IDENTIFICATION OF IRRADIATED SAGE TEA (Salvia officinalis L.) BY ESR SPECTROSCOPY
Semra Tepe Çam1,*, Birol Engin2
1Gazi University, Faculty of Medicine, Biophysics Department, 06500 Beşevler, Ankara,
Turkey
2Turkish Atomic Energy Authority, Sarayköy Nuclear Research and Training Center, 06983
Ankara, Turkey
Abstract
The use of electron spin resonance (ESR) spectroscopy to accurately distinguish irradiated from unirradiated sage tea was examined. Before irradiation, sage tea samples exhibit one asymmetric singlet ESR signal centered at g=2.0037. Beside this central signal, two weak satellite signals situated about 3 mT left and right to it in radiation-induced spectra. Irradiation with increasing doses caused a significant increase in radiation-induced ESR signal intensity at g=2.0265 (the left satellite signal) and this increase was found to be explained by a polynomial varying function. The stability of that radiation-induced ESR signal at room temperature was studied over a storage period of 9 months. Also, the kinetic of signal at g = 2.0265 was studied in detail over a temperature range of 313-353 K by annealing samples at different temperature for various times.
Keywords: Sage tea; ESR; Irradiation; Food irradiation; Kinetics; Cellulose; Annealing.
1. Introduction
Food irradiation is used as an important tool for preservation in many countries. This can be utilized for various purposes such as killing bacteria, viruses, insects or to delay the ripening of some fruits. The permitted limit of irradiation dose in foods was stated as 10 kGy (WHO,1981; FAO/WHO,1984). To develop reliable methods for the control of irradiated foods is very important from the aspect of view of legal controls, labeling regulations and, of course, customer confidence. More analytical techniques have been developed for detecting irradiated foods and these techniques complement each other and allow determining irradiation treatment (Schreiber et al., 1993). Frequently used physical techniques are thermoluminescence (TL) (Khan and Bhatti, 1999; Engin, 2004; 2007) and electron spin resonance (ESR) (Raffi and Agnel, 1989; Raffi et al., 1994, 2000; Kwon et al., 2000; Bayram and Delincée, 2004;Yordanov et al., 1998;Yordanov and Gancheva, 2000). ESR spectroscopy is a powerful and practical technique, so European standards have been released concerning food containing bone (EN 1786, 1997), crystalline sugar (EN 13708, 2001) and cellulose (EN 1787, 2000). The last standard covers spices, herbs, shells and stones of the foods. In this category, Sage tea, Salvia officinalis L., is one of the popular plants since it has antioxidant activity and therapeutics advantage. Salvia officinalis is used in traditional herbal medicine in the form of herbal infusion or essential oil. The most common form of consumption of this herb is sage tea due to its therapeutic effect as soothing and antiseptic effect on mucus (Patenkovic et al., 2009).
The present paper reports the first results obtained by ESR technique on sage tea (Salvia officinalis L.).In this work, we aimed i) to investigate the feasibility of the application of EN 1787 standard to sage tea, ii) to investigate decay characteristics of radiation-induced ESR signals at room and high temperatures and lastly iii) to construct dose-response curve of radiation-induced signals.
2. Experimental
Sage tea used in this work was provided from local markets in Ankara (Turkey) and it had not been irradiated. Sage tea samples were used in the form of powder as provided. Their water content was determined by thermogravimetric measurements and found to be less than 10 ℅ by weight. It was irradiated with 60Co gamma rays at ambient conditions using gamma cell
with a dose rate of 1.024 kGy/h at the Sarayköy Establishment of the Turkish Atomic Energy Authority in Ankara. The radiation doses were 0.5, 1, 2, 3, 4, 5, 7 and 10 kGy in order to get
dose-response curve. The uncertainty in radiation doses was nearly 3%. The absorbed dose at the sample location was checked by Fricke chemical dosimeter. An unirradiated sage tea (control) sample was also prepared for comparison purposes. The samples were protected from light during irradiation and transport to the measurement laboratory and then stored in the closed bags in the dark. All ESR measurements were done at normal laboratory conditions (about 21 ± 2 °C and 25 ± 3 ℅ relative humidity) about 24h after the irradiation to avoid any interference of radiation-induced short-lived free radicals. The sample weight was ~ 130 mg for all the ESR measurements and all the samples were positioned in exactly the same manner in the cavity to avoid any errors in the g-factor and signal intensities arising from changes in the cavity-filling factor. A long term radical decay feature at room temperature was performed over a period of 33 days using a sample irradiated at a dose of 7 kGy. Decay kinetics of the radiation-induced radicals at four different temperatures [room (293 K), 313, 333 and 353 K] was performed by using the samples irradiated at a dose of 4 kGy. The sage tea samples were
transferred after irradiation process to water baths at temperatures mentioned above, then their ESR spectra were recorded regularly over a time interval of 0-90 min after cooling them to room temperature following predetermined heating times (2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90 min). The activation energies of the involved radical species were calculated from Arrhenius plots.
