ple was placed into 10 ml aluminium sample cartridge. The
solvent flow (CO2 + ethanol) was downward with a rate of
2 g/min. The restrictor temperature was 80 ◦ C. The extract was
collected in ethanol and was diluted to a constant volume of
3 ml.
 İ.H. Adil et al. / J. of Supercritical Fluids 43 (2007) 55–63 57

2.3. Solvent extraction
Ethanol was used for solvent extraction of dried pomaces.
Mixtures with solid to solvent ratio 0.05 g/ml were prepared by
adding 0.2 g sample in 4 ml solvent. The mixtures were kept
at room temperature in dark for 24 h [14,34]. The total pheno-
lic contents and antiradical efficiencies of the resulting extracts
and those obtained by subritical (CO2 + ethanol) extraction were
compared.
2.4. Determination of total phenolic content (TPC)
Folin-Ciocalteu method was used for the determination of
TPC [35]. This method is based on the color change determined
at 740 nm (Pharmacia LKB-Novaspec II model spectrophotome-
ter, UK) caused by reduction of the Folin-Ciocalteau reagent
by phenolates produced in the presence of sodium carbonate.
TPC was expressed as gallic acid equivalent using the standard
curve prepared at different concentrations of gallic acid. Three
replications were done for each analysis.
2.5. Determination of antioxidant activity
Antioxidant activities of the extracts were determined
by detecting the scavenging of DPPH• (1,1-diphenyl-2-
picrylhydrazyl) radical [36]. Ethanol in the extracts was
evaporated before the assay. The assay is based on the color
change determined at 515 nm (Pharmacia LKB-Novaspec II
model spectrophotometer, UK) caused by reduction of the
DPPH• radical. The reaction time for the assay was deter-
mined as 60 min based on the reduction of the DPPH• radical
by gallic acid. The percentage of remaining DPPH• was
determined by using the standard curve prepared by gallic
acid (0.01–0.1 g gallic acid/g DPPH• ). These were plotted
against the sample concentration to determine EC50 (efficient
concentration of the sample to decrease the initial DPPH• con-
centration by 50%). Three replications were done for each
analysis. The antioxidant activity was expressed in terms
of antiradical efficiency (AE) [37–40] which is defined as,
AE = 1/EC50 .
2.6. Experimental design
The three level Box–Behnken design [41,42] with four
independent variables was used. The independent variables
were pressure, temperature, ethanol concentration in CO2
and extraction time. Uncoded and coded values of the inde-
pended variables, the experimental points used according to the
Box–Behnken design are shown in Table 2.

Table 2
Coded and uncoded levels of independent variables for Box–Behnken design and, TPC and AE of the extracts
Experimenta X1 X2 X3 X4 Pressure Temperature Ethanol concentration Time Apple Peach
no. (MPa) (◦ C) (wt.%) (min) pomace pomace
TPCb AEc TPCb AEc

1 +1 +1 0 0 60 60 17 25 0.238 1.999 0.150 0.792

2 +1 −1 0 0 60 40 17 25 0.281 1.362 0.160 0.760
3 −1 +1 0 0 20 60 17 25 0.168 1.298 0.130 0.629
4 −1 −1 0 0 20 40 17 25 0.143 0.879 0.090 0.405
5 0 0 +1 +1 40 50 20 40 0.383 2.573 0.241 1.232
6 0 0 +1 −1 40 50 20 10 0.297 1.477 0.187 0.773
7 0 0 −1 +1 40 50 14 40 0.238 0.435 0.150 0.613
8 0 0 −1 −1 40 50 14 10 0.146 0.582 0.092 0.222
9 0 0 0 0 40 50 17 25 0.340 1.981 0.215 0.913
10 +1 0 0 +1 60 50 17 40 0.356 2.767 0.214 1.275
11 +1 0 0 −1 60 50 17 10 0.222 0.725 0.143 0.334
12 −1 0 0 +1 20 50 17 40 0.222 1.121 0.140 0.604
13 −1 0 0 −1 20 50 17 10 0.122 0.751 0.077 0.346
14 0 +1 +1 0 40 60 20 25 0.365 1.783 0.210 1.079
15 0 +1 −1 0 40 60 14 25 0.206 1.245 0.130 0.666
16 0 −1 +1 0 40 40 20 25 0.326 0.485 0.238 1.053
17 0 −1 −1 0 40 40 14 25 0.097 0.499 0.071 0.368
18 0 0 0 0 40 50 17 25 0.355 2.029 0.227 0.935
19 +1 0 +1 0 60 50 20 25 0.427 2.193 0.250 1.335
20 +1 0 −1 0 60 50 14 25 0.246 1.043 0.145 0.573
21 −1 0 +1 0 20 50 20 25 0.265 1.249 0.184 0.718
22 −1 0 −1 0 20 50 14 25 0.146 0.586 0.092 0.276
23 0 +1 0 +1 40 60 17 40 0.502 2.246 0.225 1.169
24 0 +1 0 −1 40 60 17 10 0.194 0.781 0.075 0.362
25 0 −1 0 +1 40 40 17 40 0.235 0.950 0.138 0.751
26 0 −1 0 −1 40 40 17 10 0.160 0.889 0.117 0.321
27 0 0 0 0 40 50 17 25 0.333 1.962 0.198 0.904
a Experiments were performed in random order.
