EPR investigations of gamma-irradiated ground black pepper

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Radiation Physics and Chemistry 75 (2006) 309–321 www.elsevier.com/locate/radphyschem

EPR investigations of gamma-irradiated ground black pepper Martin Polovkaa, Vlasta Brezova´b,, Andrej Stasˇ kob, Milan Mazu´rb, Milan Suhajc, Peter Sˇimkoc a

Department of Chemical Technology of Wood, Pulp and Paper, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinske´ho 9, SK-812 37 Bratislava, Slovak Republic b Department of Physical Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinske´ho 9, SK-812 37 Bratislava, Slovak Republic c Food Research Institute, Priemyselna´ 4, P.O. Box 25, SK-824 75 Bratislava, Slovak Republic Received 4 January 2005; accepted 22 July 2005

Abstract The g-radiation treatment of ground black pepper samples resulted in the production of three paramagnetic species (GI–GIII) which arise from a different origin and have different thermal behavior and stability. The axially symmetric spectra can be characterized by the spin Hamiltonian parameters: GI (g? ¼ 2:0060, gk ¼ 2:0032; A? ¼ 0:85 mT, Ak ¼ 0:70 mT) and GII (g? ¼ 2:0060, gk ¼ 2:0050; A? ¼ 0:50 mT, Ak ¼ 0:40 mT) assigned to carbohydrate radical structures. The parameters of EPR signal GIII (g? ¼ 2:0029, gk ¼ 2:0014; A? ¼ 3:00 mT, Ak ¼ 1:80 mT) possessed features characteristic of cellulose radical species. The activation energies, evaluated by Arrhenius analysis, are in order Ea(GI)oEa(GIII)oEa(GII). The EPR measurements performed 20 weeks after radiation process confirmed that a temperature increase from 298 to 353 K, caused a significant decrease of integral EPR signal intensity for g-irradiated samples (
40%), compared to the reference (non-irradiated) ground black pepper, where a decrease of
13% was found. The influence of g-radiation treatment on the radical-scavenging activities of aqueous and ethanol extracts of black pepper were investigated by both an EPR spin trapping technique and DPPH assay. No changes were detected in either the water or ethanol extracts for a g-irradiation dose of 10 kGy. r 2005 Elsevier Ltd. All rights reserved. Keywords: Black pepper; g-irradiation; EPR spectroscopy; Free radicals; DPPH; Spin trapping; DMPO

1. Introduction Black pepper—the dried, unripe berry of Piper nigrum L., is a widely used spice which possesses interesting pharmacological/toxicological properties and clinical applications. The main alkaloid contributing towards its pungency is piperine which constitutes from 2% to 7.4% of P. nigrum cultivars (Ravindran, 2000). Piperine Corresponding author. Tel.: +421 2 5932 5666; fax: +421 2 5292 6032. E-mail address: [email protected] (V. Brezova´).

has anti-inflammatory properties and has been tested in vitro as a protection agent in the oxidative damage by inhibiting or quenching free radicals, reactive oxygen species and hydroxyls radicals (Mittal and Gupta, 2000). Studies on both animals and humans have demonstrated that this alkaloid can significantly increase the bioavailability of numerous drugs and nutritional supplements (Atal et al., 1985; Badmaev et al., 1999; Badmaev et al., 2000; Bano et al., 1991; Dhuley et al., 1993; Prasad et al., 2004; Vijayakumar et al., 2004). On the negative side it can potentially produce mutagenic and carcinogenic effects under specific conditions, e.g., when piperine

