76

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

Original article
Antioxidant capacity of fresh, sun- and sulphited-dried Malatya
apricot (Prunus armeniaca) assayed by CUPRAC, ABTS/TEAC and
folin methods
zyurek, Saliha E. Karademir & Resat Apak*
Kubilay Guclu, Mehmet Altun, Mustafa O
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey
(Received 4 May 2005; Accepted in revised form 6 January 2006)

Summary

Apricots as ve varieties of Malatya region have been assayed as fresh, sun- and sulphited-dried samples,
using the antioxidant capacity measurement methods Cupric Ion Reducing Antioxidant Capacity
(CUPRAC) and 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and total polyphenol
measurement method Folin. The novel reagent for the CUPRAC total antioxidant capacity assay,
bis(neocuproine)copper(II) chloride, was easily accessible, stable, selective and responding to all antioxidants. Sulphite (normally contributing to the colour formed in the CUPRAC assay) was removed prior to
assay on a strongly basic anion exchanger at pH 3 in the form of HSO
3 . The CUPRAC ndings correlated
well with the results of ABTS/TEAC and Folin assays (r 0.93), all being electron-transfer-based
antioxidant capacity assays. The calibration lines of pure avonoids individually and in standard-added
apricot extracts were parallel, indicating the additivity of absorbances in CUPRAC. This work reports for
the rst time the use of a novel spectrophotometric method (CUPRAC) for the assay of both total
antioxidant capacity and sulphite levels of diverse apricot samples.

Keywords

ABTS assay, antioxidant capacity, apricot (Prunus armeniaca), CUPRAC assay, folin assay, polyphenolic content, sulphite.

Introduction

When natural antioxidant defences of the organism (of

enzymatic, non-enzymatic or dietary origin) are overwhelmed by an excessive generation of reactive oxygen
species, a situation of oxidative stress occurs, in which
cellular and extracellular macromolecules (proteins,
lipids and nucleic acids) can suer oxidative damage,
causing the tissue injury (Halliwell & Gutteridge, 1989;
Halliwell & Aruoma, 1991). Consumption of foods
naturally bearing antioxidant activity (e.g. fruits and
vegetables) is the most ecient way of combating such
tissue injuries, undesired transformations and health
risks.
It is noteworthy that in many studies about plant
antioxidant research, it has been clearly indicated that
the measured antioxidant activity is too dependent on
the type of assay selected (Dorman et al., 2003; Trouillas et al., 2003; Miliauskas et al., 2004), and that the
observed antioxidant activity (or capacity) is not fully
correlated to total polyphenolics content of the plant
*Correspondent: Fax: +90-212-473 7180;
e-mail: rapak@istanbul.edu.tr

extracts (Kahkonen et al., 1999; Dorman et al., 2003;

Miliauskas et al., 2004), the linear correlation coecients either between two given antioxidant activity
assays or between antioxidant capacity and polyphenolics content of herbal extracts not being as close to 1 as
expected. However, electron transfer-based antioxidant
capacity assays involving one redox reaction with the
oxidant (also the probe for monitoring the reaction) as
an indicator of the reaction end-point:
Probe (oxidant) e (from antioxidant)
! reduced probe oxidised antioxidant
where the change in colour of the probe is proportional
to the total antioxidants concentration, may yield results
that show good correlations among themselves (Huang
et al., 2005). In this regard, Folin (FCR), ABTS/TEAC,
FRAP and DPPH assays are all classied as electrontransfer assays, and it is emphasised that the reaction
rate dierences between antioxidants and oxidants are
not reected in the ABTS/TEAC values because the
TEAC assay is an end-point assay (Huang et al., 2005).
The diverse antioxidant activity/capacity assay methods
existing in literature depending on the consumption of
chromogenic radicals, i.e. ABTS (Miller et al., 1993) and

doi:10.1111/j.1365-2621.2006.01347.x
 2006 Institute of Food Science and Technology Trust Fund

Antioxidant capacity of apricot K. Guclu et al.

DPPH (Sanchez-Moreno et al., 1998), oxygen radical

absorption capacity: ORAC (Cao et al., 1995), or ferricreducing ability: FRAP (Benzie & Strain, 1996), have
been extensively criticised for their inadequacies (Apak
et al., 2004). Ou et al. (2002) noticed that the antioxidant
activities of common vegetables in the US market (total
sample size: 927) analysed by ORAC and FRAP assays
did not correlate well, and concluded that there is no
total antioxidant as a nutritional index available for
food labelling because of the lack of standard quantitation methods. Therefore, the selected chromogenic redox
reagent for the assay of plant material should be easily
accessible, stable, selective, respond to all types of known
antioxidants regardless of chemical type or hydrophilicity; the concerned redox reaction should be rapid and the
resulting colour should be stable for a reasonable period
of time. Such a photometric reagent tting the above
purposes, bis(neocuproine)copper(II) chloride, which
had been introduced by our research group for various
reducing agents as a mild oxidant (Tutem et al., 1991),
and used to determine the biochemically important
reductants such as cysteine (Tutem & Apak, 1991) and
vitamin E (Tutem et al., 1997), and ascorbic acid (Guclu
et al., 2005), has been recently developed by our group
for measuring the total antioxidant capacity in plant
extracts and human serum via cupric ion reducing
potency (Apak et al., 2004, 2005), and this method
named as the Cupric Ion Reducing Antioxidant Capacity
(CUPRAC) method has been utilised in this work for
the antioxidant assay of apricot extracts, and the results
expressed as trolox (TR) equivalents were compared
with the ndings of ABTS (Re et al., 1999) and Folin
(Singleton et al., 1999) spectrophotometric methods.
As fruits and vegetables contain dierent varieties of
antioxidant compounds such as vitamins (especially
vitamins C and E), avonoids and carotenoids, a diet
rich in these is assumed to oer protection against
cardiovascular diseases, cancer and age-related degenerative transformations, as shown by epidemiological
studies (Schieber et al., 2001). Van der Sluis et al. (2001)
have reported that the antioxidant activity of fruits
could be inuenced by geographical origin, cultivar and
the conditions of harvest and storage. Scalzo et al. found
the following order of antioxidant activities for a
number of fruits: wild strawberries > cultivated strawberries > kiwifruit  apples  apricots  peaches
(Scalzo et al., 2005). According to Guo et al., the
antioxidant capacity (FRAP) rank of apricot was 24th
among twenty-eight fruits (Guo et al., 2003), while
Pellegrini et al. ranked apricot the 19th (TRAP), 23rd
(FRAP), and 24th (TEAC) among thirty fruits, depending on the method selected (Pellegrini et al., 2003). This
work aims to establish the antioxidant capacity of
apricots collected from Malatya (the leading apricot
producer city) of Turkey with respect to variety and
form using the reliable assays such as CUPRAC and