b Total phenolic content (mg gae/g sample).
c Antiradical efficiency (mg DPPH• /g sample).
58 İ.H. Adil et al. / J. of Supercritical Fluids 43 (2007) 55–63

2.7. Data analysis
Second-order polynomial equations were used to express
TPC (mg gallic acid equiv./g sample) and AE (mg DPPH• /g
sample) of the extracts (Y) as a function of the coded independent
variables, where X1 , X2 , X3 and X4 represent the code of pres-
sure, temperature, ethanol concentration in CO2 and extraction
time, respectively:
Y = a0 + a1 X1 + a2 X2 + a3 X3 + a4 X4 + a11 X12 + a22 X22
+a33 X32 + a44 X42 + a12 X1 X2 + a13 X1 X3 + a14 X1 X4
+a23 X2 X3 + a24 X2 X4 + a34 X3 X4
Experimental data were analyzed by multiple regression to fit
the second order models to the dependent variables. The pack-
age program MINITAB 13.20 was used to perform regression
analysis. The models were used to plot response surfaces using
Statistica 5.0 (1995) and contour plots using Surfer 6.01 by keep-
ing two independent variables constant. Contour plots obtained
for TPC and AE were superimposed to estimate the optimum
extraction conditions.
3. Results and discussion
Preliminary experiments (data not shown here) were per-
formed in order to determine the levels of the design variables.
A wide pressure range was covered by selecting the pressure
between 20 and 60 MPa. Extractions performed at 60 MPa,
60 ◦ C, using 2 g/min solvent flow rate for 30 min showed that
supercritical (CO2 + ethanol) extraction by 0–10 wt.% ethanol
addition resulted in extracts with low TPC and AE compared
to subcritical (CO2 + ethanol) extraction by 10–20 wt.% ethanol
addition [43]. Higher ethanol concentrations were not used in
order to avoid saturation of CO2 with ethanol and formation
of two phases (use of co-solvent concentrations above 30% is
not recommended by ISCO Inc., Lincoln, NE, USA). The time
levels were determined by performing extractions at 60 MPa,
60 ◦ C, using 2 g/min solvent (with 20 wt.% ethanol) flow rate
for 120 min. It was observed that TPC significantly increased in
the first period of the extraction. Then the extraction rate slowed
down and after about 40 min TPC almost reached to a plateau
where the increase in TPC was not significant compared to the
first period [43]. Similar mechanism was observed during the
supercritical CO2 extraction of apricot kernel oil. In the first
period solubility of the solute in CO2 , in the second period dif-
fusion inside the particles govern the mass transfer [44]. The
extraction time was selected to be maximum 40 min to assure
that TPC of the extracts increase significantly with extraction
time before reaching to a plateau, at all extraction conditions.