0969-806X/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2005.07.007

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reacts with nitrites, which are also present in food (Shenoy and Choughuley, 1992). The samples of black pepper are often microbiologically contaminated and contain various dangerous microbes and spores (Huis in ‘t Veld, 1996). The girradiation treatment of foods and plant products is nowadays accepted as a standard and safe sterilization technique which lowers the risk of microbiological contaminations and prolongs the durability of products (Oh et al., 2003). However, efficient customer protection requires unambiguous determination as to whether nutrition products have been exposed to g-irradiation even for long periods after the radiation process (Delince´e and Ehlermann, 1989; Delince´e, 2002; Desrosiers, 1996; Chabane et al., 2001; Esteves et al., 1999; Haire et al., 1997; Raffi et al., 1994; Raffi et al., 2000). Consequently the European Committee for Standardization (CEN) in EN 1787 (2000) proposed an EPR method for detection of foods containing irradiated cellulose and later standardized four additional methods for the detection of irradiated foods, (i.e. EN 13708, 2001; EN 13751, 2002; EN 13783, 2001; EN 13784, 2001). EPR spectroscopy is a unique technique for the detection of paramagnetic species which are formed during the g-radiation process but its application is limited by the lifetime of radiolytically produced free radicals (Bayram and Delince´e, 2004; Delince´e and Soika, 2002; Formanek et al., 1999; Raffi et al., 2000; Yordanov and Gancheva, 2000; Yordanov et al., 1998). The g-radiation treatment of plant products containing cellulose leads to the generation of a typical three-line EPR signal, characterized by a g-value of 2.00607 0.0005 and hyperfine splittings about 370.05 mT (Raffi et al., 2000; Yordanov and Gancheva, 2000; Yordanov et al., 1998). For the purpose of post-radiation identification of cellulose containing foods, the presence of relatively weak satellite lines of this ‘‘cellulosic’’ radical species was accepted as unambiguous evidence for g-radiation treatment (Yordanov and Gancheva, 2000; Yordanov et al., 1998). However, it was observed that the EPR intensity of the ‘‘cellulosic’’ triplet signal gradually decreased with storage time and that the rate of disappearance was dependent on temperature, humidity, presence of oxygen and on further factors (Bayram and Delince´e, 2004; Yordanov and Aleksieva, 2004; Yordanov and Gancheva, 2000; Yordanov et al., 1998). In general the EPR signal disappeared within 70–90 days after irradiation process (Raffi et al., 2000). Consequently the method was deemed to have failed in the verification of g-radiation treatment and consequently it was recommended that thermoluminescence techniques should be used (Delince´e, 2002; Bayram and Delince´e, 2004; Raffi et al., 2000; Yordanov et al., 1998). Recently, Yordanov and Gancheva (2000) pointed to the different thermal behavior of EPR signals of non-

irradiated and g-radiation-treated foods containing cellulose, even after long storage period after radiation, when the specific ‘‘cellulosic’’ EPR signal is extremely low, and recommended this technique as a method to identify g-radiation-processed foods. The main aim of the presented investigation was the detailed study of radical species produced upon gradiation treatment of ground black pepper samples using EPR spectroscopy. In addition the influence of gradiation on the radical-scavenging activity was determined using both EPR spin trapping technique and DPPH assay.

2. Experimental 2.1. Chemicals and materials For the EPR investigations a ground black pepper (density 550 g dm3) from Vietnam was used. This spice was irradiated using a 60Co source at doses of 5, 7.5, 10, 20, and 30 kGy according to commercial practices at Artim, Ltd., Prague, Czech Republic. The mean dry matter content of the ground black pepper after irradiation was of 87.9%. All samples were stored in closed bags in the dark at 6 1C and at a relative humidity of 60%. The EPR measurements were performed 1, 5, 10, 14 and 20 weeks after g-radiation treatment. The spin trapping agent 5,5-dimethyl-1-pyrroline-Noxide (DMPO, Scheme 1), freshly distilled before use and stored at 18 1C under argon, and K2S2O8 were purchased from Aldrich. The stable free radical 1,1diphenyl-2-picrylhydrazyl (DPPH, Scheme 1), from Sigma Chemicals, was used as supplied. Ethanol, of spectroscopic grade (Mikrochem, Slovak Republic), phosphate buffer, prepared according to So¨rensen

N N

.

O2N

H3C H3C

NO2

+ N O DMPO

NO2 DPPH

Scheme 1. Structures of DMPO spin trapping agent and DPPH free radical.

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(by mixing 4 ml of 0.0667 M KH2PO4 with 6 ml 0.0667 M Na2HPO4, pH ¼ 6.98), and distilled water were used in the experiments. The aqueous and ethanol black pepper extracts were prepared by dispersing 1 g of the ground black pepper sample in either 10 ml of distilled water or ethanol, respectively. The suspension was then gently stirred and stored in darkness for 24 h at room temperature. Subsequently, the pepper particles were removed by filtration and the colored liquid extracts were used in the EPR and UV/vis measurements.

