Separation and Purification Technology 162 (2016) 68–76

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Effect of solvent, time and temperature on the extraction of phenolic

compounds and antioxidant capacity of peach (Prunus persica L.) fruit
Abderrahmane Mokrani ⇑, Khodir Madani
Laboratoire de Biomathématiques, Biophysique, Biochimie et Scientométrie (L3BS), Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000 Bejaia, Algeria

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the present study is to investigate the effects of solvent type (ethanol, methanol, acetone and
Received 4 November 2015 water), acetone concentration (20–100%, v/v), solvent acidity (0–2 N), time (30–450 min) and tempera-
Received in revised form 25 January 2016 ture (25–70 °C) on the extraction of total phenolic compounds (TPC), total flavonoid compounds (TFC)
Accepted 28 January 2016
and on the antioxidant capacity: 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity (DPPH-RSA)
Available online 29 January 2016
and ferric reducing power (FRP) of peach fruit (Prunus persica L.) using single factor experiments
approach. All the studied extracting conditions showed significant effect (p < 0.05) on TPC, TFC, DPPH-
Keywords:
RSA and FRP. On the basis of TPC and antioxidant activity parameters, the best extraction conditions were
Peach (Prunus persica L.)
Phenolic compounds extraction
60% acetone without acidification for 180 min at 25 °C. Based on these optimized conditions, high content
Antioxidant activity of TPC, DPPH-RSA and FRP of peach extracts were obtained with values of 363 GAE/100 g, 48% percentage
Solvent of inhibition and 317 AAE/100 g, respectively. Good, positive or negative, Pearson correlation coefficients
Time were found between TPC, TFC DPPH-RSA and FRP of peach extracts, especially under the influence of sol-
Temperature vent type, solvent concentration and acidity extraction conditions.
Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction
Polyphenols represent the major part of antioxidants present in
the diet. Their total dietary ingestion could be more than 1 g/day,
which is much higher than that of all other classes of phytochem-
icals and known dietary antioxidants [1]. As antioxidants, polyphe-
nols are reducing agents, and jointly with other nutritional
reducing agents, such as vitamins (C and E) and carotenoids, may
protect cell constituents against oxidative damage limiting there-
fore the risk of various degenerative diseases associated to oxida-
tive stress. Several studies have shown that polyphenols limit the
development of cancers, cardiovascular and neurodegenerative
diseases, diabetes and osteoporosis [2,3].
Fruits and beverages are the main sources of polyphenols. Pea-
ches are a well-liked summer fruit and there has been a growing
attention in their nutritional value [4]. Peach (Prunus persica L.)
belongs to the Rosaceae family and is associated to stone fruits
such as plums, cherries and almonds. The peach is a native from
China where it means long life. It can be eaten either fresh or
canned and its flavor can be incorporated into many beverages
[5]. Peaches and nectarines production has an important place in
the world (21.6 million tons in 2013) with a cultivated area of
⇑ Corresponding author.
E-mail address: damane80@yahoo.fr (A. Mokrani).
around 1.5 million ha. China was the first country producing
peaches and nectarines in the world (more than 11.9 million tons
in 2013) followed by Italy (1.4 million tons) and Spain (1.3 mil-
lion tons) [6].
Phenolic compounds represent the main antioxidant
phytochemicals present in peaches [7]. However, vitamin C and
carotenoids also contribute to their antioxidant capacity [8–10].
Peach phenolics have been shown to display several biological
activities such as antioxidant activity [11,12], anti-allergic and
anti-inflammatory activities [13], antibacterial activity [14], hep-
atoprotective activity [15], nephroprotective activity [16] antipro-
liferative [17], chemopreventive and anticancer activities [18,19].
Extraction of phenolic compounds from plant matrix is influ-
enced by several factors such as their chemical nature, the extrac-
tion method used, the size of the particles, time and storage
conditions and the presence of interfering substances [20]. Pheno-
lic compounds may have different hydroxyl groups which can be
conjugated to sugars, acids or alkyl groups. Consequently, the
polarities of phenolic compounds greatly vary and it is hard to
develop a single method for efficient extraction of all phenolic
compounds. Therefore, optimization of the extraction process is
fundamental for an accurate assessment of phenolic compounds
from different food matrices [21].
Although the phenolic profile of the peach published by some
authors [7,22,23], there are no studies on the effect of extracting

http://dx.doi.org/10.1016/j.seppur.2016.01.043
1383-5866/Ó 2016 Elsevier B.V. All rights reserved.
 A. Mokrani, K. Madani / Separation and Purification Technology 162 (2016) 68–76
parameters on the recovery of phenolic compounds from peach
fruit. To the best of our knowledge, no data was reported about
the effect of extracting parameters on the survey of phenolics from
peach.
The aim of the present study is to investigate the effects of sol-
vent type (ethanol, methanol, acetone and water), acetone concen-
tration (20%, 40%, 60%, 80% and 100%, v/v), solvent acidity (0, 0.1,
0.5, 1, 1.5 and 2 N), time (30, 120, 180, 270, 360 and 450 min)
and temperature (25, 37.5, 50, 60 and 70 °C) on the extraction of
antioxidant phenolic compounds (total phenolic compounds; TPC
and total flavonoid compounds; TFC) and on the antioxidant capac-
ity (DPPH radical-scavenging activity; DPPH-RSA and ferric reduc-
ing power; FRP) of peach (Prunus persica L.) fruit using single factor
experiments approach.
2. Materials and methods
2.1. Chemicals
Folin–Ciocalteu reagent was provided from Biochem, Che-
mopharma (Montreal, Quebec). Sodium carbonate (Na2CO3),
disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen
phosphate (NaH2PO4), acetone, ethanol and methanol were
obtained from Prolabo (made in CE). Potassium ferricyanide
(C6N6FeK3), ferric chloride (FeCl3
6H2O), trichloroacetic acid,
gallic acid and quercetin from Biochem-chemopharma (UK).
1,1-diphenyl-2-picrylhydrazyl (DPPH°) was obtained from
Sigma–Aldrich (Steinheim, Germany).