Tables 2 and 3 show the experimental data and the regres-
sion coefficients obtained by fitting experimental data to the
second order response models for TPC and AE of the extracts,
respectively. t-Tests for p ≤ 0.05 show that all independent vari-
ables were significant for TPC and AE of the extracts from
both pomaces. Pressure–time interactions were significant for
only AE but temperature–time interactions were significant for
both TPC and AE of the extracts from apple pomace. Simi-
larly, temperature–time interactions were significant for both

Table 3
Second order response model constants and regression analysis for TPC and AE of the extracts
Term Coefficienta Apple pomace Peach pomace

TPCb AEc TPCb AEc

Intercept a0 0.3427** 1.9907** 0.2133** 0.9173**

X1 (pressure) a1 0.0587** 0.3504** 0.0291** 0.1742**
X2 (temperature) a2 0.0359* 0.3573** 0.0088* 0.0866**
X3 (ethanol concentration) a3 0.0820** 0.4475** 0.0525** 0.2893**
X4 (time) a4 0.0662** 0.4072** 0.0347** 0.2738**
X12 (pressure) a11 −0.0662** −0.2453 −0.0368** −0.1468**
X22 (temperature) a22 −0.0563* −0.4399** −0.0419** −0.1083**
X32 (ethanol concentration) a33 −0.0280 −0.4732** −0.0100 −0.0392
X42 (time) a44 −0.0361 −0.3298* −0.0338** −0.1524**
X1 (pressure) × X2 (temperature) a12 −0.0170 0.0545 −0.0125 −0.0480
X1 (pressure) × X3 (ethanol concentration) a13 0.0155 0.1217 0.0032 0.0800*
X1 (pressure) × X4 (time) a14 0.0085 0.4180* 0.0020 0.1707**
X2 (temperature) × X3 (ethanol concentration) a23 −0.0175 0.1380 −0.0217** −0.0680
X2 (temperature) × X4 (time) a24 0.0582* 0.3510* 0.0322** 0.0942*
X3 (ethanol concentration) × X4 (time) a34 −0.0015 0.3108 −0.0010 0.0170
R2 0.900 0.891 0.978 0.980
F 7.72 7.04 38.24 42.14
Sig F 0.001 0.001 0.000 0.000
S.E. 0.04692 0.3321 0.01224 0.06844
a Y = a0 + a1 X1 + a2 X2 + a3 X3 + a4 X4 + a11 X12 + a22 X22 + a33 X32 + a44 X42 + a12 X1 X2 + a13 X1 X3 + a14 X1 X4 + a23 X2 X3 + a24 X2 X4 + a34 X3 X4 .
b Total phenolic content (mg gae/g sample).
c Antiradical efficiency (mg DPPH• /g sample).
* Significant at p ≤ 0.05.
** Significant at p ≤ 0.01.
 İ.H. Adil et al. / J. of Supercritical Fluids 43 (2007) 55–63
Fig. 1. Effects of pressure (X1 ) and temperature (X2 ) on (a) TPC and (b) AE of the extracts from apple pomace (X3 = X4 = 0).
Table 4
Optimum extraction conditions
Apple pomace
Coded values
Pressure (X1 ) 0.73–0.85
Temperature (X2 ) 0.57–0.84
Ethanol concentration (X3 ) 1
Time (X4 ) 1
TPC and AE of the extracts from peach pomace. Additionally,
temperature–ethanol concentration interactions were significant
for TPC, and pressure–ethanol concentration, pressure–time
interactions were significant for AE of the extracts from peach
pomace (Table 3).
Fig. 1 shows that the effect of pressure on TPC and AE of the
extracts from apple pomace was positive up to about 50 MPa.
Similar response surfaces (not shown here) were obtained for
extracts from peach pomace, as well [43]. This is mainly due
to the increase in the density of CO2 , i.e. increase in the sol-
vating power with increasing pressure [10,45]. This is parallel
to the solubility behavior of hydroxycinnamic acids in super-
critical CO2 at 8.5–50 MPa and 40–60 ◦ C [19,20], and the
solubility behavior of catechin [22] and epicatechin [23] in
supercritical or subcritical (CO2 + ethanol) at 8–12 MPa and
40 ◦ C. The solubility of epicatechin in CO2 containing 20%
ethanol increased almost four fold by increasing pressure from
8 to 10 MPa at 40 ◦ C [23]. Similar results were obtained dur-
ing the extraction of phenols from grape seeds [15]. However,
Fig. 2. Effects of ethanol concentration (X3 ) and extraction time (X4 ) on (a) TPC and (b) AE of the extracts from peach pomace (X1 = X2 = 0).