 2006 Institute of Food Science and Technology Trust Fund

ABTS, and to compare the results with the ndings of

Folin assay for total phenolics.
Apricots (Prunus armeniaca L. or Armeniaca vulgaris
Lam.) are members of the rose family (rosaceae), and
closely related to plum, peach, cherry and almond. As
apricots ripen early, they require certain climatic conditions, mainly distinctly dierent seasons comprised of
a fairly cold winter and moderately high temperatures in
the spring and early summer. Such conditions are found
in a cultivation band stretching from Turkey through
Iran and Himalayas to China and Japan, southern
Europe and North Africa, South Africa, Australia and
California. In the pulp of apricots, vitamin C contribution to the observed total antioxidant capacity is
thought to be <20% (Institute of Nutrition and Food
Safety, China CDC, 2000). In addition to carotenoid
pigments, the major antioxidant compounds present in
apricot are: isochlorogenic acid, caeic acid, 4-methylcatechol, chlorogenic acid, catechin, epicatechin,
pyrogallol, catechol, avonols and p-coumaric acid
derivatives. The CUPRAC method that we used for
total antioxidant assay was previously shown to respond
to all these antioxidant compounds, namely vitamin C,
avonoids and phenolic (hydroxycinnamic) acids, in a
dose-dependent manner (Apak et al., 2004).
Overall, dried apricots have a far greater nutritional
value (e.g. especially in terms of vitamin A and minerals)
than the fresh ones because all nutrients are concentrated. Apricots are often treated with sulphur dioxide
(also known as SO2-fumigation) as synthetic antioxidant before being sun dried. Fumigation is carried out in
rooms of approximately 2.4 2.4 2.2-m dimensions;
the duration of fumigation is about 812 h, and the
average consumption of sulphur in this process is 24 kg
S per tonne material. Those apricots that have not been
treated with this preservative become darker in colour,
with a carmelised, almost g-like avour. Sulphur
dioxide fumigation of apricots provides protection
against enzymatic (polyphenol oxidase-catalysed)
browning. As high concentrations of sulphite may cause
allergic reactions in sensitive individuals, the residual
sulphur dioxide levels in dried fruits has been set as
2000 ppm (US Federal Register, 1988). High contents of
sulphites in exported fruits and vegetables may be
subject to food alert notications by the European
Community member countries based on the regulation
(EC) no. 178/2002, laying down the general principles
and requirements of food law. Processes have been
designed by food technologists for desulphiting some
over-fumigated and dried apricots, e.g. by hot air ow
(Ozkan & Cemeroglu, 2002). Therefore, sulphite content
of SO2-fumigated and dried apricots should be precisely
determined along with their total antioxidant content.
About one-third of the dehydrated apricots used in
the US market are imported, the majority from Turkey.
Turkish apricots are a dierent variety and the product

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

77

78

Antioxidant capacity of apricot K. Guclu et al.

is markedly dierent from the US apricots in colour,

avour, sweetness and acidity. Turkey is the leading
fresh apricot producer of the world; the annual production may reach 500 000 t depending on the season (dried
apricot export amounting to 50 000 t), and in the
Eastern Anatolia city of Malatya producing almost half
of this amount, about 9095% of all apricot gardens are
devoted to the production of fruits that would be sold in
dried form. As dierent species, approximately 73% of
total production comes from Hacihaliloglu, 17% from
Kabaasi and the rest from Soganci, Hasanbey, Cataloglu and Zerdali (wild apricot, <1%) apricot trees.
As geographical regions of economical importance in
Malatya city, Darende, Akcadag, Battalgazi, Kale,
Merkez, Yazihan and Hekimhan are in the front. The
earliest harvest of the season is made at Kale, followed
by Battalgazi, Merkez, Yazihan, Akcadag and Darende,
the period between the earliest and latest harvests
reaching 2025 days (Asma, 2000). The apricots ripened
in July are either sun-dried without additives or
predominantly subjected to SO2-fumigation in sulphiting rooms, and then dried. Sun drying permits the
production of apricots with a rich orange colour,
translucent appearance and desirable gummy texture.
After being sulphuretted, the apricots are spread on
cloth or nylon, and dried for 23 days under open air
and sunlight, and each pit is removed by hand keeping
the fruit whole. Once the pits are removed, the fruits are
re-dried for 23 more days producing Sekerpare (Guner
& Keskin, 2004) up to a nal moisture content of 20
23% (by weight) for sulphuretted products and 1015%
for sun-dried only (gun kurusu) products. The dried
apricots are classied with respect to size, packaged and
exported (Asma, 2000).
Materials and methods

Chemicals, solutions and instruments

Neocuproine (2,9-dimethyl-1,10-phenanthroline) and

FolinCiocalteau phenol reagent (Sigma Chemical
Company, Steinheim, Germany), TR [()-6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid] (Aldrich
Chemicals Company, Steinheim, Germany), ABTS [2,2azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt] (Fluka Chemicals, Steinheim, Germany),
ammonium acetate, copper(II) chloride, potassium
persulphate, sodium hydroxide, copper(II) sulphate, sodium
carbonate, sodium potassium tartarate, 96% ethyl alcohol
and methanol (E. Merck, Darmstadt, Germany).
CuCl2 solution, 1.0 10)2 m, was prepared by dissolving 0.4262 g CuCl22H2O in water, and diluting to
250 mL. Ammonium acetate buer at pH 7.0, 1.0 m
was prepared by dissolving 19.27 g NH4Ac in water
and diluting to 250 mL. Neocuproine (Nc) solution,
7.5 10)3 m was prepared daily by dissolving 0.039-g Nc

in 96% ethanol, and diluting to 25 mL with ethanol.

