Effect of ionising radiation on polyphenolic content and antioxidant potential of parathion-treated sage (Salvia officinalis) leaves

Food Chemistry 141 (2013) 1398–1405

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Effect of ionising radiation on polyphenolic content and antioxidant potential of parathion-treated sage (Salvia officinalis) 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 officinalis

a b s t r a c t The c-irradiation effects on polyphenolic content and antioxidant capacity of parathion-pretreated leaves of Salvia officinalis plant were investigated. The analysis of phenolic extracts of sage without parathion showed that irradiation decreased polyphenolic content significantly (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 significantly (p < 0.01) at 2 and 4 kGy, by 11–20% and 40–44%, respectively. The results obtained with a pure chlorogenic acid solution confirmed the degradation of phenols; however, its TEAC was significantly (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 significant losses in polyphenols. Ó 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 significantly altered following irradiation >10 kGy (Koseki et al., 2002), whereas, the capsaicinoids increased significantly, 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: [email protected] (I. Ben Salem). 1 These authors contributed equally to the work. 0308-8146/$ - see front matter Ó 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 chiefly 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 flavouring 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 officinalis, is one of the most wellknown aromatic herbs. Sentences such as ‘‘Why should a man die while sage grows in his garden?’’ reflect the importance of this plant in traditional medicine (Ramos, Azqueta, Pereira-Wilson, &

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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 beneficial 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-inflammatory, 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; Pérez, 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 Sigma–Aldrich (St. Louis, MO). Folin–Ciocalteu 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 filtered through a Milli-Q apparatus before use. 2.2. Biological material Fresh young sage leaves harvested in February–March 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 filter papers, and then divided into six batches (5 leaves each batch).

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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 Folin–Ciocalteu method, as described by Dhaouadi et al. (2013) with slight modifications. Briefly, 100 ll of diluted sample were added to 400 ll of 1:10 diluted Folin–Ciocalteu 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 (10–100 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 first 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 fifth (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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equivalent antioxidant capacity (TEAC) calculated from the equation determined from linear regression after plotting known solutions of Trolox (0.02–0.8 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 ¼ ½ðcontrol OD  sample ODÞ=control OD  100

Chlorogenic and parathion spectra (200–800 nm) were obtained with an S-22 UV/vis spectrophotometer (Boeckel + Co (GmbH + Co), Hamburg, Germany).

2.10. UV–vis spectroscopy

2.7. RP-HPLC–ESI-MS Conditions 2.11. Statistical analysis Phenolic compounds were analysed using a modification 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 flow rate was 1 ml/min, and the injection volume was 20 ll. The monitoring wavelengths were 280 nm and 350 nm. A liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS) operated under previously described conditions (Dhaouadi et al., 2013) was used for the identification and confirmation of the phenolic compounds based on their specific 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 modified 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 filtered using a CHROMABOND vacuum SPE manifold (Macherey-Nagel, Düren, 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 Hewlett–Packard 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 film thickness, of 5% phenyl–methyl 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 flow was helium (He) at 2.6 ml min1; anode gas: detector gas (N2) flow 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 identified in comparison to authentic standards and by mass spectrometry (GC–MS) 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 Dunnett’s post-test was performed using GraphPad Prism version 5.04 for Windows (GraphPad Software, San Diego, CA). Statistical significance 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 Folin–Ciocalteu method, decreased significantly (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 20–25 kGy (Kitazuru, Moreira, Mancini-Filho, Delincée, & Villavicencio, 2004). Calucci et al. (2003) evaluated the effects of gamma irradiation (0–10 kGy) on a collection of aromatic herbs and spices (basil, bird pepper, black pepper, cinnamon, nutmeg, oregano, parsley, rosemary, and sage) and noticed a significant 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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Fig. 1. Effect of ionising radiation (2–4 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-inflammatory, 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 finding in agreement with Zimmermann, Walch, Tinzoh, Stuhlinger, and Lachenmeier (2011). In order to confirm 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 UV–vis 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 confirmed this finding 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. Pérez et al. (2011) found a significant 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 significant 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 LC–ESI-MS characteristics of the identified polyphenols in Salvia officinalis extract. Data presented are means ± standard deviation (n = 3). NQ: not quantified. 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

1

6.68

263

153

2 3 4

10.59 11.79 23.93

326 324 287

353 179 537

5

26.27

265

447

6

26.41

252

593

7

29.94

252

593

8

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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Fig. 2. Spectrum of non-irradiated (0 kGy) and irradiated (2–4 kGy) chlorogenic acid prepared at 540 ppm. No absorbance was recorded in the interval 400–800 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 modification 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 (1–10 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 benzenesulfinate (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).