59
Peach pomace
Uncoded values Coded values Uncoded values
54.6–57 (MPa) (X1 ) 0.53–0.55 50.6–51 (MPa)
55.7–58.4 (◦ C) (X2 ) 0.09–0.23 50.9–52.3 (◦ C)
20 (wt.%) (X3 ) 1 20 (wt.%)
40 (min) (X3 ) 1 40 (min)
in this study, above 50 MPa the effect of pressure on TPC and
AE of the extracts was not significant (Fig. 1) since the increase
in the density of CO2 with pressure is not as high as lower
pressures. The increase in oil yield was not significant above
45 MPa during supercritical CO2 extraction of apricot kernel
oil for the same reason [44]. In fact, optimum pressures were
found to be between 54.6–57 and 50.6–51 MPa for subcritical
(CO2 + ethanol) extraction of polyphenols from apple and peach
pomaces, respectively (Table 4).
Temperature also had a positive effect on TPC and AE of
the extracts up to about 50 ◦ C (Fig. 2). In literature, the effect of
temperature was reported to be negative on the supercritical CO2
extraction of phenolic compounds at low pressures between 10
and 15 MPa [19,46] and beyond this pressure the effect of tem-
perature becomes positive. This pressure is called cross-over
pressure and is explained by the solubility being controlled by a
balance between the solvent density and the change in the solute
vapor pressure with increasing temperature [47]. The cross-over
effect was not seen in this study because the extraction pressures
60 İ.H. Adil et al. / J. of Supercritical Fluids 43 (2007) 55–63
were beyond the possible cross-over pressure. It was reported
that an increase was observed in the recovery of phenolic com-
pounds from grape seeds with near critical CO2 with an increase
in temperature from 35 to 55 ◦ C with 10% methanol [14]. In this
study, above 50o C, TPC and AE of the extracts decreased with
further increase in temperature (Fig. 1). The optimum tempera-
tures were found to be between 55.7–58.4 and 50.9–52.3 ◦ C for
subcritical (CO2 + ethanol) extraction of polyphenols from apple
and peach pomaces, respectively (Table 4). This was possibly
due to the degradation of polyphenols at high temperatures and
it was consistent with the findings of Alonso-Salces et al. [12]
who observed a sharp decrease in the recovery of apple polyphe-
nols especially catechins above 60 ◦ C during pressurized liquid
extraction.
Fig. 2 shows that ethanol concentration increased TPC
and AE of the extracts from peach pomace by increasing
the solvating power of CO2 . Similar response surfaces (not
shown here) were obtained for extracts from apple pomace,
as well [43]. Solubilities of quercetin, catechin and epicate-
chin increase with increasing ethanol concentration from 5 to
30% [21–23]. The solubility increase of polyphenols in CO2
with the amount of ethanol added depends on the interac-
tions between the solute and the co-solvent. The solubility of
quercetin in (CO2 + ethanol) increases with ethanol concentra-
tion due to increased phenol-alcohol interactions [21]. Although
catechin presents a lower melting point than its isomer epicate-
chin, at 9 MPa and 40 ◦ C, epicatechin have higher solubility in
(CO2 + ethanol) than catechin since its polar nature provide more
hydrogen-bonding or dipole–dipole interactions with ethanol
than catechin [23]. Therefore, optimum ethanol concentrations
for subcritical (CO2 + ethanol) extraction of polyphenols from
both apple and peach pomaces were found to be 20% (Table 4)
which was the upper level of the experimental design.
The TPC and AE of the extracts increased with extraction
time and likely to remain constant close to 40 min (Figs. 2). This
was expected since the maximum extraction time was selected
to be 40 min according to the preliminary experiments to assure
that TPC of the extracts increase significantly with extraction
time at all extraction conditions. Moreover, it was found that
exhaustive extractions of phenolic compounds from olive leaves
were obtained after 140 min at 33.4 MPa, 100 ◦ C, using 10%
methanol during supercritical CO2 extraction [18]. The optimum
times for subcritical (CO2 + ethanol) extraction of polyphe-
nols from both apple and peach pomaces were found to be
40 min (Table 4) which was the upper level of the experimental
design.
