Food Chemistry 141 (2013) 13981405

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effect of ionising radiation on polyphenolic content and antioxidant

potential of parathion-treated sage (Salvia ofcinalis) leaves
Issam Ben Salem a,b,c,,1, Sana Fekih a,b, Haitham Sghaier b, Mehrez Bousselmi a,b, Mouldi Saidi b,
Ahmed Landoulsi c, Sami Fattouch a,1
a

Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB), National Institute of Applied Sciences and Technology (INSAT), University of Carthage, Tunis, Tunisia
Research Unit, Application of Nuclear Techniques in the Fields of Health, Agriculture, and Environment, National Centre for Nuclear Science and Technology (CNSTN), Sidi Thabet
Technopark, 2020 Ariana, Tunisia
c
Biochemistry and Molecular Biology Laboratory, Faculty of Science of Bizerte, University of Carthage, Tunis, Tunisia
b

a r t i c l e

i n f o

Article history:
Received 4 February 2013
Received in revised form 3 April 2013
Accepted 6 April 2013
Available online 18 April 2013
Keywords:
Antioxidant activity
Ionising radiation
Medicinal plant
Parathion
Polyphenols
Salvia ofcinalis

a b s t r a c t
The c-irradiation effects on polyphenolic content and antioxidant capacity of parathion-pretreated leaves
of Salvia ofcinalis plant were investigated. The analysis of phenolic extracts of sage without parathion
showed that irradiation decreased polyphenolic content signicantly (p < 0.05) by 30% and 45% at 2
and 4 kGy, respectively, compared to non-irradiated samples. The same trend was observed for the Trolox
equivalent antioxidant capacity (TEAC), as assessed by the anionic DPPH and cationic ABTS radical-scavenging assays. The antioxidant potential decreased signicantly (p < 0.01) at 2 and 4 kGy, by 1120% and
4044%, respectively. The results obtained with a pure chlorogenic acid solution conrmed the degradation of phenols; however, its TEAC was signicantly (p < 0.01) increased following irradiation. Degradation products of parathion formed by irradiation seem to protect against a decline of antioxidant capacity
and reduce polyphenolic loss. Ionising radiation was found to be useful in breaking down pesticide residues without inducing signicant losses in polyphenols.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Ionising radiation has been demonstrated to be very effective
for pathogen inactivation in both raw and cooked foods (Farkas,
1998). The International Consultative Group of Food Irradiation
(ICGFI) concluded that irradiation of food at a dose level of
10 kGy or below was toxicologically safe and nutritionally adequate (WHO, 1981). During the last decade, several studies have
shown varying sensitivities of particular food ingredients and
nutrients to radiation treatment (Sommer, Schwartz, Solar, & Sontag, 2009). The phenolic content of rosemary was signicantly altered following irradiation >10 kGy (Koseki et al., 2002), whereas,
the capsaicinoids increased signicantly, by about 10%, in sundried and dehydrated paprika samples irradiated at a dose of
10 kGy (Topuz & Ozdemir, 2003).
In addition to the occurrence of degradative biochemical reactions and spoilage microorganisms in food products, the presence
of environmental toxicants, particularly pesticides, in freshly
harvested material has attracted great attention in scientists. In
Corresponding author at: National Centre for Nuclear Science and Technology
(CNSTN), Sidi Thabet Technopark, 2020 Ariana, Tunisia. Tel.: +216 71 537410; fax:
+216 71 537555.
E-mail address: issamcnstn@yahoo.fr (I. Ben Salem).
1
These authors contributed equally to the work.
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.04.008