2.2. Methods and apparatus 2.2.1. EPR measurements of powder samples The homogenized ground black pepper powders (100 mg) were placed in thin-wall quartz EPR tubes (internal diameter of 3 mm, length of 150 mm, and wall thickness about 0.1 mm) to produce cylindrical samples with identical dimensions (sample column height 2.670.2 cm). The sample was then inserted into a standard TE102 (ER 4102 ST) rectangular cavity of an EMX X-band EPR spectrometer (Bruker, Germany) and the EPR spectrum was recorded at various temperatures. Temperature control was achieved using a Bruker temperature control unit ER 4111 VT. The careful filling procedure of EPR cells resulted in good reproducibility between samples with a standard deviation in the relative EPR intensity of 75% for five independent measurements. The EPR spectrometer settings were as follows: microwave frequency, 9.45 GHz; microwave power, 0.63–31.73 mW; center field, 335.4 mT; sweep width, 20–500 mT; gain, 5  105; modulation amplitude, 0.05 mT; modulation frequency, 100 kHz; sweep time, 84 s; time constant, 40.96 ms, number of scans, 5; temperature, 298–373 K. The gvalues were determined with uncertainty of 70.0005 by simultaneous measurement of a reference sample containing DPPH fixed on EPR cell. The EPR instruments settings for quantitative evaluation were examined by DPPH standard. The experimental EPR spectra processing and simulation was carried out using WIN EPR and SimFonia programs (Bruker). The integral intensities of the EPR signals were obtained by double integration of the spectrum. The multi-component experimental EPR spectra were evaluated as a linear combination of individual EPR spectra simulations using a least-squares minimization procedure with the Scientist Program (MicroMath). The statistical parameters of the calculation procedure (R2, coefficient of determination and correlation) serve as a determination of the simulation quality, i.e. correlation of the experimental and simulated spectra. The relative concentration of the individual paramagnetic species was evaluated from the

311

contributions of the individual simulations to experimental spectrum after double integration. 2.2.2. Radical-scavenging activity of pepper extracts The ability of prepared black pepper aqueous/ethanol extracts to terminate radical species was monitored using EPR spin trapping technique using DMPO with either K2S2O8 or the stable free radical DPPH. 2.2.2.1. K2S2O8. In spin trapping EPR experiments free radicals were generated in situ by the thermal decomposition of persulfate ions at 333 K (Stasˇ ko et al., d – 2000). As the generated free radicals (SOd 4 and OH) have a very short lifetime, their stationary state concentrations are very low and cannot be detected directly; consequently spin trapping agent DMPO was used. In the presence of DMPO the transient free radicals Rd are added to the double bond of spin trapping agent forming a longer-lived nitroxide radical adducts dDMPO–R, readily detectable using the EPR technique (Janzen et al., 1994; Li et al., 1988). In these experiments, aqueous solutions of 0.2 mol dm–3 DMPO (50 ml), phosphate buffer (pH ¼ 6.98; 150 ml), 0.01 mol dm–3 K2S2O8 (50 ml) and aqueous pepper extracts (50 ml) were mixed under air, then transferred into a flat (4 mm wide) EPR cell and inserted in the standard TE102 rectangular cavity. The temperature was raised to 333 K (60 1C) and simultaneously the time-course of EPR spectra was monitored for 630 s giving a set of ten EPR spectra. Each individual EPR spectrum represents accumulation of three scans measured with 21 s sweep time. Control experiments were performed by replacing the spice extract with 50 ml of distilled water. The experiments with the ethanol extracts were performed in slightly altered solutions prepared by the addition of 0.2 mol dm–3 DMPO (50 ml), phosphate buffer (pH ¼ 6.98; 150 ml), 0.01 mol dm–3 K2S2O8 (50 ml) and ethanol pepper extract (200 ml). Again control experiments were performed replacing the spice extract by 200 ml of ethanol. 2.2.2.2. DPPH. In a further series of experiments the DPPH free radical, stable at room temperature, was used. After mixing pepper extract and DPPH solution in ethanol, the decrease of radical concentration resulting from termination reactions between the free radical and antioxidants present in the extract was monitored by EPR spectrometry. The DPPH solution in ethanol was placed in a syringe (1 ml, cDPPH ¼ 1  1024 mol dm23 ), and a second syringe was filled with 1 ml of the diluted ethanol black pepper extract. Both syringes were attached to a micro-mixing chamber and connected to a flat cell (8 mm wide) inserted into the TM-110 (ER 4103 TM) cylindrical cavity of the EPR spectrometer. After the simultaneous injection of both solutions into the flat cell, the time-development of the DPPH EPR

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spectra was monitored for 630 s obtaining the set of ten EPR spectra. (Individual EPR spectrum represents accumulation of three scans measured with 21 s sweep time.) Control experiments were performed by replacing the spice extract with 1 ml of ethanol. The EPR spectrometer settings in experiments with aqueous/ethanol extracts of black pepper samples were as follows: microwave frequency, 9.45 GHz (rectangular TE102 resonator) or 9.78 GHz (cylindrical TM resonator); microwave power, 10.03 mW; center field, 335.4 mT (TE102) or 348.2 mT (TM); sweep width, 8–10 mT; gain, 2  105; modulation amplitude, 0.1 mT; sweep time, 21 s; time constant, 40.96 ms, number of scans, 3; temperature, 293 K. In general the reproducibility of the EPR measurements for five independent, individual, samples was 75%.