2.2. Plant material
Peach fruits used in this study were purchased from local mar-
ket at Bejaia city (Algeria) at July 2011. After transferring at the
laboratory, some tests were done on fruits in order to determine
their physicochemical characteristics. Peach fruits presented an
average weight of 93.4 g, a height of 58.6 mm and a width of
52.3 mm, pH of 3.7, titratable acidity of 0.57, dry matter of 15.26
and total soluble solids content (Brix) of 13.04. The fruits were
washed, dried, pitted and then homogenized in a blender before
being freeze-dried in a lyophilizer (Alpha1-4 LDplus, Christ, Oster-
ode, Germany). The freeze-dried peach samples obtained were
kept at 4 °C prior to analysis.
2.3. Plant material extraction
500 mg of peach fruit dried powder were weighted in a glass
vial and extracted with 15 mL of extracting solvent. The extraction
process was done in a water bath shaker at different temperatures
and times. The mixture was centrifuged at 5000 rpm for 20 min
(NF 200, Nuve, Turkey) and then filtered through a Watman filter
paper. The extraction process was carried out in duplicate. The
obtained filtered extracts were used for determination of total phe-
nolic compounds (TPC), total flavonoid compounds (TFC) and
antioxidant activities: DPPH° radical-scavenging activity (DPPH-
RSA) and ferric reducing power (FRP) measurements.
2.4. Experimental design
In the present study, single factor experiments was used to
determine the optimum conditions for extracting antioxidant phe-
nolic compounds from peach fruits. A total of five parameters
namely extraction solvent (ethanol, methanol, acetone and water),
acetone concentration (20–100%; v/v), solvent acidity (0–2 N),
extraction time (30–450 min) and extraction temperature (25–
70 °C) were studied on which one parameter was varied at a time
69
while the other parameters were fixed. The optimal extracting con-
ditions were selected on the basis of the TPC, DPPH-RSA and FRP
measurements.
2.4.1. Solvent extraction
By setting extraction time with extraction temperature to
180 min and 25 °C, respectively, peach samples were extracted
using different types of solvents: 60% (v/v) acetone, 60% ethanol,
60% methanol and water.
2.4.2. Solvent concentration
Using the best extraction solvent determined previously, sam-
ples were extracted with different concentrations of solvent (20,
40, 60, 80 and 100%, v/v) fixing extraction time and extraction tem-
perature at 180 min and 25 °C, respectively.
2.4.3. Solvent acidity
Peach samples were extracted using the best solvent type and
the best solvent concentration. The extraction was repeated by
varying the concentrations of HCl acidity (0.1, 0.5, 1, 1.5 and 2 N)
mixed with the best extracting solvent at the ratio of 85% of solvent
and 15% of HCl (85:15, v/v). The extraction time and temperature
were fixed at 180 min and 25 °C, respectively.
2.4.4. Extraction time
Peach samples were extracted using the best solvent concentra-
tion and the best solvent acidity determined previously. Extraction
time was varied from 30 to 450 min (30, 120, 180, 270, 360 and
450 min). Extraction temperature was fixed at 25 °C.
2.4.5. Extraction temperature
Using the best parameters determined above (solvent type, sol-
vent concentration, solvent acidity and extraction time), peach
samples were extracted at different temperatures ranging from
25 to 70 °C (25, 37.5, 50, 60 and 70 °C).
2.5. Total phenolic compound (TPC) determination
The amount of TPC in peach extracts was determined according
to the Folin–Ciocalteu method [24]. Samples (200 lL) were intro-
duced into test tubes in which 1.0 mL of Folin–Ciocalteu’s reagent
(previously diluted 10 with water) and 0.8 mL of sodium carbon-
ate (7.5%, w/v) were added. The tubes were mixed and allowed to
stand in darkness at room temperature for 30 min. Absorption at
765 nm against a blank was measured using an UV/VIS Spec-
trophotometer (Shimadzu UV mini1240, Suzhou Jiangsu, China).
The total phenolic content was expressed as mg gallic acid equiv-
alents per 100 g of dry weight (mg GAE/100 g DW) using a calibra-
tion curve. All measurements were carried out in triplicate.
2.6. Total flavonoid compound (TFC) determination
Total flavonoid content was estimated according to the proce-
dure of Santas et al. [25] based on the aluminum chloride complex
formation. To 1 mL of peach extract, 1 mL of 2% (w/v) AlCl3
methanolic solution was added. The mixture was allowed to react
for 10 min at room temperature and the absorbance was read at
410 nm against a blank. Total flavonoid content was calculated as
quercetin from a calibration curve and results were expressed as
mg quercetin equivalent per 100 g of dry weight (mg QE/100 g
DW). All measurements were done in triplicate.
2.7. DPPH° radical-scavenging activity (DPPH-RSA)
In the 1,1-diphenyl-2-picrylhydrazyl (DPPH°) assay, antioxidants
were capable to reduce the stable radical DPPH° to the yellow
70 A. Mokrani, K. Madani / Separation and Purification Technology 162 (2016) 68–76
colored diphenylpicrylhydrazine (DPPH-H). The test is based on
the reduction of an alcoholic solution of DPPH° in the presence of
a hydrogen donating antioxidant due to the formation of the
non-radical form DPPH-H [26]. The DPPH° radical-scavenging
activity of the peach fruit extracts was estimated as described by
Blois [27]. Briefly, 0.1 mL of each sample extract was mixed with
0.9 mL of 0.04 mg/mL methanolic solution of DPPH°. The mixtures
were left for 20 min at room temperature and its absorbance then
measured at 517 nm against a blank. All measurements were car-
ried out in triplicate. The percentage of DPPH radical-scavenging
activity was calculated using the following equation:
%DPPH radical-scavenging ¼ ½ðAc  As Þ  100
where Ac was the absorbance of the negative control (contained
extraction solvent instead of the sample), and As was the absor-
bance of the samples.
2.8. Ferric reducing power (FRP)
The ferric reducing power of the peach fruit extracts was per-
formed by using the potassium ferricyanide–ferric chloride
method [28]. Substances, with reduction potential, react with
potassium ferricyanide (Fe3+) to form potassium ferrocyanide
(Fe2+), which subsequently reacts with ferric chloride to form ferric
ferrous complex who has an absorption maximum at 700 nm [29].
1 mL of extract was mixed with 2.5 mL phosphate buffer (0.2 M, pH
6.6) and 2.5 mL of 1% (w/v) potassium ferricyanide. The mixtures
were incubated at 50 °C for 20 min, after that 2.5 mL of 10% (w/
v) trichloroacetic acid were added. 2.5 mL of the mixture were
taken and mixed with 2.5 mL of distiller water and 0.5 mL of
0.1% FeCl3 (w/v) and thoroughly mixed. The intensity of blue green
color was measured at 700 nm against a blank. Ferric reducing
activity was determined as mg ascorbic acid equivalents per
100 g of dry weight (mg AAE/100 g DW). The values are presented
as the means of triplicate analyses.