agricultural phytosanitary practises, a small fraction of the used

pesticide amount is directly involved in the pesticide action, and
most of these chemicals remain as residues, and may exert adverse effects on both target and non-target organisms (Fattouch
et al., 2010). To our knowledge, despite several studies addressing
the problems of pesticide residues toxicity in food, limited works
have investigated the interactions between hazardous residues
and health-promoting phytochemicals, particularly plant polyphenols (Rung & Schwack, 2005). The protective effect of the latter
compounds is chiey attributed to their antioxidant potential by
scavenging free radicals, chelating metals in foods, activating antioxidant enzymes and inhibiting enzymes that cause oxidation
reactions (Heim, Tagliaferro, & Bobilya, 2002).
During the last decades, medicinal and aromatic plants have
been extensively studied and found to be excellent sources of
bioactive and health-promoting compounds. In addition to their
aromatic and avouring properties, medicinal and aromatic plants
have been used as additives to prevent the oxidative deterioration
and microbial proliferation in several perishable food products
(Fattouch, Sadok, Raboudi-Fattouch, & Slama, 2008; Sarkardei &
Howell, 2008). Sage, Salvia ofcinalis, is one of the most wellknown aromatic herbs. Sentences such as Why should a man
die while sage grows in his garden? reect the importance of this
plant in traditional medicine (Ramos, Azqueta, Pereira-Wilson, &

I. Ben Salem et al. / Food Chemistry 141 (2013) 13981405

Collins, 2010). Native to southern Europe, it is largely cultivated in

the Mediterranean countries, including Tunisia, where it is used as
a common ornamental species as well as for its medicinal and aromatic properties. Salvia leaves are recognised as a source of benecial phenolic compounds and natural potent antioxidants (Lu &
Yeap Foo, 2001; Matsingou, Petrakis, Kapsokefalou, & Salifoglou,
2003; Ramos et al., 2010). In folk medicine, sage is used as an herbal tea and for healing wounds, as well as for alleviating stomach,
liver, and rheumatic pain (Kelen & Tepe, 2008; Sokovic, Tzakou,
Pitarokili, & Couladis, 2002). Moreover, this Lamiaceae has interesting pharmacological properties, such as antioxidant, anti-inammatory, analgesic, antipyretic, homeostatic, hypoglyacemic, and
antitumour activities (Fiore et al., 2006). While the phenolic composition of this plant has been already described, few works have
focused on the irradiation of ground or powdered sage leaves
(Brandstetter, Berthold, Isnardy, Solar, & Elmadfa, 2009; Nagy, Solar, Sontag, & Koenig, 2011; Prez, Banek, & Croci, 2011) and no
studies on the c-irradiation effect on whole sage leaves have been
reported. Thus, in the present study, the effect of ionising radiation
of sage leaves on their total phenolic content and antioxidant potential was examined. In addition, parathion-pretreated leaves of
this medicinal plant were investigated in comparison to control
non-pretreated samples, in order to check the effectiveness of the
irradiation treatment on parathion residues.
2. Materials and methods
2.1. Chemicals and reagents
Polyphenolic standards, DPPH (2,2-diphenyl-1-picrylhydrazyl),
ABTS [2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)],
Trolox
(6-hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic
acid), parathion, paraoxon, aminoparathion and p-aminophenol
were from SigmaAldrich (St. Louis, MO). FolinCiocalteu reagent
(100%), methanol and formic acid were from Fluka (St. Louis,
MO). Stock solutions of analytical standards (100 ppm) were prepared in acetone and stored in darkness at 20 C. The solvents
used were of analytical grade. Water was distilled and ltered
through a Milli-Q apparatus before use.
2.2. Biological material
Fresh young sage leaves harvested in FebruaryMarch were
kindly provided by the National Institute of Agriculture of Tunisia
(INAT). The leaves were homogeneously sampled based on their
weight and size with variation not exceeding 10%. The leaves were
washed in distilled water for 3 min, dried between Whatman lter
papers, and then divided into six batches (5 leaves each batch).