Trolox, 1.0 10)3 m, was prepared in 96% ethanol. The
chromogenic radical reagent ABTS, at 7.0 mm concentration, was prepared by dissolving 0.1920 g of the
compound in water, and diluting to 50 mL. To this
solution was added 0.0331 g K2S2O8 such that the nal
persulphate concentration in the mixture be 2.45 mm.
The resulting ABTS radical cation solution was left to
mature at room temperature in the dark for 1216 h, and
then used for TEAC assays. The solutions used in the
Folin assay of polyphenolics were prepared as follows:
Lowry A: 2% aqueous Na2CO3 in 0.1-m NaOH; Lowry
B: 0.5% CuSO4 aqueous solution in 1% NaKC4H4O6
solution; Lowry C: prepared freshly as mixture (50-mL
Lowry A + 1 mL Lowry B); FolinCiocalteau reagent
was diluted with H2O at a volume ratio of 1:3 prior to
use. All percentages are given as (w/v), and distilled and
de-aerated (N2-bubbled) water was used throughout.
All spectrophotometric measurements were made with
a pair of matched quartz cuvettes using a CARY 1E
UV-Vis spectrophotometer. The pH measurements
were made with the aid of a E512 Metrohm Herisau
pH-meter using a glass electrode; the centrifugations were
performed with an Adams Dynac Centrifuge apparatus.
The deep-freezed apricot samples were homogenised
with the aid of a CAT X-620 Ultra-Torrax homogeniser.
Procedures

CUPRAC assay of total antioxidant capacity

To a test tube were added 1 mL of CuCl2 solution

(1.0 10)2 m), 1 mL of neocuproine alcoholic solution
(7.5 10)3 m), and 1-mL NH4Ac buer solution, and
mixed; (x) millilitres of apricot (fresh, sun-, sulphiteddried and desulphited samples) extract followed by
(1.1 ) x) millilitres of water were added (total volume 4.1 mL), and mixed well. Absorbance against a
reagent blank was measured at 450 nm after 30 min. As
the molar absorptivity of TR in the CUPRAC method is
e 1.67 104 L mol)1 cm)1, and the calibration curve
for pure TR is a line passing through the origin, the TR
equivalent molar concentration of the apricot extract
sample in nal solution may be found by dividing the
observed absorbance to the e for TR (optical cuvette
thickness 1 cm). The TR equivalent antioxidant
capacity may be traced back to the original extract
considering all dilutions, and proportionated to the
initial mass of apricot sample taken to nd a capacity in
the units of micromoles TR per gram dry matter. When
the technique of standard additions was used for apricot
extracts, (x 0.10.6) millilitres of extract was taken
such that the initial CUPRAC absorbance of the
solution was approximately 0.2. Varying volumes
between 50 and 250 lL of 1.0 10)3 m TR stock
solution and enough water were added to make the

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

 2006 Institute of Food Science and Technology Trust Fund

Antioxidant capacity of apricot K. Guclu et al.

nal total volume 4.1 mL, and the absorbance reading was made as described for direct measurements. The
standard additions technique was performed for conrming the validity of the principle of additivity in
absorbance measurements, i.e. for showing the parallelism of calibration lines of TR in pure solution and in TR
standard-added extracts.
ABTS assay of total antioxidant capacity

Capacityin lmol TR=g Af =eTR Vf =Vs rVi =m103

Treatment of apricot samples

The matured ABTS radical solution of bluegreen

colour was diluted with 96% ethanol at a ratio of
1:10. The absorbance of the 1:10 diluted ABTS+ radical
cation solution was 1.28 0.04 at 734 nm. To 1 mL of
the radical cation solution, 4 mL of ethanol was added,
and the absorbance at 734 nm was read at the end of the
rst and sixth minute. The procedure was repeated for
the unknown extract by adding 1 mL of the radical
cation solution to (x) millilitre of apricot extract
(x varied between 0.1 and 0.5 mL) and (4.0 ) x)
millilitre of ethanol and recording the absorbance
readings at the end of rst and sixth minutes. The
absorbance dierence (DA) was found by subtracting
the extract absorbance from that of the reagent blank
(pure radical solution), and this was correlated to TR
equivalent antioxidant concentration with the aid of a
linear calibration curve (usually the absorbance decrease
at the sixth minute was used for calculations).
Folin method of total phenolics assay

To (x) millilitres of the apricot extract (such that x

varied between 0.1 and 0.5 mL) was added (2 ) x)
millilitres of H2O. An aliquot of 2.5 mL of Lowry C
solution was added, and the mixture was let to stand for
10 min. At the end of this period, 0.25 mL of Folin
reagent was added, and thirty more min was allowed for
stabilisation of the blue colour formed. The absorbance
against a reagent blank was measured at 750 nm.
Calculation of total antioxidant capacity and
phenolics content

The molar absorptivity of TR in the above methods

were as follows:
eTR 1:67  104 L mol1 cm1 (CUPRAC method);
eTR 2:6  104 L mol1 cm1 (ABTS method);
eTR 4:65  103 L mol1 cm1 (Folin method):
If a fruit extract obtained from fresh, sun-, sulphiteddried and desulphited apricot samples (initial volume
Vi) prepared from (m) grams of solid matter was diluted
(r) times prior to analysis, and a sample volume of (Vs)
was taken for analysis from the diluted extract, and

 2006 Institute of Food Science and Technology Trust Fund

colour development (after addition of reagents) was

made in a nal volume of (Vf) to yield an absorbance of
(Af), then the TR equivalent antioxidant capacity of the
apricot sample (in lmol TR per gram of solid matter, or
simply lmol TR per gram) was found using the
equation:

Fresh apricots as ve dierent species (Hacihaliloglu,

Cologlu, Kabaasi, Soganci and Zerdali) were collected
from the gardens of Malatya Bahce Bitkileri Arastirma
Enstitusu (Malatya Garden Plants Research Institute).
The collected samples were subjected to pre-treatment
procedures according to the traditional practice of the
region under the supervision of the Institute, and
classied into three forms as fresh, sun- and sulphiteddried samples. Fresh samples were suitably packaged (in
1-kg packs), and immediately brought to our laboratories (in Istanbul University) by air transport, and deepfreezed at )25 C prior to analysis. A part of the fresh
samples were deseeded and sun-dried on concrete oor.
Another part of the fresh samples were fumigated with
sulphur dioxide in sulphiting rooms, then sun-dried and
deseeded. Both sun- (without additive) and sulphiteddried samples in 1-kg packs were deep-freezed as
described.
Solvent extraction of apricot samples and
separation/analysis of sulphite

Three dierent weighings (each approximately 4 g) of

each form of the apricots were taken and extracted;
three measurements were made for each extract. The
extraction procedure was as described by Garcia-Alonso
et al. (2004). Briey, the deep-freezed samples were
homogenised in cold methanol (3 25 mL), and centrifuged at 6 150 g for 10 min; the centrifugates were
ltered through an ordinary lter paper into a 100-mL
ask, and the settled pulp at the bottom of the centrifuge
tube was taken up with methanol containing some
water, ltered into the same ask, and diluted to a nal
volume of 100-mL CUPRAC, ABTS/TEAC and Folin
assays were conducted immediately on these extracts.
For desulphiting, the sulphited-dried sample extracts,
25 mL of the extract adjusted to pH 3 (by adding 0.1
0.3 mL of 1-m HCl) was passed thrice through a 5-g
resin bed of Dowex-X8 (Cl) form) strongly basic anion
exchanger placed in a 25-mL burette and thoroughly
pre-washed with water. The bed height of the anionic
resin was approximately 10 cm, and the ow rate was
1.6 mL min)1. The resin-retained sulphite (as HSO
3)
was eluted with 0.1-m NaOH, and analysed with
the same CUPRAC reagent (at pH 7); the molar

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

79

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

5.72
3.98
6.67
0.35
0.47

83.35
99.77
87.69
78.52
84.39
0.80
0.47
0.69
0.83
0.59

22.97
31.41
25.45
15.38
18.66
5.24
0.49
2.31
0.62
3.52

37.84
52.47
38.83
31.25
35.22
0.47
1.70
3.85
1.15
0.58

40.59
43.16
39.99
34.48
43.13
0.54
0.30
0.69
1.50
0.73

14.45
15.58
12.45
10.27
16.24
13.52 0.56
14.84 0.29
12.78 1.72
9.68 0.47
17.00 0.90
2.37
5.25
4.25
8.09
0.87

35.20
40.37
33.82
28.38
43.33
16.16 0.13
16.46 0.59
12.29 0.45
9.45 1.56
15.81 2.44
15.30 0.70
15.14 0.42
13.55 0.17
9.32 0.19
17.92 3.05

0.54
0.24
0.17
0.08
0.14

3.50
4.46
3.18
2.86
3.37

1.14
0.93
0.29
0.16
0.35
3.92
4.23
3.22
2.67
4.06
Hacihaliloglu
Cologlu
Kabaasi
Soganci
Zerdali

11.38 1.47
10.73 2.61
9.04 1.24
8.47 1.93
10.90 1.49

TEACCUPRAC TEACABTS/TEAC TEACFOLIN

TEACCUPRAC TEACABTS/TEAC TEACFOLIN
TEACCUPRAC TEACABTS/TEAC TEACFOLIN
TEACCUPRAC TEACABTS/TEAC TEACFOLIN
Type of
apricot

Desulphited apricot (lmol TR per g)

In accord with the literature procedures, methanol was

selected as the hydrophilic extraction solvent for apricot,
because the lipophilic contribution to overall antioxidant capacity is much lower (Scalzo et al., 2005). Methanol has also been claimed to be a good extraction
solvent for low-polymerised avonols (Kallithraka
et al., 1995).
Table 1 shows the CUPRAC, ABTS/TEAC, and
Folin assay results of ve varieties of Malatya apricots
in four dierent forms (fresh, sun-, sulphited-dried and
desulphited). The antioxidant capacities are given as TR
equivalents, in the units of lmol TR per gram solid
matter. As other reducing agents as well as phenolics
react with the Folin reagent in a molybdenum blue-type
heteropoly acid reaction (Santos-Buelga & Scalbert,
2000), the results of the Folin assay may also be reported
as TR equivalents, because TR is a reference antioxidant
compound in these assays, and Folin assay is actually an
electron-transfer-based antioxidant capacity assay as it
responds to other antioxidants as well as to phenols
(Huang et al., 2005). However, the Folin results are
signicantly greater than those of CUPRAC and ABTS,
because polyphenolics are included along with the
reducing compounds (antioxidants). It is apparent
from Table 1 that the hierarchy for antioxidant capacity of apricot forms was: fresh < desulphited sun
dried < sulphited-dried, with sulphite contributing
to the observed capacity. The hierarchy of varieties
was: Soganci < Kabaasi < Hacihaliloglu Cologlu
Zerdali (wild apricot), the last three showing close
values.
Generally, it may be said that there is very little
information in peer-reviewed literature references about
the antioxidant activity of apricots from various geographical origins and genotypes (Scalzo et al., 2005).
Comparing with the ndings of other researchers, Scalzo
et al. (2005) reported TEAC capacities of 0.81.4-lmol
TR per gram fresh-weight for apricot varieties (plants
grafted on ve rootstocks, Italy) with an average value
of 1.2 (for the hydrophilic component), but could not
correlate TEAC data of fruits with Folin results of total
phenolics. Garcia-Alonso et al. (2004) used TBARS and
TEAC tests to rank fresh apricot as 2324/28 among
various fruits, but also could not correlate antioxidant
activity results with avonol contents. Guo et al. (2003)
gave the FRAP (as Fe2+-equivalents) values of 3.4, 7.9

Sun-dried apricot (lmol TR per gram)

Results and discussion

Fresh apricot (lmol TR per gram)

absorptivity of the CUPRAC reagent for sulphite was

esulphite 4.92 103 L mol)1 cm)1, and the equation
for the linear calibration of sulphite was: ACUPRAC
4.92 103 Csulphite + 0.0223. The desulphited apricot
extracts were brought to a pH of 7 by the addition of
0.080.2 mL of 1.2-m NaOH, and analysed for their
antioxidant content.