2.9. Statistical analysis
The results are presented as the means ± standard deviation
(SD) of duplicate extractions and three replicate assays. Significant
differences were determined by one-way analysis of variance
(ANOVA) completed by Tukey’s test. The Pearson correlation test
was used to compare the different values of assays obtained in
our extracts. Significant levels were defined at p < 0.05, p < 0.01
and p < 0.001. All these analyses was performed by GraphPad
Prism (version 5).
3. Results and discussion
3.1. Solvent extraction
Historically, recovery of phenols from fruits by liquid extraction
using hot or cold solvents was common. Suitable solvents for this
use are aqueous mixtures with ethanol, methanol, acetone and
dimethylformamide [30].
In this study, diverse solvents, in mixtures with water, were
used to extract antioxidant phenolic compounds from freeze-
dried peaches. Results showed that all the peach extracts con-
tained phenolic compounds and the content of these compounds
varied according to each solvent used for extraction (Fig. 1). Among
the tested solvents, 60% acetone was significantly (p < 0.05) the
most efficient for extracting TPC from peach (363 mg GAE/100 g),
approximately more than 2 times the one of water (182 mg
GAE/100 g) and 60% ethanol (178 GAE/100 g) which represent
the lowest value. However, 60% ethanol was significantly the best
solvent for extracting TFC from peach with yield of 57 mg
QE/100 g, followed by 60% methanol, 60% acetone and water with
values of 47 mg QE/100 g, 38 mg QE/100 g and 17 mg QE/100 g,
respectively.
Generally, acetone is the best solvent for extracting proantho-
cyanidins and tannins; ethanol efficiently extracts flavonoids and
their glycosides, catechols and tannins; whereas phenolic acids
and catechin were better extracted with methanol. These facts
are in agreement with polarity of the solvent used for the extrac-
tion and solubility of phenolics in them since the polarity of ace-
tone, ethanol and methanol is 0.355, 0.654 and 0.762,
respectively. [31]. Therefore, there is no single solvent able to
extract all of the classes of phenolic compounds from a sample,
simultaneously.
Acetone is a more efficient solvent for extracting phenolic com-
pounds with a high molecular weight such as condensed tannins. It
is strongly believed that the higher the molecular weight of the sol-
vent, the lower the polarity which enable other substances of
about the same molecular weight to be easily extracted. This can
be associated to ‘‘like dissolves like” or ‘‘polarity versus polarity”
principle as both acetone and tannins are of high molecular weight.
Acetone has the lowest polarity but contains the highest total phe-
nolic compounds value [32,33]. This would explain why acetone
was found to be more efficient for extracting phenolics from peach
since flavanols are the major phenolic compounds class present in
this fruit [7,23].
Acetone has been also demonstrated to be more effective than
other organic solvents for extracting antioxidant phenolics from
different raw materials such as berries and apples [34], buckwheat
[35], star fruits [36], strawberries [37], onions [38], barley seeds
[39], beach peas [40], eggplant byproducts [41], pistachio byprod-
ucts [42], fenugreek [43], fig [44] and soybean [45].
The antioxidant activity of the peach phenolic extracts was
determined by two methods, namely the 1,1-diphenyl-2-
picrylhydrazyl radical-scavenging activity (DPPH-RSA) and the fer-
ric reducing power (FRP) assays, which have been widely used for
the assessment of antioxidant capacity of various plant extracts
and natural products.
Determination of scavenging stable DPPH° free radical is a very
quick way to evaluate the antioxidant activity of the extracts in a
very short time. With this method, it was possible to assess the
antiradical ability of an antioxidant by measuring the reduce in
the absorbance of DPPH° at 517 nm. As a result of a color change
from violet to yellow, the absorbance diminishes when the DPPH
° radical was scavenged by an antioxidant through hydrogen dona-
tion to form a stable DPPH-H molecule. In the radical form this
molecule had an absorbance at 515 nm which disappeared by
receiving an electron or hydrogen from an antioxidant to become
a stable diamagnetic molecule [46].
Acetone was also observed to be the solvent presenting the
highest antioxidant activity (Fig. 1). The percentage of the DPPH°
radical-scavenging activity (DPPH-RSA) of acetone was 48%, more
four times than water (11%), which represents the lowest value.
Intermediate DPPH-RSA percentages were found for ethanol
(37%) and methanol (40%), whose values were not significantly dif-
ferent at p < 0.05.
Ferric reducing power (FRP) of peach extracts was measured by
the direct reduction of Fe3+(CN–)6 to Fe2+(CN–)6 and was deter-
mined by measuring absorbance of the resulting Perl’s Prussian
blue complex formed after the addition of ferric ions (Fe3+). In this
method, the yellow color of the test solution changes to various
shades of green and blue depending on the content of reductants
(antioxidants) in the sample. These reducing agents reduce the
Fe3+/ferricyanide complex to the ferrous form. Thus, Fe2+ can be
monitored by measuring the formation of Perl’s Prussian blue at
700 nm [47,48].
 A. Mokrani, K. Madani / Separation and Purification Technology 162 (2016) 68–76
Fig. 1. Effect of solvent type on the extraction of TPC (A), TFC (B), DPPH-RSA (C) and FRP (D) from peach (n = 2)⁄. Values are presented as means ± SD of six measurements.
Values marked with the different lower case letters (a–d) are significantly (p < 0.05) different. ⁄Replication of extractions.
The same thing as DPPH-RSA, 60% acetone extract was observed
to have significantly the highest FRP (317 mg AAE/100 g), more
than five times the one of water (60 mg AAE/100 g), which repre-
sents the lowest value. 60% ethanol and 60% methanol showed
no significant differences with intermediate values of 265 and
260 mg AAE/100 g, respectively.
Our results are in accordance of those of González-Montelongo
et al. [49] who shown that 50% acetone was more efficient for
extracting banana peel phenolics and therefore, produced extracts
with higher antioxidant activity. Thus, by compromising between
the yield of TPC and antioxidant activities (DPPH-RAS and FRP),
acetone was chosen as the best solvent to optimize the following
extraction conditions.