1399

with a diameter of 9.7 mm and an overall length of 452 mm. The

starting activity of the source was 99.162 kCi. The installation is
equipped with a stainless steel telescopic source rack that allows
obtaining a linear source of approximately 900 mm height. The
source pencils are distributed circularly on a diameter of 140 mm
for the upper source rack and of 80 mm for a lower one. The source
rack comprises 20 housings allowing sources loading for several
years. These sources are stored in dry conditions in a cylindrical
shield container in which they were transported. Salvia leaf samples were exposed to gamma radiation to an overall average range
of doses between 0 and 4 kGy at a dose rate of 22.21 Gy/min and at
room temperature (27 2 C).
2.4. Irradiation of parathion and chlorogenic acid standards
Parathion standard preparation (1 ppm) was obtained by diluting stock solutions in water. Chlorogenic acid was prepared in
water at the equivalent concentration (540 ppm) of the polyphenolic extract of the sage leaves neither pretreated with parathion nor
irradiated (0 kGy). Samples were exposed to gamma radiation at
dose levels of 2 and 4 kGy. Non-irradiated (0 kGy) solutions were
kept at room temperature and used as a control for comparative
analysis. Each experiment was done in triplicate. In order to avoid
compounds hydrolysis, the samples were immediately used for
analytical investigation.
2.5. Polyphenols extraction and content estimation
One gram of sage leaf was ground in the presence of 10 ml of
cold acetone/water (3:1 v/v, kept at 20 C). The mixture was sonicated for 20 min, and then centrifuged at 4000 rpm for 15 min at
room temperature. The supernatant was collected, and acetone
was evaporated at 40 C on a rotary evaporator (Fattouch et al.,
2007). To prevent oxidation of the polyphenols, extraction was
achieved rapidly and extracts were immediately used or conserved
in darkness at 20 C until further use.
Prior to HPLC analysis, the total phenolic content (TPC) of the
Salvia leaves extracts was estimated spectrometrically by the
FolinCiocalteu method, as described by Dhaouadi et al. (2013)
with slight modications. Briey, 100 ll of diluted sample were
added to 400 ll of 1:10 diluted FolinCiocalteu reagent. After
5 min, 500 ll of 10% (w/v) sodium carbonate solution were added.
Following 1 h of incubation at room temperature, the absorbance
at 765 nm was measured in triplicate. TPC was calculated from
the equation determined from linear regression after plotting
known solutions of gallic acid (10100 ppm). Results are expressed
in mg of gallic acid equivalent (GAE) per gram of fresh weight (fw)
of plant material.
2.6. Assessment of antioxidant capacity

2.3. Pretreatment with parathion and c irradiation

Three batches were treated by presoaking leaves in a freshly
prepared parathion solution diluted in water at recommended
concentration (1 ppm) for agricultural uses. The rst and second
batches were respectively irradiated at 2 and 4 kGy, while the third
was non-irradiated and used as a control. The fourth and fth (irradiated with 2 and 4 kGy, respectively) as well as the sixth (nonirradiated) batches were presoaked in distilled sterile water instead of parathion. Prior to irradiation, all batches were gravitationally drained for 1 h, and then transferred to labelled glass
test tubes (one leaf per tube). During the irradiation step, the
non-irradiated samples were kept at room temperature (27 2 C).
The Tunisian gamma irradiation facility (at Sidi Thabet) is designed for the preservation of foodstuff and sterilisation of medical
devices. The source consists of eight encapsulated 60Co pencils

The antioxidant activity of the polyphenolic extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a free radical
(Najjaa, Zerria, Fattouch, Ammar, & Neffati, 2011). The DPPH scavenging reaction was performed in polypropylene tubes at room
temperature. One millilitre of a 4  105 M methanolic solution of
DPPH was added to 25 ll of the sample. The mixture was shaken
vigorously and left in the dark at room temperature for 60 min.
The absorbance of the resulting solution was measured at
517 nm. Methanol was used as a blank solution, and DPPH solution
added to 25 ll of distilled water served as control. The antiradical
activity was also assessed using a second functional test based on
the ABTS scavenging capacity as described by Dhaouadi et al.
(2013). The absorbance of the reactive mixture was measured at
734 nm and compared to the antioxidant potency of Trolox used
as a reference. The results were expressed in terms of Trolox

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I. Ben Salem et al. / Food Chemistry 141 (2013) 13981405

equivalent antioxidant capacity (TEAC) calculated from the

equation determined from linear regression after plotting known
solutions of Trolox (0.020.8 mM). The anti-radical activity was
also expressed as the inhibition percentage as calculated using
the following formula:

HP 5890 chromatograph connected to an HP 5970 mass detector

with an electron energy of 70 eV.