Sulphited-dried apricot
(lmol TR per gram)

Antioxidant capacity of apricot K. Guclu et al.

Table 1 Cupric Ion Reducing Antioxidant Capacity (CUPRAC), 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid)/Trolox Equivalent Antioxidant Capacity (ABTS/TEAC),
and Folin assay TEAC results of ve varieties of Malatya apricots in four dierent forms

80

 2006 Institute of Food Science and Technology Trust Fund

Antioxidant capacity of apricot K. Guclu et al.

 2006 Institute of Food Science and Technology Trust Fund

CUPRAC value (mol TR g1) of apricot solutions

20

16

12

0
0

10

20

30

40

50

Total phenolics concentration (mol TR g1)

of apricot solutions
Figure 1 The correlation of Cupric lon Reducing Antioxidant Capacity
(CUPRAC) assay results with Folin total phenolics content.

20

ABTS value (mol TR g1) of apricot solutions

and 7.2 lmol g)1, respectively, for pulp, peel, and seed
of apricots. As Fe2+ is a 1 e-reductant in the CUPRAC
assay while TR is a 2 e-reductant (Apak et al., 2004),
e.g. an FRAP value of 3.4 for pulp should correspond to
approximately 1.7-lmol TR per gram. In their comprehensive work for vegetables and fruits, Pellegrini et al.
(2003) ranked apricot as 2324/30 fruits with FRAP
4.02 lmol (Fe2+) per gram and TEAC 1.44-lmol TR
per gram fresh apricot. Halvorsen et al. (2002) reported
FRAP values of 5.2 lmol (Fe2+) per gram for fresh
fruit, and 32.5 lmol (Fe2+) per gram for the dried fruit,
corresponding to approximately 2.6 and 16.2 TEAC
values, respectively, which are well in accord with our
CUPRAC values for fresh and sun-dried apricots, given
in Table 1. Munzuroglu et al. (2003) speculates that for
fruits grown at high altitudes, natural factors such as
radiation, temperature dierence between day and
night, limited water and mineral sources, and wind
may force the metabolism of plants to a level where the
plants resist to environmental stress by increasing the
production of several vitamins and phenolic constituents
contributing to overall antioxidant potency. Thus
Malatya apricots, including all varieties, show signicantly higher antioxidant capacity (both as CUPRAC
and ABTS/TEAC) than analogues from other parts of
the world (See Table 1). It is known that in apricot pulp,
vitamin C contribution to the overall antioxidant
capacity is <20% (Guo et al., 2003). The vitamin C
contents of Malatya apricots as reported by Munzuroglu et al. (2003) is around 150-lg ascorbic acid per
gram, which, when compared with our average
TEACCUPRAC value of 3.6-lmol TR per gram, is also
roughly of the same contribution level, as both ascorbic
acid and TR exhibit similar molar absorptivities in the
CUPRAC spectrophotometric method, being both 2
e-reductants (Apak et al., 2004). Further, Munzuroglu
et al. (2003) states that cultivated varieties (Hacihaliloglu, Kabaasi, Cologlu and Soganci) have signicantly
higher vitamin C contents than the wild types (Zerdali),
while our ndings for total antioxidant capacity shows
that Zerdali is among the highest three varieties for
antioxidant capacity. Thus, there should be a component of antioxidant capacity for apricot that should
highly compensate for its deciency in vitamin C
content. That component may be total phenolics content, as observed for fruit nectars by Tosun & Ustun
(2003) who reported average FRAP values of 5.7 for
apricot nectars close to that of orange nectars (6.5), also
pointing out to the fact that Turkish apricots may have
higher antioxidant capacity than analogues from other
climatic zones. For fruits relatively poor in vitamin C,
signicant antioxidant activities are generally thought to
arise from avonoids and phenolic acids (Guo et al.,
1997). When analysed with the aid of linear regression,
both our CUPRAC and ABTS/TEAC assays correlated
well with Folin phenolics content (Figs 1 and 2). Thus,

16

12

0
0

10

20

30

40

50

Total phenolics concentration (mol TR g1)

of apricot solutions
Figure 2 The correlation of 2,2-azinobis (3-ethylbenzothiazoline-6-

sulphonic acid) (ABTS) assay results with Folin total phenolics content.

the higher antioxidant capacity of Malatya apricots may

result from their high total phenolics and not from their
ascorbic acid content. The TR-equivalent antioxidant
capacities (as stoichiometric TEAC coecients in
the original CUPRAC method) of most phenolic

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

81

Antioxidant capacity of apricot K. Guclu et al.