Solubility of phenolic compounds depends widely on the nature
of the solvent (polarity) used, their degree of polymerization, as
well as their interaction with other food components and the for-
mation of insoluble complexes [20]. Differences observed in the
antioxidant activity (DPPH-RSA and FRP) of peach extracts could
be due the variation of the quantity and quality of phenolic com-
pounds present in the different extracts. Since acetone was the sol-
vent presenting the highest yield of TPC and antioxidant activities
(DPPH-RSA and FRP) simultaneously, it is believed that phenolics
contributing efficiently to the total antioxidant activity of peach
extract are phenolic compounds with higher molecular weight
and lower polarity, according to ‘‘like dissolves like” principle.
3.2. Solvent concentration
Phenolic compounds of peach were extracted using different
concentrations of acetone ranging from 20% to 100% (v/v). Results
showed that all acetone concentrations were capable of extracting
phenolics with significant differences (p < 0.05). The highest yield
of TPC was recorded for 60% acetone (363 mg GAE/100 g), more
seven times the one of pure acetone (49 mg GAE/100 g) which
71
represents the lowest value (Fig. 2). Other concentrations (40%,
80% and 20% acetone) showed intermediate values of 328 mg
GAE/100 g, 305 mg GAE/100 g and 287 mg GAE/100 g, respectively.
However, 20% and 100% acetone were more efficient for the extrac-
tion of TFC from peach with values of 76 mg QE/100 g and 73 mg
QE/100 g, respectively. Intermediate values of 63 and 56 mg
QE/100 g were obtained for 40% and 80% acetone, respectively.
The lowest yield of TFC (38 mg QE/100 g) was obtained using
60% acetone.
Acetone concentration had also significant effect (p < 0.05) on
DPPH-RSA and FRP of peach extracts (Fig. 2). The highest values
were obtained at 60% acetone with 48% and 317 mg AAE/100 g
for DPPH-RSA and FRP, respectively. Thus, a concentration of 60%
acetone was chosen as best acetone gradient for the following
steps.
The use of organic solvents, in mixtures with water, contributes
to the creation of a moderately polar medium that enhance the
extraction of polyphenols. However, the use of water as single sol-
vent provides an extract with a high content of impurities (organic
acids, sugars, soluble proteins), that could interfere in the phenolic
identification and quantification [50].
Similar to the present study, acetone:water mixtures (40–70%
v/v) have been reported to be one of the most effective solvents
for extracting phenolics from different natural sources. Tan et al.
[31] found that the high TPC extraction yield from henna (Lawsonia
inermis) stems was obtained using 40% acetone. Al-Farsi and Lee
[51] found that 50% acetone was the most efficient solvent for phe-
nolic extraction from date seeds. Meneses et al. [52] demonstrated
that 60% acetone was the best solvent for extracting antioxidant
phenolic compounds from brewer’s spent grains. Acetone:water
mixture is capable to break polyphenol–protein complexes. This
fact would explain the high efficiency of this solvent to extract
phenolic compounds. Downey and Hanlin [53] demonstrated that
mixtures of acetone ranging from 50% to 70% are more effective
72 A. Mokrani, K. Madani / Separation and Purification Technology 162 (2016) 68–76
Fig. 2. Effect of acetone concentration on the extraction of TPC (A), TFC (B), DPPH-RSA (C) and FRP (D) from peach (n = 2)⁄. Values are presented as means ± SD of six
measurements. Values marked with the different lower case letters (a–d) are significantly (p < 0.05) different. ⁄Replication of extractions.
in extracting condensed tannins from grape skin. Kallithraka et al.
[54] found that 70% acetone was the best solvent for the extraction
of grape seed phenolics especially proanthcynidins.
3.3. Solvent acidity
The effect of acid concentration (0–2 N) on the extraction of
TPC, TFC and antioxidant activity of peach was investigated.
Results showed that the yield of TPC, TFC and DPPH-RSA increased
gradually when the acid concentration increased. However, FRP of
peach extracts significantly decreased when acid was added to sol-
vent (Fig. 3).
The addition of acid in the extraction solvent could act by rising
the stability of the phenolics (e.g. anthocyanins) and increasing
their dissolution. These compounds being initially part of polymers
or linked to other cell wall components, via a hydrolysis mecha-
nism, improve the disintegration of cell walls and therefore
facilitate solubilization and diffusion of phenolic compounds from
the plant material [50]. However, strong acid concentration may
cause partial hydrolysis and degradation of anthocyanin [55,56].
This might explain the decrease in the FRP of peach extracts by
increasing acid concentration. Thus, neutral solvent (60% acetone
without acidification) was chosen for the following steps.
3.4. Extraction time
Extraction time is essential in economizing energy and cost of
the extraction process. Results showed that TPC, DPPH-RSA and
FRP increased when extraction time was increased from 30 min
to 180 min (Fig. 4). This duration allowed extraction of 363 mg
GAE/100 of TPC from peach, which corresponded to respective
antioxidant activities of 48% percentage inhibition for RSA and
317 mg AAE/100 g for FRP. After 180 min, increasing the extraction
time did not improve the recoveries, except for TFC. This could be
explained by the Fick’s second law of diffusion witch predicts a
final equilibrium between the concentrations of solute in the solid
matrix and in the bulk solution after a certain time. Therefore, a
longer time is not required to extract more phenolics [57]. Our
results are in accordance with those of Yap et al. [58] and Chan
et al. [59] who demonstrated that 180 min was the best time for
extracting phenolics from star fruit (Averrhoa carambola L.)
residues and limau purut (Citrus hystrix) peels, respectively.
Furthermore, longer extraction times increase the possibility of
phenolics oxidation [60]. This oxidation might be prevented by
addition of reducing agents to the solvent system [61]. Naczk
et al. [62] reported that the optimum extraction time and temper-
ature to extract phenolics from canola meal is 2 min (2  1 min) at
room temperature.
Taking into account these facts, an extraction time of 180 min
was selected as the best extraction time for extracting phenolic
compounds and antioxidant activities of peach.