%inhibition control OD  sample OD=control OD  100

Chlorogenic and parathion spectra (200800 nm) were

obtained with an S-22 UV/vis spectrophotometer (Boeckel + Co
(GmbH + Co), Hamburg, Germany).

2.10. UVvis spectroscopy

2.7. RP-HPLCESI-MS Conditions

2.11. Statistical analysis
Phenolic compounds were analysed using a modication of a
previously described reversed-phase high-performance liquid
chromatography (RP-HPLC) technique (Fattouch et al., 2007). A
Beckman system having a UV detector model D 166 was used.
The data were processed with Gold Analysis Chromatography Data
Station Software V1.5. The separation was achieved on a C18 column at ambient temperature. The mobile phase comprised (A)
1% formic acid in Milli-Q water and (B) methanol, which were previously degassed. The solvent gradient started at 100% A, reaching
70% A at 10 min, 55% A at 30 min, 55% A at 35 min, 50% A at 40 min,
45% A at 45 min, 30% A at 50 min, 25% A at 53 min, 20% A at 56 min,
and 95% A at 60 min, followed by a post-time isocratic for 10 min
at 95% A before the next injection. The ow rate was 1 ml/min,
and the injection volume was 20 ll. The monitoring wavelengths
were 280 nm and 350 nm. A liquid chromatographyelectrospray
ionisation mass spectrometry (LCESI-MS) operated under previously described conditions (Dhaouadi et al., 2013) was used for
the identication and conrmation of the phenolic compounds
based on their specic and characteristic molecular ions.
2.8. Extraction of parathion and derivatives
The extraction and analysis of parathion, paraoxon, aminoparathion and p-aminophenol were performed using the downscaled
and slightly modied method described by Salghi et al. (2012). A
representative sample (2 g) of parathion-treated sage leaf was
ground and mixed thoroughly with 1.5 ml of acetone prior to
extraction. The mixture was sonicated for 30 min and then the
extract was ltered using a CHROMABOND vacuum SPE manifold
(Macherey-Nagel, Dren, Germany). Then, the acetone extract
was partitioned with a saturated sodium chloride solution
(0.3 ml) and dichloromethane (0.7 ml) following centrifugation at
8000 rpm. The dichloromethane fraction was evaporated using a
rotary evaporator at 40 C. The extract was transferred to a Florisil
clean-up column. The elution was achieved with a mixture of
diethyl ether/n-hexane (6:4, v/v).
2.9. Gas chromatography (GC/ECD)
The eluted parathion and derivatives were appropriately
diluted with n-hexane to be analysed by gas chromatography. GC
was performed using a HewlettPackard 6890 gas chromatograph
(Palo Alto, CA), equipped with an electron capture detector (ECD)
and a capillary column of 25 m  0.32 mm i.d.  0.52 lm lm
thickness, of 5% phenylmethyl polysiloxane HP-5. The oven temperature was programmed to start at 80 C, followed by an increase
of 15 C/min to 250 C, held for 10 min. Injector and detector temperatures were set at 250 C; injection volume 1 ll. The carrier gas
ow was helium (He) at 2.6 ml min1; anode gas: detector gas (N2)
ow was 10 ml min1 and the make-up (N2) was at 60 ml min1.
The freshly working standard solutions were obtained by dilutions
with n-hexane. The detector response for the tested compounds
was linear in the range of the concentrations studied (0.001
1 ppm). Peaks were also identied in comparison to authentic
standards and by mass spectrometry (GCMS) analysis using an