(a)

(b)

2.2.3. UV/vis experiments UV/visible spectra of aqueous/ethanol black pepper extracts were recorded using UV/vis spectrometer PC 2000 (Sentronic, Germany) with a DH 2000 lamp.

3.1. EPR spectra of ground black pepper samples Fig. 1 shows the X-band EPR spectra of the reference (non-irradiated) sample together with sample g-irradiated at 30 kGy dose measured at 298 K 1 week after g-radiation treatment. The EPR spectra recorded using a broad magnetic field sweep width (500 mT) clearly demonstrated a broad singlet line with unresolved hyperfine splittings attributed to Mn(II) ions (Lozak et al., 2002; Morsy, 2002; Morsy and Khaled, 2001; Morsy and Khaled, 2002; Polat and Korkmaz, 2003; Polovka et al., 2003) upon which is superimposed a sharp EPR signal, whose intensity increases with increasing g-radiation dose. The relative EPR intensity of g-radiation-induced radical species is significantly related to the g-radiation dose as depicted in Fig. 2. The dotted line represents an exponential growth curve 1 I EPR ¼ I EPR max ð1  expðk  DÞÞ with k ¼ 0:06 kGy . Simulation analysis of the detailed reference sample EPR spectra (magnetic field sweep of 20 mT) confirmed the presence of two individual paramagnetic species (PI and PII) superimposed on the Mn(II) broad line species as illustrates Fig. 3. The first EPR signal PI represents sharp singlet line characterized by giso ¼ 2:0050 and DBpp ¼ 0:55 mT which can be attributed to semiquinone radicals produced by the oxidation of polyphenolic compounds present in plants (Jezierski et al., 2002; Morsy and Khaled, 2001; Morsy and Khaled, 2002; Pedersen, 2002; Polovka et al., 2003; Ukai and Shimoyama, 2003; Merdy et al., 2002). The second EPR signal PII is described by an axially symmetric line

100

150

200

250

300 350 400 Magnetic field, mT

450

500

550

Fig. 1. X-band EPR spectra of ground black pepper samples before (a) and after g-radiation treatment with dose 30 kGy (b), measured at 298 K using microwave power 0.633 mW and magnetic field sweep width of 500 mT. (EPR spectra were recorded 1 week after g-radiation.)

EPR integral intensity

3. Results and discussion

0

5

10

15

20

25

30

Dose, kGy Fig. 2. The dependence of relative integral EPR intensity of black pepper samples on g-radiation dose evaluated by EPR spectra measured at 298 K using 0.633 mW microwave power. (EPR spectra were recorded 1 week after g-radiation.)

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experiment 0 kGy

313

experiment 30 kGy

PI

PI

PII PII Mn (II) Mn (II) GI

GII

GIII

simulation 30 kGy

simulation 0 kGy

330

335 340 Magnetic field, mT

345

330

335 340 Magnetic field, mT

345

Fig. 3. Experimental X-band EPR spectra of ground black pepper samples before (0 kGy) and after g-radiation treatment with dose 30 kGy measured at 298 K using microwave power 0.633 mW and magnetic field sweep width of 20 mT. (EPR spectra were recorded 1 week after g-radiation.) Simulated spectra were calculated as the linear combination of individual EPR signals shown using the following relative concentrations (in %): simulation 0 kGy—PI 2; PII 7; Mn(II) 91 and R2 ¼ 0:966; simulation 30 kGy—PI 2; PII 6; Mn(II) 80; GI 5; GII 1; GIII 6 and R2 ¼ 0:979.

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Table 1 Spin Hamiltonian parameters of g-radiation-induced EPR signals GI–GIII observed in ground black pepper samples g-irradiated at doses from 5 to 30 kGy, along with the activation energy and half-time values evaluated from experimental spectra EPR signal (origin)

g-Value

Hyperfine splittings (mT)

DBpp (mT)

Activation energy (kJ mol–1)

Half-time (week)

GI (carbohydrate)

G ? ¼ 2:0060 G k ¼ 2:0032 g? ¼ 2:0060 gk ¼ 2:0050 G ? ¼ 2:0029 gk ¼ 2:0014

A? ¼ 0:85 Ak ¼ 0:70 (2H) A? ¼ 0:50 Ak ¼ 0:40 (1H) A? ¼ 3:00 Ak ¼ 1:80 (2H)