3.5. Extraction temperature
Applying the best solvent (60% acetone) and the best extraction
time (180 min), the uses of different extraction temperatures have
significantly (p < 0.05) affected the extraction of TPC, TFC and
DPPH-RSA of peach (Fig. 5). The results showed that TPC yield
decreased when the temperature increased from 25 °C to 70 °C
with values of 363 mg GAE/100 g and 284 mg GAE/100 g, respec-
tively. However, increasing temperature extraction from 25 °C to
60 °C proportionally increased the yield of TFC from 38 mg
QE/100 g to 81 mg QE/100 g, respectively. Applying >60 °C of heat
during the extraction has significantly reduced the TFC.
Heating might soften the plant tissue and weaken the phenol–
protein and phenol–polysaccharide interactions, therefore more
polyphenols would migrate into the solvent [63]. This might
explain the increase in TFC yield when increasing temperature
 A. Mokrani, K. Madani / Separation and Purification Technology 162 (2016) 68–76 73

Fig. 3. Effect of acetone acidification on the extraction of TPC (A), TFC (B), DPPH-RSA (C) and FRP (D) from peach (n = 2)⁄. Values are presented as means ± SD of six
measurements. Values marked with the different lower case letters (a–d) are significantly (p < 0.05) different. ⁄Replication of extractions.

Fig. 4. Effect of time on the extraction of TPC (A), TFC (B), DPPH-RSA (C) and FRP (D) from peach (n = 2)⁄. Values are presented as means ± SD of six measurements. Values
marked with the different lower case letters (a–d) are significantly (p < 0.05) different. ⁄Replication extractions.

extraction since flavonoid compounds are usually found as glyco- 48% and 39%, respectively. However, no significant difference was
sides [21]. found for the percentage of DPPH-RSA between 37.5 °C and
The DPPH-RSA was reduced when the extraction temperature 70 °C. Increasing temperature extraction from 25 °C to 70 °C had
increased from 25 °C to 37.5 °C with percentages inhibition of no significant effect on FRP of peach extracts.
74 A. Mokrani, K. Madani / Separation and Purification Technology 162 (2016) 68–76

Fig. 5. Effect of temperature on the extraction of TPC (A), TFC (B), DPPH-RSA (C) and FRP (D) from peach (n = 2)⁄. Values are presented as means ± SD of six measurements.
Values marked with the different lower case letters (a–d) are significantly (p < 0.05) different. ⁄Replication extractions.

Cacace and Mazza [64] reported that temperature affected the
extraction of anthocyanins and increasing the temperature over
30–35 °C resulted in the decomposition of anthocyanins. This
could be explained by a higher vulnerability of anthocyanins to
high temperature.
Several studies reported that heat improved the extraction effi-
ciency of phenolic compounds and enhance antioxidant activity of
phenolic extracts [65–68]. This was probably due to the increased
phenolic solubility, faster diffusion rate, better mass transfer and
extraction yield and reduced solvent viscosity and surface tension
[69]. However, it should be noted that increasing temperature
extraction above certain values may promoting possible concur-
rent degradation of phenolic compounds which were previously
mobilized at lower temperature or even the decomposition of
residual phenolics remaining in the plant matrix. Furthermore,
high temperature may enhance solvent loss through vaporization
and augment the extraction process cost from the industrialization
point of view [59]. Xu et al. [70] demonstrated that heat treatment
of peel citrus increases the free fraction of phenolic acids and
therefore increases the total antioxydant activity of extract. How-
ever, ester, glycoside, and ester bound fractions and total flavanone
glycosides were decreased.
Since the highest TPC value was extracted at the temperature of
25 °C with an extraction yield of 363 mg GAE/100 g, this tempera-
ture was chosen as the best temperature for extracting phenolic
compounds from peach fruit.
3.6. Pearson correlation analysis
In order to more appreciate the relationships between antioxi-
dant capacities and phenolic content of peach extracts, correlations
between assays under different extracting conditions were
analyzed.
Under the parameter of solvent type (Table 1), correlations
between TPC, TFC and antioxidant assays (DPPH-RSA and FRP)
were positively high (0.64 < r < 0.97, P < 0.001). We can suggest
that the hydrogen electron donating abilities of peach extracts
were directly proportional to the concentration of total phenolics.
Under the influence of solvent concentration (Table 1), TPC was
correlated positively with DPPH-RSA (r = 0.76) and FRP (r = 0.96)
assays at P < 0.001. However, TFC was correlated negatively with
DPPH-RSA and FRP assays with Pearson correlations coefficient of
0.82 and 0.68, respectively. It could be concluded that there
are other phenolic compounds other than flavonoids witch

Table 1
Pearson correlations coefficient between different assaysa under influence of extraction conditions (n = 2)b.

Solvent type Acetone concentration Acetone acidification Extraction time Extraction temperature
TFC RSA FRP TFC RSA FRP TFC RSA FRP TFC RSA FRP TFC RSA FRP
TPC 0.09ns 0.72*** 0.64*** 0.57*** 0.76*** 0.96*** 0.54*** 0.73*** 0.48** 0.14ns 0.58*** 0.01ns 0.41* 0.25ns 0.45*
TFC 0.68*** 0.76*** 0.82*** 0.68*** 0.88*** 0.88*** 0.21ns 0.27ns 0.27ns 0.09ns
RSA 0.97*** 0.82*** 0.85*** 0.07ns 0.18ns
a
TPC, total phenolic compounds; TFC, total flavonoid compounds; RSA, DPPH radical scavenging activity; FRP, ferric reducing power.
b
Replication of extraction.
ns
Not significant.
*
Significant at p < 0.05.
**
Significant at p < 0.01.
***
Significant at p < 0.001.
 A. Mokrani, K. Madani / Separation and Purification Technology 162 (2016) 68–76
contribute to the DPPH-RSA and FRP of peach extracts. Addition-
ally, a synergism of phenolics, with one another, and other com-
pounds with an antioxidant activity present in the extract may
contribute to the overall observed antioxidant capacity.
About the parameter of solvent acidity, TPC and TFC were
observed to be positive significantly (P < 0.001) correlated with
DPPH-RSA assay with Pearson correlation coefficients of 0.73 and
0.88, respectively (Table 1). However, they negatively correlated
with FRP assay with Pearson correlation coefficient of 0.48
(P < 0.01) for TPC and 0.88 (P < 0.001) for TFC. Previous studies
showed that antioxidant activity of phenolic compounds depends
widely on their structure. Therefore, we believe that solvent acidity
did not affect the capacity of peach phenolics to scavenge free rad-
icals. However, it might reduce their reducing capacity.