Table 5
TPC and AE of the extracts obtained by ethanol and subcritical (CO2 + ethanol) extraction at optimum conditions
Apple pomace
TPCa
Ethanol extraction 1.71
Subcritical (CO2 + ethanol) extraction 0.47
a Total phenolic content (mg gae/g sample).
b Antiradical efficiency (mg DPPH• /g sample).
c TPC/AE (mg DPPH• /mg gae).
Apple and peach contain other antioxidative compounds,
ascorbic acid and carotenoids that might contribute to TPC
and AE of the extracts. Folin-Ciocalteu reagent interacts with
other different reducing nonphenolic substances and leads to
overestimation of TPC. In fact, for apple purees and juices,
the contribution of the interfering substances was reported to
be between 31 and 48%. Among the interfering substances
ascorbic acid has the major contribution and a correction in
Folin-Ciocalteu assay is required [48]. However, ascorbic acid
is not considerably soluble in supercritical CO2 or ethanol
and there are no investigations available in literature related
with supercritical CO2 extraction of ascorbic acid from natu-
ral materials. Ascorbic acid is heat sensitive [49]. In addition,
a statistically significant decrease in ascorbic acid was reported
in freeze dried berries [50]. The already low ascorbic acid con-
tents [50] of apple and peach were probably decreased during
juice production (especially during heat treatment of peach mash
before pressing) and freeze drying of both pomaces prior to
extraction. Therefore, Folin-Ciocalteu assay was used withouth
ascorbic acid correction in this study. Furthermore, the contri-
bution of ascorbic acid to antioxidant activity was reported to be
negligible [51], as low as 0.4% and 0.76% in apple and peach,
respectively [25]. On the other hand it is known that carotenoids
(especially ␤-carotene, [52]) are soluble in supercritical CO2 .
Recently, ␤-carotene was extracted from apricot pomace at opti-
mum extraction conditions of 31.1 MPa, 69 ◦ C with addition
of 27.4% ethanol [53]. Apple and peach contain majorly ␤-
carotene but its concentrations in apple and peach are almost
85 and 25 times lower than that of apricot, respectively [54].
Furthermore, the concentration of carotenoids (␤-carotene and
␤-cryptoxantin) is much less (level of ␮g/kg) than the amount of
polyphenols (level of mg/kg) in peach and their contribution to
antioxidant activity is not significant [51]. There was a signifi-
cant relationship (p ≤ 0.01) between TPC and AE of the extracts
with high correlations for subcritical (CO2 + ethanol) extraction
of polyphenols from apple and peach pomaces. The correlation
coefficients (r), 0.78 for apple and 0.92 for peach, prove the
validity of above discussion about the insignificant contribution
of nonphenolic antioxidants in apple and peach pomaces to TPC
and antioxidant assays.
Table 5 gives TPC and AE of the extracts at optimum
extraction conditions. TPC and AE of the extracts from apple
pomace were higher than those from peach pomace. Although
solute–solute interactions might vary the individual solubil-
ity in multicomponent systems, an explanation might be made
based on the limited solubility data of polyphenols in subcritical
Peach pomace
AEb AE/TPCc TPCa AEb AE/TPCc
9.3 5.44 0.81 6.21 7.67
3.30 7.02 0.26 1.5 5.77
 İ.H. Adil et al. / J. of Supercritical Fluids 43 (2007) 55–63
(CO2 + ethanol) that are available in literature. Hydroxycin-
namic acids are soluble in supercritical CO2 (8.5–50 MPa,
30–40 ◦ C), ferulic acid is the most soluble and caffeic acid
is the least soluble one, where p-coumeric acid has solubil-
ity between the two [19]. Quercetin, catechin and epicatechin
are soluble in supercritical or subcritical (CO2 + ethanol) (up to
30%). Above 100 MPa and at 40 ◦ C, epicatechin is more solu-
ble than quercetin, and quercetin is more soluble than catechin
[21–23]. In apple and peach, quercetin present as quercetin gly-
cosides [12,27–29,31,32]. Since the associated group is a moiety
of sugar, the solubilities of these glycosides might not be as high
as solubility of quercetin in subcritical (CO2 + ethanol) or might
not behave the same way as quercetin. Although the concentra-
tion of quercetin glycosides in apple is higher [12,27,28] than
the concentration of those in peach [32], their solubility behavior
was not included in this discussion. In apple 95% of flavan-3-ols
is (−)epicatechin and 5% is (+)catechin [29]. However, in peach
the concentration of epicatechin is less than the concentration of
catechin [32]. Also, the concentrations of ferulic and p-coumeric
acids in peach are less than the concentrations of those in apple
[26]. Furthermore, anthocyanins are slightly soluble in subcrit-
ical (CO2 + ethanol) [43]. Therefore, low TPC and AE of the
extracts from peach pomace could be explained by low concen-
trations of more soluble hydroxycinnamic acids, epicatechin and
presence of anthocyanins in peach.