All determinations were obtained from triplicate measurements

and averaged. Presented data were expressed as mean standard
deviation. One-way analysis of variance with Dunnetts post-test
was performed using GraphPad Prism version 5.04 for Windows
(GraphPad Software, San Diego, CA). Statistical signicance was
declared at p < 0.05 or p < 0.01.
3. Results and discussions
3.1. Effect of ionising radiation on polyphenols
Food irradiation is a controlled application of ionising radiation
to improve hygiene, safety and reduce microbial load, thus extending the shelf life of perishable food products (Alothman, Bhat, &
Karim, 2009). The herein obtained phenolic contents of the nontreated sage leaves (36.5 2.35 mg GAE/g fw) are in agreement
with those reported for another Salvia species, Salvia sclarea, commonly known as Clary sage, where the total phenolic content ranged from 38.3 to 97.8 mg GAE/g (Tulukcu, Sagdic, Albayrak, Ekici, &
Yetim, 2009). The used cold aqueous-acetone extraction had been
previously shown to provide a good extraction of the main polyphenols from quince peel and pulp matrices (Fattouch et al.,
2007). This procedure permits the removal of contaminant substances, and particularly precipitates proteins and peptides that
could interfere with polyphenols in HPLC analysis when absorbing
at 280 nm. In addition, the procedure reduces the number of
extraction steps, particularly avoiding preparative chromatographic SPE or SPME that is required when more polar solvents
(water or methanol) are used for extraction. The herein used rapid
process should preserve the native form of the polyphenols, which
is indispensable in the assessment of their functional potentials,
such as antiradical capacity. The low phenolic content of the prepared sage extracts in the present work could be expected when
using young leaves which did not yet accumulate phenols. The
polyphenolic content (Fig. 1) of the extracts obtained from Salvia
leaves irradiated at 2 kGy, as determined by the FolinCiocalteu
method, decreased signicantly (p < 0.05) by 30% (25.5
1.79 mg GAE/g fw) compared to the non-irradiated samples. This
decrease is more pronounced for the samples treated at 4 kGy
reaching 45% (20.2 0.92 mg GAE/g fw). Yalcin, Ozturk, Tulukcu,
and Sagdic (2011) studied the irradiation effects on Clary sage (S.
sclarea L.) seeds and found that the total phenolic content of the
samples decreased at 5.5 kGy irradiation, whereas it was slightly
increased at 2.5 and 7.5 kGy. Similar decrease of the polyphenols
content was reported in the literature for cinnamon following irradiation at 2025 kGy (Kitazuru, Moreira, Mancini-Filho, Delince, &
Villavicencio, 2004). Calucci et al. (2003) evaluated the effects of
gamma irradiation (010 kGy) on a collection of aromatic herbs
and spices (basil, bird pepper, black pepper, cinnamon, nutmeg,
oregano, parsley, rosemary, and sage) and noticed a signicant decrease in phenolic compounds. These reports explained the loss of
phenols by the damaging effect of ionising radiation on the phenolic compounds (Sato, Hiraoka, & Sakumat, 1993) which may be also
happening in our study.

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I. Ben Salem et al. / Food Chemistry 141 (2013) 13981405

Fig. 1. Effect of ionising radiation (24 kGy) on the polyphenolic content of aqueous-acetone extracts of Salvia leaves pretreated or not with parathion. The data are presented
as means of three independent experiments (SD < 10%).