0.67

22.5

10

0.40

35.7

36

1.20

29.1

4

GII (carbohydrate) GIII (cellulose)

(g? ¼ 2:0062, gk ¼ 2:0052; DBpp ¼ 5:0 mT) and can be probably assigned to the mechanically induced free radicals producing during the grinding process. The formation of mechanically induced radicals from the grinding of red pepper was evidenced previously by Korkmaz and Polat, 2001. Additionally, the grinding process of sugars producing free radical species was described by Yordanov and Georgieva (2004) and the effects of mechanical treatment on radical production in lactose and carboxymethylcellulose was described by Raffi et al. (2002). In order to confirm the presence of mechanically induced paramagnetic species, firstly the EPR spectra of commercially available black peppercorns were measured, and the monitored EPR signal was fully compatible with PI described above. Subsequently, the black peppercorns were ground in a pepper grinder, and the so obtained material was further carefully homogenized in an agate mortar. The EPR spectra measured using the so prepared black pepper powder are characterized with low intensity signal PII, superimposed on PI singlet line. Accordingly, the grinding procedure might probably cause the formation of radical species PII. However, further explanations of EPR signal PII sources should be taken into account. The EPR spectra of g-irradiated ground black pepper samples revealed the formation of three radiationinduced EPR signals (GI–GIII) attributed to a combination of carbohydrates radical structures (GI and GII) (Korkmaz and Polat, 2001; Vanhaelewyn et al., 2000), and a typical ‘‘cellulosic’’ signal GIII (Bayram and Delince´e, 2004; Kispe´ter et al., 2003; Yordanov and Aleksieva, 2004; Yordanov and Gancheva, 2000; Yordanov et al., 1998). Additionally, the intensity of signal PI increased upon g-radiation treatment in good accordance with previously published data (Raffi et al. 2000; Desrosiers, 1996; Yordanov et al., 1998; Korkmaz and Polat, 2001). Fig. 3 shows the simulation of multicomponent experimental EPR spectrum of black pepper sample g-irradiated at a dose of 30 kGy, representing the linear combination of individual paramagnetic species. The spin Hamiltonian parameters of these g-radiation-

induced radical species, obtained by simulation of experimental EPR spectra, are summarized in Table 1. The simulated spectrum of the reference sample was calculated as a linear combination of the individual signals PI and PII (R2 ¼ 0.966), and the simulation of gradiation-treated sample corresponded to the linear combination of PI and PII with GI, GII and GIII signals (R2 ¼ 0.979), both superimposed on Mn(II) broad EPR signal. It should be noted here that the changes in EPR spectra of g-radiation-treated samples induced by the temperature increase discussed below, allowed the better identification of individual EPR signals in the multi-component experimental EPR spectra, due to the limited thermal stability of GI and GIII signals. In order for the data to be used quantitatively the instrumental settings must be such that line broadening due to the magnetic field modulation induced by the Helmholtz coils and power saturation due to the strength of the microwave field is negligible. The values of magnetic field modulation used herein are low enough that it will not cause line broadening. Power saturation can influence significantly the line width and signal intensity, in relation to the relaxation time of the paramagnetic species under study. Consequently we have studied the power saturation characteristics of the radicals. The investigations of the saturation behavior of EPR signals PI and PII confirmed that the growth of microwave power caused no saturation, as the intensity of both signals increased monotonically with increasing power. Fig. 4 illustrates the dependence of integral EPR intensity on the square root of microwave power evaluated for non-irradiated and g-irradiated samples, respectively. The experimental data were fitted using least-square analysis to the mathematical pffiffiffiffi model of saturation, I EPR ¼ I EPR ð1  expða  PÞÞ in accord max with data published by C - olak and Korkmaz (2003). The effect of a temperature increase on the black pepper EPR spectra of non-irradiated and g-irradiated sample (dose 30 kGy) measured 1 week after radiation

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Integral EPR intensity

M. Polovka et al. / Radiation Physics and Chemistry 75 (2006) 309–321

1

2 3 4 (Microwave power, mW)1/2

5

6

Fig. 4. The dependence of relative integral EPR intensity on microwave power square-root evaluated for non-irradiated ground black pepper sample (J) and g-irradiated samples (dose in kGy): (K) 10; (’) 20; (m) 30. (EPR spectra were recorded 1 week after g-radiation.) Marked symbols represent the experimental data and dashed lines their mathematical simulations using least-square analysis.