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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 confirmed 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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Thus, the gamma radiolytic treatment could be considered as an efficient 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 beneficial, 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 significant 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 confirmed by HPLC analysis. Consequently, the presence of this pesticide on Salvia leaves prior to irradiation treatment was beneficial 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, like organophosphorus pesticides, supports the usefulness of this technological preservative method applied in food processing. 3.5. c-Radiation effects on antioxidant potential in the presence of parathion Polyphenols, natural antioxidants present in plant extracts, play a key role in anti-oxidative defence mechanisms in biological systems and act as free radical scavenging agents (Engelhard, Gazer, & Paran, 2006). Polyphenols present in the plant S. officinalis are known for their therapeutic effects (Kasimu et al., 1998). The results obtained from the measurement of the antiradical activity of Salvia polyphenolic extracts confirm that there is no significant (p > 0.05) difference between the leaves pre-treated and those not pretreated with parathion at 0 and 2 kGy (Figs. 3 and 4). However, the presence of parathion on leaves irradiated at 4 kGy reduced the expected decrease of anti-oxidative activity following irradiation process by 23% and 30% in DPPH and ABTS tests, respectively. These results confirm 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 financial support from the Ministry of Higher Education and Scientific Research (Tunisia). This work was financially supported in part by the Tunisian-Moroccan scientific research projects (11/MT/54; 22/MT/06; 38/MT/08).