Previous analytical investigations showed that health-promoting potentials of sage leaves, i.e. anti-inammatory, anti-allergic,
anti-fungal and antiseptic properties, are thought to be due to their
biologically active phenols. Qualitative and quantitative analysis of
sage leaves polyphenolic extracts (Table 1) showed that the main
compounds were represented by luteolin and rosmarinic, fumaric,
chlorogenic, caffeic and carnosic acids, a nding in agreement with
Zimmermann, Walch, Tinzoh, Stuhlinger, and Lachenmeier (2011).
In order to conrm the irradiation effect on phenolic compounds,
an aqueous solution of the standard chlorogenic acid prepared at
540 ppm, equivalent to that determined for this compound in the
phenolic extract of the sage leaf (Table 1), was irradiated at 2
and 4 kGy. For clarity, in Fig. 2, only two superimposed UVvis
spectra of each triplicated radiation treatment of the standard
chlorogenic acid were illustrated. The non-irradiated compound,
as expected under the above-described experimental conditions,
showed two peaks at 287 nm and 326 nm; while the spectrum of
the irradiated samples at 2 and 4 kGy presented only one peak at
280 mm (Fig. 2). The characteristic 325 nm peak of the chlorogenic
acid disappeared at 2 and 4 kGy suggesting a degradative process.
This compound is composed of two molecules, caffeic acid and quinic acid (Clifford, Wu, Kirkpatrick, & Kuhnert, 2007). As suggested
in previous reports (Sato et al., 1993), the irradiation treatment
caused the hydrolysis of chlorogenic acid into its two derivatives
that absorb at 280 nm. RP-HPLC analysis conrmed this nding
and allowed us to estimate the rate of chlorogenic acid degradation
to 57% and 84% at 2 and 4 kGy, respectively. The non-irradiated
preparations did not show the peaks of caffeic and quinic acids,
which were only detected in the irradiated samples, supporting
the purity of the starting preparation and the non-spontaneous
degradation of the chlorogenic acid under the experimental conditions. The data found in the literature about the susceptibility of
polyphenols to irradiation are disparate, maybe due to different
phenolic compositions of the studied samples. Prez et al. (2011)
found a signicant irradiation effect on the phenolic content of
the powdered oregano extracts as well as their antioxidant potential, whereas they did not notice these facts when irradiating powdered sage extracts. Nagy et al. (2011) examined phenolic
components in dried and ground spices, including sage, and found
that irradiation did not induce the cleavage of a glycoside bond or
the release of caffeic acid from rosmarinic acid or their derivatives.
In contrast, Krimmel, Swoboda, Solar, and Reznicek (2010) investigated caffeic acid and derivatives in aqueous solution and noticed

that irradiation initiated the release of caffeic acid from chlorogenic acid as well as from rosmarinic acid.
3.2. Effect of ionising radiation on the antioxidant capacity
Based on the TEAC analysis, the determined antioxidant activities of the studied samples showed that the extracts of the non-irradiated sage leaves exhibited 21.2 0.93 and 76.4 4.03 lmol TEAC/
100 g DW as assessed by DPPH and ABTS assays, respectively. In the
case of the 2 kGy irradiated samples, this potential decreased, in
DPPH (Fig. 3) and ABTS (Fig. 4) tests, by 20% and 11%, respectively;
while for the samples exposed to 4 kGy, this decrease reached 40%
and 44% (p < 0.01). Using the standard chlorogenic acid, a signicant increase (p < 0.01) of the antioxidant activity was observed
as the irradiation treatment increased. This observation is probably
due to newly formed compounds and derivatives produced by
Table 1
LCESI-MS characteristics of the identied polyphenols in Salvia ofcinalis extract.
Data presented are means standard deviation (n = 3). NQ: not quantied.
Peak
number

Retention
time (RT)

kmax

[MH]

Compound

Content
(mg/g fw)

Protocatechuic
acid
Chlorogenic acid
Caffeic acid
Salvianolic acid I
isomer
Luteolin-7-oglucoside
Luteolinrutinoside
Luteolinrutinoside
isomer
Luteolin-7-oglucuronide
Rosmarinic acid
Apigeninrutinoside
Salvianolic acid B
Salvianolic acid K
Rosmanol isomer
Carnosic acid
Carnosic acid
isomer