compounds (quercetin, catechins, gallic acid, etc.) were

greater than two (Apak et al., 2004), meaning that each
phenolic or avonoid behaved as a reducing agent in the
redox reaction of concern capable of donating electrons
greater than or equal to two molecules of TR or ascorbic
acid, contributing with a greater share to the overall
TEAC antioxidant capacity.
The optimal pH of sulphite removal was chosen as pH
3.0, because this pH is well above the pKa1 of sulphurous acid (H2SO3), i.e. 1.8, where the predominant form
of sulphite is HSO
3 , which can easily be retained by the
strongly basic anion exchanger. This was also a suitable
pH for ascorbic acid, as this antioxidant compound is in
molecular form at this pH, and therefore would not be
retained by the anionic resin, preventing a loss in
observed antioxidant capacity of the sulphited-dried
apricot extract. The CUPRAC reagent reacted easily
with sulphite (esulphite 4.92 103 L mol)1 cm)1), and
this work is the rst report of the use of the CUPRAC
reagent simultaneously for antioxidant assay and for
determining sulphite content of sulphited-dried fruit
extracts. The sulphite content of Zerdali, Soganci,
Kabaasi, Hacihaliloglu and Cologlu varieties were
found as 479, 602, 744, 732 and 948 p.p.m. SO2
3 ,
respectively, well below the maximum residual sulphur
dioxide limit of 2000 p.p.m. set by Food and Drug
Administration (FDA) for dried fruits. The sulphite
contents of sulphited-dried apricots correlated well
with the dierence in CUPRAC antioxidant capacity
between the sulphited-dried and desulphited (by anionexchange) apricot extracts (r 0.97) (Table 1). As
Asma described for Malatya apricots (Asma, 2000),
sulphite-drying causes a weight reduction factor of
about 3.84, which more or less explains our increased
antioxidant capacities as a result of drying (Table 1). As
the Turkish food codex requires that the maximum
moisture content of dried apricots be 25% (by weight)
and the remaining mass ratio of dried apricots drops to
c. 25% after 17 days of open drying in drying modelling
experiments (Sarsilmaz et al., 2000), roughly a fourfold
increase in antioxidant capacity (Table 1) of dried
apricots compared to that of the fresh ones is an
indication that antioxidant values of apricots are
preserved during drying. This is not always true for
other fruits as Halvorsen et al. (2002) reported that
dried apricots and prunes contained 26 times higher
levels of antioxidants than the fresh fruits, while raisins
contained substantially lower levels compared with fresh
grapes, a fact attributable to the nature of the drying
process. Vinson et al. (2005) reported a similar nding
in that processing to produce the dried fruit signicantly
decreased the phenols in most fruits on a dry weight
basis, while dried gs and plums essentially maintained
their nutritional values, including antioxidants. When
sulphited-dried apricots were subjected to the separation
of HSO
3 with the aid of an ion exchange column, the

remaining antioxidant capacities in the samples

(Table 1) more or less reected the weight losses upon
drying, conrming that preliminary fumigation treatment with SO2 helped preserve the samples against
oxidation. A dierent fact worthy of mention about SO2
sulphuretting and drying is that SO2 probably caused
more expansion of the apricot pores, giving rise to faster
heat and mass transfer between apricot surface and the
surrounding air during drying (Togrul & Pehlivan,
2004), thereby enabling a shorter drying time with the
potential advantage of preserving antioxidant values.
Another possibility associated with the ndings of
Halvorsen et al. (2002) for sulphited-dried fruits is a
slight positive error, as sulphite is a reducing agent
which may react with the FRAP reagent to produce the
coloured Fe(II)-tripyridyltriazine chelate. In further
tests of antioxidant assay with dried fruits, this point
should always be borne in mind.
Trolox was added to apricot (fresh, sun-, sulphiteddried and desulphited samples) extracts in the standard
addition mode to see if the original calibration line of
TR individually would dier in slope from that in the
complex solution (fruit extract). The parallelism of
calibration lines of TR in pure solution and in extracts
(Figs 36) conrmed that there were no chemical
interactions among the complex antioxidant compounds
of the apricot extract and TR (as standard compound)
causing either an intensication or quenching of overall
CUPRAC absorbance, and that the CUPRAC absorbances were additive. Thus, the CUPRAC method may
be safely used for the total antioxidant capacity assay of
apricot extracts.
1.40

1.20

1.00

Absorbance

82

0.80

0.60

0.40

TR alone
TR in fresh apricot solution of
initial A450 = 0.22

0.20

0.00
0

CTR 105, M
Figure 3 Calibration curve of trolox (TR) as added standard in
fresh apricot extract.

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

 2006 Institute of Food Science and Technology Trust Fund

1.40

1.40

1.20

1.20

1.00

1.00

Absorbance

Absorbance

Antioxidant capacity of apricot K. Guclu et al.

0.80

0.60

0.60

0.40

0.40

0.20

0.20

TR alone
TR in sun-dried apricot solution of
initial A450 = 0.24

TR alone
TR in desulphited apricot solution
of initial A450 = 0.22

0.00

0.00
0

Figure 4 Calibration curve of trolox (TR) as added standard in

sun-dried apricot extract.

1.60

1.40

1.20

1.00

0.80

0.60

0.40
TR alone
TR in sulphited-dried apricot
solution of initial A450 = 0.58

0.20

0.00
0

CTR 105, M
Figure 5 Calibration curve of trolox (TR) as added standard in
sulphited-dried apricot extract.

The method selection for total antioxidant capacity

assay is very important here for obtaining relatively
objective results. Our CUPRAC assay measures the
total reducing power of the sample like FRAP and bears
all the advantages of FRAP, added to the fact that it
employs a pH of 7.0 close to that of the physiological

 2006 Institute of Food Science and Technology Trust Fund

CTR 105, M

CTR 105, M

Absorbance

0.80

Figure 6 Calibration curve of trolox (TR) as added standard in

desulphited apricot extract.

pH, unlike the acidic and therefore unrealistic pH of

FRAP (pH 3.6). Furthermore, the FRAP reagent does
not attack SH compounds like glutathione (Apak
et al., 2004) while the CUPRAC reagent rapidly reacts
with thiols. Guo et al. (2003) claims that this disadvantage may be unimportant for FRAP, because only
limited amount of plant glutathione is absorbed by
humans (Schafer & Buettner, 2001). Adverse examples
can easily be found from the literature, e.g. Halvorsen
et al. (2002) have reported that the FRAP capacity for
garlic, a vegetable especially enriched in thiol-type
antioxidants, is much lower than that of ORAC,
because the FRAP reagent does not respond to SH
compounds. Other advantages of the CUPRAC reagent
(Apak et al., 2004, 2005) can be briey summarised as
follows:
1 As a result of reagent selectivity because of the favourable redox potential (i.e. close to that of ABTS reagent
in the TEAC method), citric acid and reducing sugars
commonly found in plant extracts are not attacked by
the CUPRAC reagent.
2 Reagent stability and accessibility (low cost).
3 Ease of applicability to conventional laboratories.
4 The relative independence of the CUPRAC reaction
from air, sunlight, humidity and pH (to a certain extent).
5 High sensitivity and perfect linearity over a wide
concentration range.
6 Simultaneous applicability to the measurement of
hydrophilic as well as lipophilic antioxidants (e.g.
b-carotene and a-tocopherol).