exposure is depicted in Fig. 5. The EPR spectra of the reference sample showed only negligible changes upon increasing temperature from 298 to 353 K (Fig. 5a). However, increasing the temperature to 373 K caused an approximately 40% growth of EPR intensity, probably due to the thermal decomposition of sample and the formation of the peroxidic radical species as proposed by Franco et al. (2004). On the other hand, the temperature dependence of EPR spectra of g-irradiated samples was quite different, because the temperature increase from 298 to 373 K caused a significant and irreversible decrease of gradiation-induced signals GI–GIII, as depicted in Fig. 5b–d for a sample treated with a dose of 30 kGy. The simulations of multi-component EPR spectra were evaluated using difference spectra, which were obtained by the subtraction of experimental spectra for the girradiated and reference samples at the corresponding temperatures. The dependence of individual signal areas, evaluated for the paramagnetic species GI–GIII, on temperature are shown in Fig. 5c. The data shows a substantial decrease of the carbohydrate (GI) and cellulose (GIII) radical species as the temperature is increased, and the activation energies are in order Ea (GI) ¼ 22.5 kJ mol–1 oEa (GIII) ¼ 29.1 kJ mol–1 oEa (GII) ¼ 35.7 kJ mol–1 (Fig. 5d).

315

The changes in the EPR spectra of non-irradiated and g-radiation-processed black pepper samples were monitored during 20 weeks of storage in the dark (temperature 6 1C; relative humidity 60%). The storage time after radiation treatment only influenced the EPR spectra of g-irradiated samples as the EPR spectra of reference sample remained unchanged. However, in all the g-radiation-treated samples an exponential decline of integral EPR intensity was observed, as is illustrated in Fig. 6a for sample treated at dose 30 kGy. The inset in Fig. 6a shows the changes in the EPR spectra observed 20 weeks after g-radiation process. Analysis of the experimental spectra showed that the decrease of EPR intensity with storage time for the g-radiation-induced signals GI–GIII can be described by a formal first-order kinetic model, and the calculated half-lives confirmed that the ‘‘cellulosic’’ triplet signal had the lowest stability (Table 1). This information is in accord with data published previously, which recommended the direct EPR identification of black pepper g-radiation treatment during 6–7 weeks after the radiation process (Delince´e, 2002; Bayram and Delince´e, 2004; Raffi et al., 2000; Yordanov et al., 1998). In addition we compared the temperature changes of the EPR spectra for non-irradiated and g-irradiated samples 20 weeks after g-radiation treatment. The loss of integral EPR intensity in non-irradiated samples monitored as the temperature was increased from 298 to 353 K represented 13%, while the same temperature changes caused a change of 40% in the g-irradiated sample (dose 30 kGy). The results obtained are fully compatible with the technique specified for the identification of food exposed to g-radiation treatment a long time after the radiation process (Yordanov and Gancheva, 2000). 3.2. Radical-scavenging properties of aqueous and ethanol black pepper extracts investigated by thermal decomposition of K2S2O8 using an EPR spin trapping technique The radical-scavenging activity of aqueous extracts prepared using ground pepper samples processed with various g-radiation doses was tested in a hydroxyl radical generating system (Stasˇ ko et al., 2000). The thermal decomposition of K2S2O8 in aquatic media produces reactive sulfate anion-radicals (Eq. (1)), which react with water generating hydroxyl radicals (Eq. (2)). In the presence of DMPO as the spin trap (Scheme 1) the radical intermediates are added to double bond of spin trap (Eqs. (3) and (4)), producing nitroxide radicals, characterized with specific hyperfine splittings and g-values (Li et al., 1988): DT

 K2 S2 O8 ! 2Kþ þ 2SOd 4 ,

(1)

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316

332

298 K

298 K

313 K

313 K

333 K

333 K

353 K

353 K

334 336 Magnetic field, mT

338

340

EPR signal area

293 (c)

332 (b)

334 336 Magnetic field, mT

338

340

ln (EPR signal area)

(a)

303

313

323 333 343 Temperature, K

353

363

0.0028

373 (d)

0.0030 1/T, K-1

0.0032

0.0034

Fig. 5. X-band EPR spectra of ground black pepper samples measured at various temperatures using microwave power of 0.633 mW: (a) reference (non-irradiated) sample; (b) g-radiation dose 30 kGy. (EPR spectra were recorded 1 week after g-radiation.) (c) Dependence of signal area for g-radiation induced EPR signals GI (’), GII (K) and GIII (m) on temperature for black pepper sample g-irradiated at dose 30 kGy. The EPR spectra simulations were calculated using difference spectra obtained by the subtraction of experimental EPR spectra of irradiated and reference samples at the corresponding temperatures. (d) Arrhenius analysis of data shown in (c).