1.35 0.15

6.68

263

153

2
3
4

10.59
11.79
23.93

326
324
287

353
179
537

26.27

265

447

26.41

252

593

29.94

252

593

30.77

255

461

9
10

31.39
35.28

329
280

359
577

11
12
13
14
15

39.26
40.76
44.26
48.24
49.45

285
287
285
283
221

717
555
345
331
285

Total

0.54 0.22
0.27 0.18
NQ
0.94 0.11
0.77 0.13
NQ

1.53 0.10
0.47 0.20
0.98 0.12
1.86 0.11
1.42 0.29
NQ
2.17 0.53
NQ
12.3 2.14

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I. Ben Salem et al. / Food Chemistry 141 (2013) 13981405

Fig. 2. Spectrum of non-irradiated (0 kGy) and irradiated (24 kGy) chlorogenic acid prepared at 540 ppm. No absorbance was recorded in the interval 400800 nm.

c-irradiation. However, in the case of sage extracts, the noticed decrease of antioxidant capacity when irradiated suggests that the
derivatives generated from the different phenols did not compensate the degradative processes, especially, the modication of the
oxidisable groups, as suggested in previous reports (Sato et al.,
1993). Using the functional DPPH and ABTS+ assays, similar trends
were obtained suggesting that irradiation affected both anionic and
cationic radical-scavenging compounds. Murcia et al. (2004) found
that irradiated (110 kGy) dessert spice samples did not show differences in TEAC values from non-irradiated ones. Conversely, in

Xanthium occidentale, c-radiation of chlorogenic acid stimulated

the formation of unstable o-quinone, as demonstrated by conversion to the stable derivative, 6-benzosulfonylchlorogenate when
reacting with benzenesulnate (Sato et al., 1993). Yalcin et al.
(2011) reported that irradiation at 2.5 and 4 kGy doses have negative effects on the antioxidant activity of Clary sage seed. The herein
observed reduction of the antioxidant activity of the irradiated sage
leaves may be due to free radicals formed as a result of irradiation
treatment which could be competitive to DPPH or ABTS radicals in
the used assays. Arici, Arslan, and Gecgel (2007) reported that

Fig. 3. Effect of ionising radiation on the DPPH scavenging capacity of the standard chlorogenic acid and the Salvia leaves extracts pretreated or not with parathion (p < 0.05).

I. Ben Salem et al. / Food Chemistry 141 (2013) 13981405

1403

3.3. Effect of c-radiation on parathion

Fig. 4. Effect of ionising radiation on the ABTS scavenging capacity of the standard
chlorogenic acid and the Salvia leaves extracts pretreated or not with parathion
(p < 0.05).

radicals and induced molecules form as a result of irradiation exposure. These free radicals can react with O2 in the long run and cause
the formation of hydroperoxides, which create alcohols, aldehydes,
aldehyde esters, and hydrocarbons.

Presently, food contamination by pesticide residues is pervasive

due to the increased use of these toxic chemicals. Parathion belongs to the family of organophosphorous compounds which are
the most widely used pesticides by virtue of their biodegradable
nature and short persistence. Until now, it is not established that
parathion is a human carcinogen; however, it has recently been reported that this substance induced mammary tumours after subcutaneous injection in rats (Cabello et al., 2001). The herein nonirradiated parathion standard showed an absorption spectrum
with a maximum at 220 nm (Fig. 5); whereas, for the c-irradiated
sample, no peak was observed neither at 2 kGy nor at 4 kGy. The
GC analysis conrmed the degradation of parathion and allowed
the clear detection of the parent compound, parathion, and its
derivatives, paraoxon, aminoparathion and p-aminophenol
(Fig. 5). The chromatographic technique allowed not only qualitative but also quantitative estimation of the peaks. At 4 kGy, only
18% of the initial parathion were present in the solution, aminoparathion was the main (57.3%) derivative present, whereas paraoxon was not detected. Even though the irradiation doses used in
this work are not that high, it seems that parathion, used at an initial concentration of 1 ppm, was considerably degraded by gamma
rays, particularly at 4 kGy. This radiolysis behaviour of parathion
was previously studied by Luchini, Peres, and Rezende (1999),
who found that the required dose for parathion radiolytic degradation in methanol was somewhat higher than in aqueous solutions.
These authors suggested that the parathion degradation by gamma
radiation was probably due to the indirect effect of the radiation on
the molecules of the pesticide, i.e., the solvent molecules are ionised by the radiation energy and then react with the pesticide.