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

83

84

Antioxidant capacity of apricot K. Guclu et al.

7 High precision, i.e. low intra- and inter-assay CV.

8 The working pH of CUPRAC (i.e. pH 7) is close to
the physiological pH; thus, the test does not underestimate or overestimate the quantity of antioxidants as
tests carried out in more acidic (e.g. FRAP) or basic (e.g.
Folin) media where the phenolic antioxidants are either
essentially protonated or fully deprotonated, respectively.
9 Even though there is a high variability even for values
obtained from the same assay because of the lack of
standardisation of the assays (Pellegrini et al., 2003),
and in spite of the fact that there are poor correlations
between the TEAC capacity and total phenolic content
of food samples (Scalzo et al., 2005), the CUPRAC
results correlate quite well with those of ABTS/TEAC
and Folin (Fig. 1, r 0.932, and Fig. 2, r 0.939),
which are of the same chemical nature, i.e. electrontransfer-based capacity assays (Huang et al., 2005).
Conclusion

In this work, apricots as ve varieties of Malatya

region have been assayed as fresh, sun- and sulphiteddried samples, using the antioxidant capacity measurement methods CUPRAC and ABTS, and total
polyphenol measurement method Folin. The same test
(CUPRAC) was used for determining the sulphite
levels of sulphited-dried samples by prior separation of
monohydrogensulphite at pH 3 on an anion exchanger
followed by spectrophotometry. The CUPRAC test
was simple, practical and rapid for total antioxidant
capacity assay of apricot methanolic extracts, using
easily accessible and stable reagents. The CUPRAC
reagent was capable of quantitatively oxidising all sorts
of antioxidants, including vitamins C and E, polyphenolics, avonoids and thiol-type compounds, with
ABTS-compatible TEAC coecients for all these
compounds. The reagent did not respond to citric acid
or reducing sugars that may be widely found in plantbased food extracts. The CUPRAC results were highly
reproducible, and correlated well with those of ABTS
and Folin assays. Turkish (Malatya) apricots generally
showed higher antioxidant capacity than those reported
in the literature from other varieties of dierent
geographical origin.
Acknowledgments

The authors would like to express their gratitude to

Istanbul University Research Fund, Bilimsel Arastirma
Projeleri Yurutucu Sekreterligi, for the funding of
Project YOP-4/27052004, and to State Planning Organisation of Turkey for the Advanced Research Project of
Istanbul University (2005K120430). Thanks are also

because of Malatya Kayisi Arastirma-Gelistirme ve

Tanitma Vak (Malatya Apricot Research-Development and Presentation Foundation) for their permission
in the use of facilities.
References
Apak, R., Guclu, K., Ozyurek, M. & Karademir, S.E. (2004). A novel
total antioxidant capacity index for dietary polyphenols, vitamin C
and E, using their cupric ion reducing capability in the presence of
neocuproine: CUPRAC method. Journal of Agricultural and Food
Chemistry, 52, 79707981.
Apak, R., Guclu, K., Ozyurek, M., Karademir, S.E. & Altun, M.
(2005). Total antioxidant capacity assay of human serum using
copper(II)-neocuproine as chromogenic oxidant: the CUPRAC
method. Free Radical Research, 39, 949961.
Asma, B.M. (2000). Kayisi Yetistiriciligi (Apricot Growing). Malatya,
Turkey: Evin Publishers.
Benzie, I.F.F. & Strain, J.J. (1996). The ferric reducing ability of
plasma (FRAP) as a measure of antioxidant power: the FRAP
assay. Analytical Biochemistry, 239, 7076.
Cao, G., Verdon, C.P., Wu, A.H.B., Wang, H. & Prior, R.L. (1995).
Automated oxygen radical absorbance capacity assay using the
COBAS FARA II. Clinical Chemistry, 41, 17381744.
Dorman, H.J., Peltoketo, A., Hiltunen, R. & Tikkanen, M.J. (2003).
Characterisation of the antioxidant properties of de-odourised
aqueous extracts from selected Lamiaceae herbs. Food Chemistry,
83, 255262.
Garcia-Alonso, M., Pascual-Teresa, S., Santos-Buelga, C. & RivasGonzalo, J.C. (2004). Evaluation of the antioxidant properties of
fruits. Food Chemistry, 84, 1318.
Guclu, K., Sozgen, K., Tutem, E., Ozyurek, M. & Apak, R. (2005).
Spectrophotometric determination of ascorbic acid using copper(II)neocuproine reagent in beverages and pharmaceuticals. Talanta, 65,
12261232.
Guner, M. & Keskin, R. (2004). Mechanical removal of sulphuretted
apricot pit. Biosystems Engineering, 88, 187192.
Guo, C., Cao, G.H., Soc, E. & Prior, R.L. (1997). High-performance
liquid chromatography coupled with coulometric array detection of
electroactive components in fruits and vegetables: relationship to
oxygen radical absorbance capacity. Journal of Agricultural and
Food Chemistry, 45, 17871796.
Guo, C., Yang, J., Wei, J., Li, Y., Xu, J. & Jiang, Y. (2003).
Antioxidant activities of peel, pulp and seed fractions of common
fruits as determined by FRAP assay. Nutrition Research, 23, 1719
1726.
Halliwell, B. & Aruoma, O.I. (1991). DNA damage by oxygen-derived
species: its mechanisms and measurement in mammalian systems.
FEBS Letters, 281, 919.
Halliwell, B. & Gutteridge, J.M.C. (1989). Free Radicals in Biology and
Medicine. Oxford, UK: Oxford University Press.
Halvorsen, B.L., Holte, K., Myhrstad, M.C.W. et al. (2002). A
systematic screening of total antioxidants in dietary plants. The
Journal of Nutrition, 132, 461471.
Huang, D., Ou, B. & Prior, R.L. (2005). The chemistry behind
antioxidant capacity assays. Journal of Agricultural and Food
Chemistry, 53, 18411856.
Institute of Nutrition and Food Safety, China CDC (2000). China
Food Composition 2002. pp. 8297. Beijing, China: Peking University Medical Press.
Kahkonen, M.P., Hopia, A.I., Vuorela, H.J. et al. (1999). Antioxidant
activity of plant extracts containing phenolic compounds. Journal of
Agricultural and Food Chemistry, 47, 39543962.
Kallithraka, S., Garcia-Viguera, C., Bridle, P. & Bakker, J. (1995).
Survey of solvents fort he extraction of grape seed phenolics.
Phytochemical Analysis, 6, 265267.