Concerning the influence of extraction time condition (Table 1),
only TPC was observed to be correlated positively with DPPH-RSA
assay (r = 0.58, P < 0.001). No significant correlations were found
between the other assays. From this correlation, we can believe
that peach phenolic compounds extracted at different times may
display different antioxidant capacities.
Under the influence of extraction temperature (Table 1), TPC
was found to be negatively correlated with FRP assay (r = 0.45,
P < 0.05). However, no significant correlations were found between
the other assays. It could be concluded that phenolic compounds
present in peach extracts had low stability at high temperatures.
4. Conclusion
In the present study, single factor experiments approach was
used to determine the optimization of the extraction process of
peach fruit phenolics, investigating some variables which such as
effect of solvent extraction (solvent type, solvent/water mixture
concentration and solvent acidity), extraction time and tempera-
ture. To the best of our knowledge, no data was reported about
the effect of extraction parameters on the recovery of phenolic
compounds from peach fruit.
Results showed that TPC, TFC, DPPH-RSA and FRP were signifi-
cantly affected by all the studied parameters. The optimal extrac-
tion conditions, selected by compromising between the rate of
total phenolic compounds (TPC) and their antioxidant activities
(DPPH-RSA and FRP), were 60% acetone without acidification for
180 min at room temperature. These conditions allowed an extrac-
tion yield of 363 mg GAE/100 g for TPC and an antioxidant activi-
ties values of 48% and 317 mg AAE/100 g for DPPH-RSA and FRP,
respectively. Good, positive or negative, Pearson correlation coeffi-
cients were found between TPC, TFC and DPPH-RSA and FRP of
peach extracts, especially under the influence of solvent type, sol-
vent concentration and solvent acidity. This study will provide
bases for future investigations on the optimization of the extrac-
tion of phenolic compounds from peach using other models such
as response surface methodology.
Since antioxidant compounds provide health benefits, peach
extracts could be of great interest for application in food and phar-
maceutical products. However, it is interesting to test other extrac-
tion methods such as microwave and ultrasound extractions,
ultrafiltration, flash distillation, supercritical fluid and subcritical
water extractions on extracting phenolic compounds from peach.
These alternative new technologies namely ‘‘green” techniques
use less solvent and energy and may increase the safety and the
quality of products.
Acknowledgements
The authors are grateful to the Algerian Ministry of Higher Edu-
cation and Scientific Research for the financial support. We also
75
thank Prof. Marc Mesnil of the STIM laboratory, University of Poi-
tiers for his providing English language help.
References
[1] A. Scalbert, I.T. Johnson, M. Saltmarsh, Polyphenols: antioxidants and beyond,
Am. J. Clin. Nutr. 81 (2005) 215–217.
[2] A. Scalbert, C. Manach, C. Morand, C. Remesy, L. Jimenez, Dietary polyphenols
and the prevention of diseases, Crit. Rev. Food Sci. Nutr. 45 (2005) 287–306.
[3] A. Scalbert, G. Williamson, Dietary intake and bioavailability of polyphenols, J.
Nutr. 130 (2000) 2073–2085.
[4] W. Abidi, S. Jimenez, M.A. Moreno, Y. Gogorcena, Evaluation of antioxidant
compounds and total sugar content in a nectarine [Prunus persica (L.) Batsch]
progeny, Int. J. Mol. Sci. 12 (2011) 6919–6935.
[5] B. Kanda, J. Boateng, L. Shackelford, S. Appiah, K.C.L.T. Walker, M. Verghese,
Effects of processed peaches (Prunus persica) in reducing azoxymethane (AOM)
induced Aberrant Crypt Foci (ACF) in fisher 344 male rats, Int. J. Cancer Res. 8
(2012) 1–14.
[6] FAOSTAT, FAOSTAT, 2015. <http://faostat3.fao.org/browse/Q/QC/E> (accessed
on 26.05.15).
[7] S. Chang, C. Tan, E.N. Frankel, D.M. Barrett, Low-density lipoprotein antioxidant
activity of phenolic compounds and polyphenol oxidase activity in selected
clingstone peach cultivars, J. Agric. Food Chem. 48 (2000) 147–151.
[8] C.M. Cantin, M.A. Moreno, Y. Gogorcena, Evaluation of the antioxidant
capacity, phenolic compounds, and vitamin C content of different peach and
nectarine [Prunus persica (L.) Batsch] breeding progenies, J. Agric. Food Chem.
57 (2009) 4586–4592.
[9] A.Z. Dalla Valle, I. Mignani, A. Spinardi, F. Galvano, S. Ciappellano, The
antioxidant profile of three different peaches cultivars (Prunus persica) and
their short-term effect on antioxidant status in human, Eur. Food Res. Technol.
225 (2006) 167–172.
[10] M.I. Gil, F.A. Tomás-Barberán, B. Hess-Pierce, A.A. Kader, Antioxidant
capacities, phenolic compounds, carotenoids, and vitamin C contents of
nectarine, peach, and plum cultivars from California, J. Agric. Food Chem. 50
(2002) 4976–4982.
[11] C. Li, M.H. Wang, Antioxidant activity of peach blossom extracts, J. Korean Soc.
Appl. Biol. Chem. (2011) 46–53.
[12] S.B. Rossato, C. Haas, M.d.C.B. Raseira, J.C.F. Moreira, J.Â.S. Zuanazzi,
Antioxidant potential of peels and fleshes of peaches from different
cultivars, J. Med. Food 12 (2009) 1119–1126.
[13] T.-Y. Shin, S.-B. Park, J.-S. Yoo, I.K. Kim, H.-S. Lee, T.K. Kwon, M.K. Kim, J.C. Kim,
S.-H. Kim, Anti-allergic inflammatory activity of the fruit of Prunus persica: role
of calcium and NF-jB, Food Chem. Toxicol. 48 (2010) 2797–2802.
[14] B.A. Cevallos-Casals, D. Byrne, W.R. Okie, L. Cisneros-Zevallos, Selecting new
peach and plum genotypes rich in phenolic compounds and enhanced
functional properties, Food Chem. 96 (2006) 273–280.
[15] C.K. Lee, K.K. Park, J.K. Hwang, S.K. Lee, W.Y. Chung, The extract of Prunus
persica flesh (PPFE) attenuates chemotherapy-induced hepatotoxicity in mice,
Phytother. Res. 22 (2008) 223–227.