Fig. 5. HPLC chromatograms of the non-irradiated (above) and irradiated at 2 Gy (below) parathion solution (1 ppm). The inset presents the spectrum of the non-irradiated
(0 kGy) and the irradiated (2, 4 kGy) parathion standard.

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I. Ben Salem et al. / Food Chemistry 141 (2013) 13981405

Thus, the gamma radiolytic treatment could be considered as an

efcient and promising process to detoxify parathion contamination, and, maybe, other structurally related organophosphates.
Rung and Schwack (2005) demonstrated that the treatment of
parathion-spiked apple fruits with simulated sunlight resulted in
nearly complete photo-degradation of the compound within 13 h.
The authors detected different photoproducts in the fruit cuticle
environment including amino-parathion. Rung and Schwack
(2005) also found that in the presence of polyphenols, amino-parathion was effectively bound to quinone intermediates formed by
both silver oxide and polyphenol oxidases. The action of polyphenol oxidases during the irradiation process should not be excluded,
since binding of pesticides having phenolic structures, including
parathion, to this enzyme has been previously reported (Fattouch
et al., 2010).

since it selectively degrades the studied toxicant organophosphorus pesticide. The same result was observed with pure chlorogenic
acid, showing that an irradiation dose of 2 kGy was benecial,
especially, as its antioxidant capacity was increased. Thus, sterilisation of food by ionising radiation should be able to break down
pesticide residues in foods without inducing signicant losses in
polyphenols.

3.4. c-Radiation effects on Salvia leaf polyphenols in the presence of

parathion

References

The obtained data showed that the phenolic content of the

extracts obtained from Salvia leaves treated with parathion was
higher than the non-treated ones (Fig. 1). While c-irradiation had
decreased polyphenolic content of sage leaves, the presence of
parathion reduced the loss of phenols during irradiation at 2 and
4 kGy in comparison to its absence. This result suggests that
parathion might have the capacity to absorb c rays, thus protecting polyphenols from radiolysis. The limitation of the loss of polyphenols present in the Salvia leaves treated with parathion was
conrmed by HPLC analysis. Consequently, the presence of this
pesticide on Salvia leaves prior to irradiation treatment was benecial for the preservation of the polyphenolic compounds of this
medicinal plant. Even though slight losses of the health-promoting
polyphenols were recorded, the induced breakdown of toxicants,
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3.5. c-Radiation effects on antioxidant potential in the presence of
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Polyphenols, natural antioxidants present in plant extracts, play
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(p > 0.05) difference between the leaves pre-treated and those
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reduced the expected decrease of anti-oxidative activity following
irradiation process by 23% and 30% in DPPH and ABTS tests, respectively. These results conrm the capacity of parathion to absorb
gamma rays and its potential to preserve polyphenols and their
antioxidant activities.
4. Conclusion
Gamma radiolysis degradation is an important process, which
has recently gained attention as a potential food safety technology.
This method has proved successful for the destruction of various
classes of organic pollutants, such as pesticides. In the current
study, we found that the polyphenols content and the antioxidant
activity of extracts of Salvia leaves pretreated with parathion were
better conserved than in leaves not pretreated with parathion. This
should support ionising irradiation as a technological food process

Acknowledgements
The authors acknowledge the nancial support from the Ministry of Higher Education and Scientic Research (Tunisia). This work
was nancially supported in part by the Tunisian-Moroccan scientic research projects (11/MT/54; 22/MT/06; 38/MT/08).

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