d SO4d þ H2 O ! HSO 4 þ OH,

(2)

  d DMPO þ SOd 4 ! DMPO  SO4 ,

(3)

DMPOþd OH!d DMPO  OH:

(4)

However, the stability of dDMPO–SO–4 adduct (aN ¼ 1:382 mT, abH ¼ 1:01 mT, agH ¼ 0:142 mT; g ¼ 2:0059) is low (21 s (Kirino et al., 1981)), consequently under given experimental conditions in the experimental EPR spectra dominated four-line signal characterized with hyperfine splittings aN ¼ 1:492 mT, aH ¼ 1:440 mT, and

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317

1 week

20 weeks

Integral EPR intensity

5 mT

0

5

10 Time after γ-irradiation, week

(a)

298 K 333 K 353 K

332

334 336 Magnetic field, mT

20

298 K 313 K 333 K 353 K

313 K

(b)

15

338

340

332 (c)

334 336 Magnetic field, mT

338

340

Fig. 6. (a) The dependence of relative integral EPR intensity of ground black pepper samples on storage time after g-radiation evaluated from EPR spectra measured at 298 K using 0.633 mW microwave power. Inset represents the experimental EPR spectra of sample girradiated at dose 30 kGy monitored 1 week and 20 weeks after radiation. Influence of increasing temperature on EPR spectra monitored for ground black pepper samples 20 weeks after treatment: (b) non-irradiated sample; (c) sample g-irradiated at dose 30 kGy.

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Relative concentration of ·DMPO-OH

318

2 mT

63

126 189 252 315 378 441 504 567 630 Time, s

Fig. 7. The dependence of relative concentration of d DMPO–OH adduct on time during thermal decomposition of K2S2O8 in phosphate buffer solutions at 60 1C in the presence of aqueous extract (50 ml) prepared from ground black pepper samples: * water without extract; J reference (non-irradiated); K 10 kGy; ’ 20 kGy; m 30 kGy. Marked symbols represent the experimental data and dashed lines their mathematical simulations using least-square analysis. Inset represents the experimental (–) and simulated (?) EPR spectra of d DMPO–OH adduct observed in system. Initial DMPO and K2S2O8 concentrations were 0.01 mol dm–3 and 1.6  10–3 mol dm–3.

a g-value of 2.0059 (inset in Fig. 7), which is representative for dDMPO-OH adducts (Li et al, 1988). Fig. 7 shows the time course of dDMPO–OH concentration upon time of thermal decomposition of K2S2O8 at 333 K in phosphate buffer solution containing DMPO after addition of 50 ml of distilled water (blank experiment), or after addition of 50 ml of aqueous extracts prepared from non-irradiated, as well as gradiation-treated samples (doses 10, 20, 30 kGy). The highest relative concentration of dDMPO–OH was observed in blank systems. The addition of extract, which contains natural black pepper antioxidants (e.g., piperine, phenolic compounds, ascorbic acid (Calucci et al., 2003; Ravindran, 2000)) led to the decrease of d DMPO–OH adduct concentration, as these active compounds terminated also hydroxyl radicals by different reaction pathways in competition to DMPO: Antioxidantsþd OH ! Products:

(5)

The experimental data of dDMPO–OH formation on time were fitted to the formal first-order kinetic model,

and the formal rate constants, as well as the formal initial rates of dDMPO–OH adduct formation, were calculated. The statistical parameters of the fitting procedure (R2, correlation, coefficient of determination) revealed good agreement of experimental data with kinetic model. The results obtained clearly demonstrated that aqueous extract of black peppers exhibited considerable radical-scavenging activity. It can be concluded that the g-radiation dose used in the black pepper treatment cause no changes of the aqueous extracts ability to terminate hydroxyl radicals. As the solubility of main black pepper alkaloid piperine in water is limited (Khajuria et al., 1998), we prepared also ethanol extracts of pepper samples. Upon thermal decomposition of K2S2O8 in aquatic buffered media containing ethanol, the produced hydroxyl radicals react with ethanol producing 1-hydroxyethyl radical (Eq. (6)), which is subsequently added to DMPO (Eq. (7)): d

OH þ CH3 CH2 OH ! CH3 d CHðOHÞ þ H2 O;

(6)