Alothman, M., Bhat, R., & Karim, A. A. (2009). Effects of radiation processing on phytochemicals and antioxidants in plant produce. Trends in Food Science and Technology, 20, 201–212. Arici, M., Arslan, F. A., & Gecgel, U. (2007). Effect of gamma radiation on microbiological and oil properties of black cumin (Nigella sativa L.). Grasas y Aceites, 58, 339–343. Brandstetter, S., Berthold, C., Isnardy, B., Solar, S., & Elmadfa, I. (2009). Impact of gamma-irradiation on the antioxidative properties of sage, thyme, and oregano. Food and Chemical Toxicology, 47, 2230–2235. Cabello, G., Valenzuela, M., Vilaxa, A., Duran, V., Rudolph, I., Hrepic, N., & Calaf, G. (2001). A rat mammary tumor model induced by the organophosphorous pesticides parathion and malathion, possibly through acetylcholinesterase inhibition. Environmental Health Perspectives, 109(5), 471–479. Calucci, L., Pinzino, C., Zandomeneghi, M., Capocchi, A., Ghiringhelli, S., Saviozzi, F., Tozzi, S., & Galleschi, L. (2003). Effects of gamma-irradiation on the free radical and antioxidant contents in nine aromatic herbs and spices. Journal of Agricultural and Food Chemistry, 51(4), 927–934. Clifford, M. N., Wu, W., Kirkpatrick, J., & Kuhnert, N. (2007). Profiling the chlorogenic acids and other caffeic acid derivatives of herbal chrysanthemum by LC–MSn. Journal of Agricultural and Food Chemistry, 55(3), 929–936. Dhaouadi, K., Raboudi, F., Funez-Gomez, L., Pamies, D., Estevan, C., Hamdaoui, M., & Fattouch, S. (2013). Polyphenolic extract of barbary-fig (Opuntia ficus-indica) syrup: RP-HPLC–ESI-MS analysis and determination of antioxidant, antimicrobial and cancer-cells cytotoxic potentials. Food Analytical Methods, 6, 45–53. Engelhard, Y. N., Gazer, B., & Paran, E. (2006). Natural antioxidants from tomato extract reduce blood pressure in patients with grade-1 hypertension: A doubleblind, placebo-controlled pilot study. American Heart Journal, 151(1), 100. Farkas, J. (1998). Irradiation as a method for decontaminating food: A review. International Journal of Food Microbiology, 44(3), 189–204. Fattouch, S., Caboni, P., Coroneo, V., Tuberoso, C. I., Angioni, A., Dessi, S., Marzouki, N., & Cabras, P. (2007). Antimicrobial activity of Tunisian quince (Cydonia oblonga Miller) pulp and peel polyphenolic extracts. Journal of Agricultural and Food Chemistry, 55(3), 963–969. Fattouch, S., Raboudi-Fattouch, F., Ponce, J. V., Forment, J. V., Lukovic, D., Marzouki, N., & Vidal, D. R. (2010). Concentration dependent effects of commonly used pesticides on activation versus inhibition of the quince (Cydonia oblonga) polyphenol oxidase. Food and Chemical Toxicology, 48(3), 957–963. Fattouch, S., Sadok, S., Raboudi-Fattouch, F., & Slama, M. B. (2008). Damage inhibition during refrigerated storage of mackerel (Scomber scombrus) fillets by a presoaking in quince (Cydonia oblonga) polyphenolic extract. International Journal of Food Science and Technology, 43, 2056–2064. Fiore, G., Nencini, C., Cavallo, F., Capasso, A., Bader, A., Giorgi, G., & Micheli, L. (2006). In vitro antiproliferative effect of six Salvia species on human tumor cell lines. Phytotherapy Research, 20(8), 701–703. Heim, K. E., Tagliaferro, A. R., & Bobilya, D. J. (2002). Flavonoid antioxidants: chemistry, metabolism and structure–activity relationships. Journal of Nutritional Biochemistry, 13(10), 572–584. Kasimu, R., Tanaka, K., Tezuka, Y., Gong, Z. N., Li, J. X., Basnet, P., Namba, T., & Kadota, S. (1998). Comparative study of seventeen Salvia plants: aldose reductase inhibitory activity of water and MeOH extracts and liquid chromatography– mass spectrometry (LC–MS) analysis of water extracts. Chemical and Pharmaceutical Bulletin, 46(3), 500–504. Kelen, M., & Tepe, B. (2008). Chemical composition, antioxidant and antimicrobial properties of the essential oils of three Salvia species from Turkish flora. Bioresources Technology, 99(10), 4096–4104. Kitazuru, E. R., Moreira, A. V. B., Mancini-Filho, J., Delincée, H., & Villavicencio, A. L. C. H. (2004). Effects of irradiation on natural antioxidants of cinnamon (Cinnamomum zeylanicum N.). Radiation Physics and Chemistry, 71, 39–41. Koseki, P. M., Villavicencio, A. L. C. H., Brito, M. S., Nahme, L. C., Sebastiao, K. I., Rela, P. R., Almeida-Muradian, L. B., Mancini-Filho, J., & Freitas, P. C. D. (2002). Effects of irradiation in medicinasl and eatable herbs. Radiation Physics and Chemistry, 63, 681–684.