[16] C.K. Lee, K.K. Park, J.K. Hwang, S.K. Lee, W.Y. Chung, Extract of Prunus persica
flesh (PPFE) improves chemotherapeutic efficacy and protects against
nephrotoxicity in cisplatin-treated mice, Phytother. Res. 23 (2009) 999–1005.
[17] M. Geng, W. Li, R. Yang, C. Cui, Xiaolin Wang, Antioxidant and antiproliferative
activity (in vitro) of polyphenols from peach blossom, J. Food, Agric. Environ.
11 (2013) 429–433.
[18] G. Noratto, W. Porter, D. Byrne, L. Cisneros-Zevallos, Identifying peach and
plum polyphenols with chemopreventive potential against estrogen-
independent breast cancer cells, J. Agric. Food Chem. 57 (2009) 5219–5226.
[19] G. Noratto, W. Porter, D. Byrne, L. Cisneros-Zevallos, Polyphenolics from peach
(Prunus persica var. Rich Lady) inhibit tumor growth and metastasis of MDA-
MB-435 breast cancer cells in vivo, J. Nutr. Biochem. 25 (2014) 796–800.
[20] M. Naczk, F. Shahidi, Extraction and analysis of phenolics in food, J.
Chromatogr. A 1054 (2004) 95–111.
[21] P. Garcia-Salas, A. Morales-Soto, A. Segura-Carretero, A. Fernandez-Gutierrez,
Phenolic-compound-extraction systems for fruit and vegetable samples,
Molecules 15 (2010) 8813–8826.
[22] C. Andreotti, D. Ravaglia, A. Ragaini, G. Costa, Phenolic compounds in peach
(Prunus persica) cultivars at harvest and during fruit maturation, Ann. Appl.
Biol. 153 (2008) 11–23.
[23] F.A. Tomás-Barberán, M.I. Gil, P. Cremin, A.L. Waterhouse, B. Hess-Pierce, A.A.
Kader, HPLC-DAD-ESIMS analysis of phenolic compounds in nectarines,
peaches, and plums, J. Agric. Food Chem. 49 (2001) 4748–4760.
[24] V.L. Singleton, J.A. Rossi, Colorimetry of total phenolics with
phosphomolybdic–phosphotungstic acid reagents, Am. J. Enol. Viticult. 16
(1965) 144–158.
[25] J. Santas, R. Carbo, M. Gordon, M. Almajano, Comparison of the antioxidant
activity of two Spanish onion varieties, Food Chem. 107 (2008) 1210–1216.
[26] I. Gülçin, Comparison of in vitro antioxidant and antiradical activities of L-
tyrosine and L-Dopa, Amino Acids 32 (2007) 431–438.
[27] M.S. Blois, Antioxidant determinations by the use of a stable free radical,
Nature 181 (1958) 1199–1200.
[28] M. Oyaizu, Studies on products of browning reaction: antioxidant activities of
products of browning reaction prepared from glucosamine, Japan. J. Nutr. 44
(1986) 307–315.
76 A. Mokrani, K. Madani / Separation and Purification Technology 162 (2016) 68–76
[29] P. Jayanthi, P. Lalitha, Reducing power of the solvent extracts of Eichhornia
crassipes (Mart.) Solms, Int. J. Pharm. Pharmaceut. Sci. 3 (2011) 126–128.
[30] M. Antolovich, P. Prenzler, K. Robards, D. Ryan, Sample preparation in the
determination of phenolic compounds in fruits, The Analyst 125 (2000) 989–
1009.
[31] M.C. Tan, C.P. Tan, C.W. Ho, Effects of extraction solvent system, time and
temperature on total phenolic content of henna (Lawsonia inermis) stems, Int.
Food Res. J. 20 (2013) 3117–3123.
[32] C. Alasalvar, M. Karamac, R. Amarowicz, F. Shahidi, Antioxidant and antiradical
activities in extracts of hazelnut kernel (Corylus avellana L.) and hazelnut green
leafy cover, J. Agric. Food Chem. 54 (2006) 4826–4832.
[33] D.B. Uma, C.W. Ho, W.M. Wan Aida, Optimization of extraction parameters of
total phenolic compounds from Henna (Lawsonia inermis) leaves, Sains
Malaysiana 39 (2010) 119–128.
[34] M.P. Kähkönen, A.I. Hopia, M. Heinonen, Berry phenolics and their antioxidant
activity, J. Agric. Food Chem. 49 (2001) 4076–4082.
[35] T. Sun, C.T. Ho, Antioxidant activities of buckwheat extracts, Food Chem. 90
(2005) 743–749.
[36] G. Shui, L.P. Leong, Residue from star fruit as valuable source for functional
food ingredients and antioxidant nutraceuticals, Food Chem. 97 (2006) 277–
284.
[37] M. Kajdzanoska, J. Petreska, M. Stefova, Comparison of different extraction
solvent mixtures for characterization of phenolic compounds in strawberries,
J. Agric. Food Chem. 59 (2011) 5272–5278.
[38] M.G. Curcic, M.S. Stankovic, I.D. Radojevic, O.D. Stefanovic, L.R. Comic, M.D.
Topuzovic, D.S. Djacic, S.D. Markovic, Biological effects, total phenolic content
and flavonoid concentrations of fragrant yellow onion (Allium flavum L.), Med.
Chem. 8 (2012) 46–51.
[39] Q. Liu, H. Yao, Antioxidant activities of barley seeds extracts, Food Chem. 102
(2007) 732–737.
[40] U.D. Chavan, R. Amarowicz, Effect of various solvent systems on extraction of
phenolics, tannins and sugars from beach pea (Lathyrus maritimus L.), Int. Food
Res. J. 20 (2013) 1139–1144.
[41] L. Boulekbache-Makhlouf, L. Medouni, S. Medouni-Adrar, L. Arkoub, K. Madani,
Effect of solvents extraction on phenolic content and antioxidant activity of
the byproduct of eggplant, Ind. Crops Prod. 49 (2013) 668–674.
[42] A. Mokhtarpour, A.A. Naserian, R. Valizadeh, M.D. Mesgaran, F. Pourmollae,
Extraction of phenolic compounds and Tannins from Pistachio by-products,
Annu. Res. Rev. Biol. 4 (2014) 1330–1338.
[43] I.M. Abeed, A.L. Mashkor, Phenolic content and antioxidant activity of
fenugreek seeds extract, Int. J. Pharmacog. Phytochem. Res. 6 (2014) 841–844.