DMPO þ CH3 d CHðOHÞ ! d DMPO2CHðOHÞCH3 : (7) d

The EPR spectrum of DMPO–CH(OH)CH3 is characterized by the hyperfine splittings aN ¼ 1:580 mT, aH ¼ 2:255 mT and g ¼ 2:0056 (Li et al., 1988). Consequently, EPR signal measured in the aqueous buffer K2S2O8 solutions after addition of 200 ml of ethanol (blank experiment) corresponded to the simultaneous formation of dDMPO–OH and d DMPO–CH(OH)CH3 adducts. However, the addition of ethanol extract prepared from non-irradiated black pepper sample caused disappearance of dDMPO–OH, due to the effective competition of pepper antioxidants with hydroxyl radicals. The analogous results were obtained also with extracts of g-irradiated samples at doses 5, 7.5 and 10 kGy. On the other hand, the ability of black pepper extracts at higher g-radiation doses (20 and 30 kGy) was lower, as the presence of dDMPO–OH adducts was monitored here. 3.3. Radical-scavenging properties of ethanol black pepper extracts investigated using DPPH free radical DPPH is a stable free radical, capable to accept electron from reactive radicals, thus behaving as a radical-scavenger (Polovka et al., 2003). Additionally, DPPH acts as an electron acceptor from antioxidants, and several electron transfer reactions of DPPH with phenols, amines and other compounds were described in the literature (Yordanov, 1996). The depletion of DPPH after addition of antioxidants can be measured by UV/ visible spectroscopy (lmax ¼ 520 nm), EPR spectroscopy, as well as other techniques (Foti and Ruberto, 2001). In our experiments ethanol solutions of DPPH

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319

DPPH relative concentration

samples at doses 5, 7.5 or 10 kGy. However, the extracts prepared from black pepper samples processed at higher doses of g-radiation (20 and 30 kGy) revealed lowered ability to terminate DPPH. The analogous results were observed, when UV/vis spectroscopy was applied in DPPH free radical concentration monitoring after mixing with ethanol extracts of black pepper. Probably, the g-radiation treatment caused the changes in the antioxidant contents of ground black pepper samples. Recently, the effect of g-irradiation on the antioxidant contents in aromatic herbs and spices was investigated and the significant losses of total ascorbate (14%) were found for the black pepper samples irradiated at dose 1071 kGy (Calucci et al., 2003).

4. Conclusions

63

126 189 252 315 378 441 504 567 630 Time after mixing, s

Fig. 8. The dependence of relative concentration of DPPH free radical on time after mixing DPPH solution in ethanol with black pepper ethanol extract (prepared from non-irradiated reference sample) evaluated for various volumes of extract in 2 ml of solution (in ml): * 0; J 100; & 200; n 250. Marked symbols represent the experimental data and dashed lines their mathematical simulations using least square analysis. Inset represents the time-course of DPPH free radical EPR spectra (magnetic field sweep width 6 mT) monitored in system containing 200 ml of extract. Initial DPPH concentration was 5  10–5 mol dm3.

were mixed with ethanol black pepper extracts prepared from non-irradiated and g-radiation-treated samples (doses 5, 7.5, 10, 20 and 30 kGy) and the decline of DPPH concentration was monitored by EPR and UV/vis spectroscopy. Under the given experimental conditions the EPR spectrum of DPPH free radical represents a five-line signal (inset in Fig. 8). Presence of specific active compounds in ethanol extract of black pepper (nonirradiated) caused the substantial decrease of DPPH relative concentration after addition of various extract volumes, comparing to blank experiment, as is shown in Fig. 8. A notable decline is monitored already in the first scan (63 s after mixing), and these data confirmed the effective termination of DPPH free radical in the ethanol solutions. Again here, the DPPH relative concentration decrease on time was fitted to the formal first-order kinetic model, and the formal rate constants, as well as the formal initial rates of DPPH termination were calculated. The values obtained confirmed that the radical-scavenging activity of ethanol extracts prepared from non-irradiated black pepper sample is indistinguishable to those prepared from g-radiation-treated

The presented EPR investigations of g-irradiated black pepper samples confirmed the limited lifetimes of three radiolytically produced paramagnetic species. The lowest stability was evaluated for cellulose EPR signal (g? ¼ 2:0029, gk ¼ 2:0014; A? ¼ 3:00 mT, Ak ¼ 1:80 mT; half-time of 4 weeks). However, the girradiated samples containing cellulose could be recognized at a well longer time after g-radiation treatment, due to the different consequence of the temperature growth from 298 to 353 K on the EPR signal intensity monitored for non-irradiated and g-radiation-processed samples. The aqueous and ethanol black pepper extracts demonstrated considerable abilities to terminate hydroxyl and DPPH radicals, and these radical-scavenging activities were not influenced by g-radiation treatment up to dose of 10 kGy.

Acknowledgments EPR group at Department of Physical Chemistry thanks Slovak Grant Agency for the financial support (Project VEGA/1/0053/03) and referee for valuable comments.

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