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

 2006 Institute of Food Science and Technology Trust Fund

Antioxidant capacity of apricot K. Guclu et al.

Miliauskas, G., Venskutonis, P.R. & van Beek, T.A. (2004). Screening
of radical scavenging activity of some medicinal and aromatic plant
extracts. Food Chemistry, 85, 231237.
Miller, N.J., Rice-Evans, C.A., Davies, M.J., Gopinathan, V. &
Milner, A. (1993). A novel method for measuring antioxidant
capacity and its application to monitoring the antioxidant status in
premature neonates. Clinical Science, 84, 407412.
Munzuroglu, O., Karatas, F. & Geckil, H. (2003). The vitamin and
selenium contents of apricot fruit of dierent varieties cultivated in
dierent geographical regions. Food Chemistry, 83, 205212.
Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A. & Deemer,
E.K. (2002). Analysis of antioxidant activities of common vegetables
employing oxygen radical absorbance capacity (ORAC) and ferric
reducing antioxidant power (FRAP) assays: a comparative study.
Journal of Agricultural and Food Chemistry, 50, 31223128.
Ozkan, M. & Cemeroglu, B. (2002). Desulphiting dried apricots by
exposure to hot air ow. Journal of the Science of Food and
Agriculture, 82, 18231828.
Pellegrini, N., Serani, M., Colombi, B. et al. (2003). Total antioxidant
capacity of plant foods, beverages and oils consumed in Italy
assessed by three dierent in vitro assays. The Journal of Nutrition,
133, 28122819.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. & RiceEvans, C. (1999). Antioxidant activity applying an improved ABTS
radical cation decolorization assay. Free Radical Biology and
Medicine, 26, 12311237.
Sanchez-Moreno, C., Larrauri, J.A. & Saura-Calixto, F. (1998). A
procedure to measure the antiradical eciency of polyphenols.
Journal of the Science of Food and Agriculture, 76, 270276.
Santos-Buelga, C. & Scalbert, A. (2000). Proanthocyanidins and
tannin-like compounds: nature, occurrence, dietary intake and
eects on nutrition and health (review). Journal of the Science of
Food Agriculture, 80, 10941117.
Sarsilmaz, C., Yildiz, C. & Pehlivan, D. (2000). Drying of apricots in a
rotary column cylindrical dryer (RCCD) supported with solar
energy. Renewable Energy, 21, 117127.
Scalzo, J., Politi, A., Pellegrini, N., Mezzetti, B. & Battino, M. (2005).
Plant genotype aects total antioxidant capacity and phenolic
contents in fruit. Nutrition, 21, 207213.
Schafer, F.Q. & Buettner, G.R. (2001). Redox environment of the cell
as viewed through the redox state of the glutathione disulde/

 2006 Institute of Food Science and Technology Trust Fund

glutathione couple. Free Radical Biology and Medicine, 30, 1191

1212.
Schieber, A., Stintzing, F.C. & Carle, R. (2001). By-products of
plant food processing as a source of functional compounds-recent
developments. Trends in Food Science and Technology, 12, 401
413.
Singleton, V.L., Orthofer, R. & Lamuela-Raventos, R.M. (1999).
Analysis of total phenols and other oxidation substrates and
antioxidants by means of FolinCiocalteu reagent. Methods in
Enzymology, 299, 152178.
Togrul, I.T. & Pehlivan, D. (2004). Modelling of thin layer drying
kinetics of some fruits under open-air sun drying processes. Journal
of Food Engineering, 65, 413425.
Tosun, I. & Ustun, N.S. (2003). An investigation about antioxidant
capacity of fruit nectars. Pakistan Journal of Nutrition, 2, 167169.
Trouillas, P., Calliste, C.-A., Allais, D.-P. et al. (2003). Antioxidant,
anti-inammatory and antiproliferative properties of sixteen water
plant extracts used in the Limousin countryside as herbal teas. Food
Chemistry, 80, 399407.
Tutem, E. & Apak, R. (1991). Simultaneous spectrophotometric
determination of cystine and cysteine in amino acid mixtures using
copper(II)-neocuproine reagent. Analytica Chimica Acta, 255,
121125.
Tutem, E., Apak, R. & Baykut, F. (1991). Spectrophotometric
determination of trace amounts of copper(I) and reducing agents
with neocuproine in the presence of copper(II). Analyst, 116, 89
94.
Tutem, E., Apak, R., Gunayd, E. & Sozgen, K. (1997). Spectrophotometric determination of vitamin E (alpha-tocopherol) by the
copper(II)-neocuproine reagent. Talanta, 44, 249255.
US Federal Register (1988). Sulting Agents In Standardized Foods;
labeling requirements; proposed rule. Washington, US Government
Printing Oce.
Van der Sluis, A.A., Dekker, M., De Jager, A. & Jongen, W.M.F.
(2001). Activity and concentration of polyphenolic antioxidants in
apple: eect of cultivar, harvest year, and storage conditions.
Journal of Agricultural and Food Chemistry, 49, 36063613.
Vinson, J.A., Zubik, L., Bose, P., Samman, N. & Proch, J. (2005).
Dried fruits: excellent in vitro and in vivo antioxidants. Journal of the
American College of Nutrition, 24, 4450.

International Journal of Food Science and Technology 2006, 41 (Supplement 1), 7685

All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.

85