[44] M. Bachir Bey, L. Meziant, Y. Benchikh, H. Louaileche, Deployment of response
surface methodology to optimize recovery of dark fresh fig (Ficus carica L., var.
Azenjar) total phenolic compounds and antioxidant activity, Food Chem. 162
(2014) 277–282.
[45] D.T.P. Lien, P.T.B. Tram, H.T. Toan, Effects of extraction process on phenolic
content and antioxidant activity of soybean, J. Food Nutr. Sci. 3 (2015) 33–38.
[46] B. Matthäus, Antioxidant activity of extracts obtained from residues of
different oilseeds, J. Agric. Food Chem. 50 (2002) 3444–3452.
[47] I. Gulcin, Antioxidant activity of eugenol: a structure–activity relationship
study, J. Med. Food 14 (2011) 975–985.
_ Gülçin, R. Elias, A. Gepdiremen, L. Boyer, Antioxidant activity of lignans from
[48] I.
fringe tree (Chionanthus virginicus L.), Eur. Food Res. Technol. 223 (2006) 759–
767.
[49] R. González-Montelongo, M. Gloria Lobo, M. González, Antioxidant activity in
banana peel extracts: testing extraction conditions and related bioactive
compounds, Food Chem. 119 (2010) 1030–1039.
[50] R. Chirinos, H. Rogez, D. Campos, R. Pedreschi, Y. Larondelle, Optimization of
extraction conditions of antioxidant phenolic compounds from mashua
(Tropaeolum tuberosum Ruíz & Pavón) tubers, Sep. Purif. Technol. 55 (2007)
217–225.
[51] M.A. Al-Farsi, C.Y. Lee, Optimization of phenolics and dietary fibre extraction
from date seeds, Food Chem. 108 (2008) 977–985.
[52] N.G.T. Meneses, S. Martins, J.A. Teixeira, S.I. Mussatto, Influence of extraction
solvents on the recovery of antioxidant phenolic compounds from brewer’s
spent grains, Sep. Purif. Technol. 108 (2013) 152–158.
[53] M.O. Downey, R.L. Hanlin, Comparison of ethanol and acetone mixtures for
extraction of condensed tannin from grape skin, S. Afr. J. Enol. Viticult. 31
(2010) 154–159.
[54] S. Kallithraka, C. Garcia-Viguera, P. Bridle, J. Bakker, Survey of solvents for the
extraction of grape seed phenolics, Phytochem. Anal. 6 (1995) 265–267.
[55] E. Revilla, J.-M. Ryan, G. Martín-Ortega, Comparison of several procedures used
for the extraction of anthocyanins from red grapes, J. Agric. Food Chem. 46
(1998) 4592–4597.
[56] L.E. Rodriguez-Saona, R.E. Wrolstad, Extraction, isolation, and purification of
anthocyanins, Curr. Protoc. Food Anal. Chem. (2001) 1–11.
[57] E. Silva, H. Rogez, Y. Larondelle, Optimization of extraction of phenolics from
Inga edulis leaves using response surface methodology, Sep. Purif. Technol. 55
(2007) 381–387.
[58] C.F. Yap, C.W. Ho, W.M. Wan Aida, S.W. Chan, C.Y. Lee, Y.S. Leong, Optimization
of extraction conditions of total phenolic compounds from star fruit (Averrhoa
carambola L.) residues, Sains Malaysiana 38 (2009) 511–520.
[59] S.W. Chan, C.Y. Lee, C.F. Yap, W.M. Wan Aida, C.W. Ho, Optimisation of
extraction conditions for phenolic compounds from limau purut (Citrus
hystrix) peels, Int. Food Res. J. 16 (2009) 203–213.
[60] M. Naczk, F. Shahidi, Phenolics in cereals, fruits and vegetables: occurrence,
extraction and analysis, J. Pharm. Biomed. Anal. 41 (2006) 1523–1542.
[61] S.K. Khanna, P.N. Viswanathan, P.S. Krishnan, G.G. Sanwal, Extraction of total
phenolics in the presence of reducing agents, Phytochemistry 7 (1968) 1513–
1517.
[62] M. Naczk, R. Amarowicz, R. Zadernowski, F. Shahidi, in: Antioxidant Capacity
of Phenolics from Canola Hulls As Affected by Different Solvents, Oxford
University Press, 2005, pp. 57–66.
[63] J. Shi, J. Yu, J. Pohorly, J.C. Young, M. Bryan, Y. Wu, Optimization of the
extraction of polyphenols from grape seed meal by aqueous ethanol solution, J.
Food Agric. Environ. 1 (2003) 42–47.
[64] J.E. Cacace, G. Mazza, Optimization of extraction of anthocyanins from black
currants with aqueous ethanol, J. Food Sci. 68 (2003) 240–248.
[65] F. Benmeziane, R. Djamai, Y. Cadot, R. Seridi, Optimization of extraction
parameters of phenolic compounds from Algerian fresh table grapes (Vitis
vinifera), Int. Food Res. J. 21 (2014) 1025–1029.
[66] E. Dorta, M.G. Lobo, M. Gonzalez, Reutilization of mango byproducts: study of
the effect of extraction solvent and temperature on their antioxidant
properties, J. Food Sci. 77 (2012) C80–C88.
[67] N. Ruenroengklin, J. Zhong, X. Duan, B. Yang, J. Li, Y. Jiang, Effects of various
temperatures and pH values on the extraction yield of phenolics from litchi
fruit pericarp tissue and the antioxidant activity of the extracted anthocyanins,
Int. J. Mol. Sci. 9 (2008) 1333–1341.
[68] T. Vatai, M. Škerget, Ž. Knez, Extraction of phenolic compounds from elder
berry and different grape marc varieties using organic solvents and/or
supercritical carbon dioxide, J. Food Eng. 90 (2009) 246–254.
[69] B.E. Richter, B.A. Jones, J.L. Ezzell, N.L. Porter, N. Avdalovic, C. Pohl, Accelerated
solvent extraction: a technique for sample preparation, Anal. Chem. 68 (1996)
1033–1039.
[70] G. Xu, X. Ye, J. Chen, D. Liu, Effect of heat treatment on the phenolic
compounds and antioxidant capacity of citrus peel extract, J. Agric. Food Chem.
55 (2007) 330–335.