I. Ben Salem et al. / Food Chemistry 141 (2013) 1398–1405 Krimmel, B., Swoboda, F., Solar, S., & Reznicek, G. (2010). OH-radical induced degradation of hydroxybenzoic- and hydroxycinnamic acids and formation of aromatic products – a gamma radiolysis study. Radiation Physics and Chemistry, 79(12), 1247–1254. Lu, Y., & Yeap Foo, L. (2001). Antioxidant activities of polyphenols from sage (Salvia officinalis). Food and Chemical Toxicology, 75, 197–202. Luchini, L. C., Peres, T. B., & Rezende, M. O. D. (1999). Degradation of the insecticide parathion in methanol by gamma-irradiation. Journal of Radioanalytical and Nuclear Chemistry, 241, 191–194. Matsingou, T. C., Petrakis, N., Kapsokefalou, M., & Salifoglou, A. (2003). Antioxidant activity of organic extracts from aqueous infusions of sage. Journal of Agricultural and Food Chemistry, 51(23), 6696–6701. Murcia, M. A., Egea, I., Romojaro, F., Parras, P., Jimenez, A. M., & Martinez-Tome, M. (2004). Antioxidant evaluation in dessert spices compared with common food additives. Influence of irradiation procedure. Journal of Agricultural and Food Chemistry, 52(7), 1872–1881. Nagy, T. O., Solar, S., Sontag, G., & Koenig, J. (2011). Identification of phenolic components in dried spices and influence of irradiation. Food Chemistry, 128, 530–534. Najjaa, H., Zerria, K., Fattouch, S., Ammar, E., & Neffati, M. (2011). Antioxidant and antimicrobial activities of Allium roseum. ‘‘lazoul’’, a wild edible endemic species in North Africa. International Journal of Food Properties, 14, 371–380. Pérez, M. B., Banek, S. A., & Croci, C. A. (2011). Retention of antioxidant activity in gamma irradiated Argentinian sage and oregano. Food Chemistry, 126, 121–126. Ramos, A. A., Azqueta, A., Pereira-Wilson, C., & Collins, A. R. (2010). Polyphenolic compounds from Salvia species protect cellular DNA from oxidation and stimulate DNA repair in cultured human cells. Journal of Agricultural and Food Chemistry, 58(12), 7465–7471. Rung, B., & Schwack, W. (2005). Aminoparathion: A highly reactive metabolite of parathion. 1. Reactions with polyphenols and polyphenol oxidase. Journal of Agricultural and Food Chemistry, 53(23), 9140–9145.

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Salghi, R., Luis, G., Rubio, C., Hormatallah, A., Bazzi, L., Gutierrez, A. J., & Hardisson, A. (2012). pesticide residues in tomatoes from greenhouses in Souss Massa Valley, Morocco. Bulletin of Environmental Contamination and Toxicology, 88, 358–361. Sarkardei, S., & Howell, N. K. (2008). Effect of natural antioxidants on stored freezedried food product formulated using horse mackerel (Trachurus trachurus). International Journal of Food Science and Technology, 43, 309–315. Sato, M., Hiraoka, A., & Sakumat, T. (1993). c-Radiation-stimulated formation of ortho-quinone from chlorogenic acid in Xanthium occidentale. Phytochemistry, 32, 281–286. Sokovic, M., Tzakou, O., Pitarokili, D., & Couladis, M. (2002). Antifungal activities of selected aromatic plants growing wild in Greece. Nahrung, 46(5), 317–320. Sommer, I., Schwartz, H., Solar, S., & Sontag, G. (2009). Effect of gamma-irradiation on agaritine, gamma-glutaminyl-4-hydroxybenzene (GHB), antioxidant capacity, and total phenolic content of mushrooms (Agaricus bisporus). Journal of Agricultural and Food Chemistry, 57(13), 5790–5794. Topuz, A., & Ozdemir, F. (2003). Influences of gamma-irradiation and storage on the carotenoids of sun-dried and dehydrated paprika. Journal of Agricultural and Food Chemistry, 51(17), 4972–4977. Tulukcu, E., Sagdic, O., Albayrak, S., Ekici, L., & Yetim, H. (2009). Effect of collection time on biological activity of Clary sage (Salvia sclarea). Journal of Applied Botany and Food Quality, 83, 44–49. WHO (1981). Wholesomeness of irradiated food: Report of a joint FAO/IAEA/WHO Expert Committee. World Health Organization Technical Report Series, 659, 1–34. Yalcin, H., Ozturk, I., Tulukcu, E., & Sagdic, O. (2011). Effect of gamma-irradiation on bioactivity, fatty acid compositions and volatile compounds of Clary sage seed (Salvia sclarea L.). Journal of Food Sciences, 76(7), 1056–1061. Zimmermann, B. F., Walch, S. G., Tinzoh, L. N., Stuhlinger, W., & Lachenmeier, D. W. (2011). Rapid UHPLC determination of polyphenols in aqueous infusions of Salvia officinalis L. (sage tea). Journal Chromatography B Analytical Technologies in the Biomedical Life and Sciences, 879(24), 2459–2464.