Solvent extraction was performed to compare TPC and AE of
the extracts obtained by solvent and subcritical (CO2 + ethanol)
extractions. Although methanol is the best solvent for polyphe-
nol extraction [18,55,56] considering the possible future
application of the extracted polyphenols in food products,
ethanol was selected as a solvent in this study due to its
lower toxicity. A low solute to solvent ratio (0.05 mg/ml) was
used to have a high yield in ethanol extraction. As shown
in Table 5, although TPC and AE of the extracts from apple
pomace obtained by subcritical (CO2 + ethanol) extraction were
lower than those obtained by ethanol extraction, AE/TPC that
is antioxidant efficiency per mg gallic acid equiv. of polyphe-
nols was high. This shows that less but more active polyphenols
were selectively extracted by subcritical (CO2 + ethanol) extrac-
tion compared to ethanol extraction. However, TPC, AE and
AE/TPC of the extracts from peach pomace obtained by subcrit-
ical (CO2 + ethanol) extraction were lower than those obtained
by ethanol extraction. This might be due to the low solubility
of anthocyanins [43] in subcritical (CO2 + ethanol) which are
among the polyphenols present in peach that are majorly differ-
ent from apple. Therefore, subcritical (CO2 + ethanol) extraction
is a feasible method to extract polyphenols from apple and peach
pomaces, especially from apple pomace.
4. Conclusions
Pressure, temperature, ethanol concentration and extraction
time affected the extraction of polyphenols from apple and
peach pomaces significantly in the ranges between 20 and
60 MPa, 40–60 ◦ C, 14–20 wt.%, and 10–40 min of extraction
time. The optimum pressure and temperature were 54.6–57 MPa
and 55.7–58.4 ◦ C for apple pomace, and 50.6–51 MPa and
61
50.9–52.3 ◦ C for peach pomace. The optimum ethanol con-
centration and extraction time were 20% ethanol and 40 min
for both pomaces. TPC and AE of the extracts at these condi-
tions were predicted to be 0.47 mg gallic acid equiv./g sample
and 3.30 mg DPPH• /mg sample for apple pomace, and 0.26 mg
gallic acid equiv./g sample and 1.5 mg DPPH• /mg sample for
peach pomace. Low TPC and AE of the extracts from peach
pomace could be contributed to low concentrations of more
soluble hydroxycinnamic acids (ferulic and p-coumeric acids),
epicatechin and presence of anthocyanins in peach. AE/TPC
of the extracts from apple pomace obtained by subcritical
(CO2 + ethanol) was higher than the extracts obtained by ethanol
extraction indicating that less but more active polyphenols were
selectively extracted by subcritical (CO2 + ethanol) extraction
compared to ethanol extraction. Identification of the polyphe-
nols in the extracts is under study in our laboratory in order to
explain the extraction mechanism better, in the future. Furher-
more, determination of antioxidant activity of the extracts by
measuring the lipid oxidation (rather than detecting the scaveng-
ing of DPPH• radical) in model food systems is also a current
project of our group.
Acknowledgements
This work was supported by the Middle East Technical
University (BAP-2002-03-14-05) and The Scientific and Tech-
nological Research Council of Turkey (TOVAG-105O009).
Supercritical carbon dioxide extractions were performed in the
Central Laboratory (R&D Training Center) of the Middle East
Technical University.
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