ESR measurements were carried out using a Bruker e-scan X-band ESR spectrometer operating at 9.8 GHz. Samples were placed in standard pyrex tubes with inner diameter 4.0 mm not exhibiting any ESR signal. The spectra were recorded at room temperature (open to air) under spectrometer operating conditions of: sweep width 20 mT, microwave power 0.1 mW, modulation frequency 86 kHz, modulation amplitude 0.5 mT, sweep time 20.97 s and time constant 328 ms. All of the experiments were repeated at least three times. A strong pitch was used as a standard sample for g-factor measurements.
3. Results and discussion
3.1. ESR spectra of unirradiated and irradiated sage tea
Before irradiation, ESR spectra of sage tea sample (control) exhibit asymmetric singlet line (Fig. 1a) centered at g = 2.0037 with peak-to-peak line width (∆Hpp) of 0.88 ± 0.02 mT. This
singlet ESR signal of the similar samples is resolved in Q-band spectra to give axially symmetric spectrum with g║ = 2.0031 ± 0.0002 and g┴ = 2.0047 ± 0.0005 (Yordanov and
attributed to semiquinone radicals produced by the oxidation of polyphenolic compounds present in plants (Raffi and Agnel, 1989; Polovka et. al., 2003; 2006). After irradiation at a dose of 4 kGy, while any change in g-factor not produced, a weakly increase in signal intensity of single central line was observedcontrary to nearly all works published up to now on studies of similar samples. Low radiation sensitivities for this line were also observed for some Mexican irradiated spices by Bortolin et al. (2006). In addition, two weak satellite lines on the sides of single central line with a separation of 6.03 mT were observed at g = 2.0265 and g = 1.9910 after irradiation (Fig. 1b). These lines, originating from a triplet spectrum (hyperfine splitting A = 3 ± 0.1 mT) whose central line is overlapped on the initial central line, are attributed to free radicals of cellulose generated by gamma irradiation (Raffi and Agnel, 1989).The detected radiation-induced ESR signals in herbs and spices are attributed to carbohydrate-like and cellulose-derived radicals according to more studies in the literature (Raffi and Agnel, 1989; Raffi et al.,2000; Polovka et al., 2003; Bayram and Delincée, 2004; Suhaj et al., 2006; Polat and Korkmaz, 2008;Yamaoki et al.,2008; Yordanov and Aleksieva, 2009;Yordanov et al., 2009). In Fig.1c, the two cellulose satellite lines spacing 6.03 mT which marked with stars are clearly visible. The presence of these two cellulosic satellite lines is considered to be unambiguous evidence of the radiation treatment of the sample, so they are used while trying to detect foods containing cellulose whether irradiated or not with respect to the EN 1787 standard.
Therefore, the left satellite ESR signal (near g=2.02) intensity was adopted throughout this work to follow the evolution of ESR spectra as a function of absorbed dose, temperature and time. A microwave power of 0.1 mW was adjusted during the experiment since these types of radicals are found to saturate around 1.5 mW at room temperature (Polat and Korkmaz, 2008).
3.2. Dose –response curve
The sample irradiated to doses of 0.5, 1, 2, 3, 4, 5, 7 and 10 kGy was used to construct the dose-response curve. In the studied dose range, γ irradiation caused an increase in the cellulose-derived radical signal intensity at g = 2.0265 of the sage tea. The other spectroscopic features, such as g-factor and peak-to-peak linewidth (ΔHpp), were found to stay constant
within our experimental error limits (Δg = ± 0.0001 and Δ (ΔHpp) = ± 0.02 mT). Fig. 2 shows
ESR signal intensity of left satellite line as a function of radiation dose. The reported ESR signal intensities are derived from peak-to-peak distance of the ESR resonance in the first-derivative spectrum. Several mathematical functions were tried to fit the experimental dose-response data to describe the variation of signal intensity with absorbed radiation dose. The
best correlation is obtained for second degree polynomial varying function (I = a + b *D _ c *
D2) given in Fig. 2. In this function, I and D stands for the ESR signal intensity and applied
radiation dose in kGy, respectively, and the parameters a, b and c are constants to be determined. The intercept (parameter a) in this function representing the ESR signal intensity at zero applied dose mean that the relative amount of free radical species of unirradiated (control) sample and parameters b and c represent the rate of radical production and/or radiation yield upon irradiation at room temperature.The parameter values of a, b and c calculated from fitting procedures were found to be 0, 1.061 and 0.045 (r2 = 0.9933),
respectively.
3.3. Fading and kinetic of the radiation-induced cellulosic ESR signal
The changes in the concentration of the free radicals with time were observed when the sample was stored at normal laboratory conditions (about 21 ± 2°C, 25 ± 3 % humidity) in the dark without any special conditioning before and after irradiation. In this work, the water content of samples was less than 10℅ by weight. This fading study covers records of ESR spectra over ~ 33 days storage period using the sample irradiated at a dose of 7 kGy. Fig. 3 shows the fading behaviour of the left satellite line (g = 2.0265). After 33 days storage at laboratory conditions signal intensity decreased to about 46% of the initial value (recorded 24 h after the irradiation) for sage tea. At the end of this storage period the remained signal intensity was approximately nine times of the unirradiated spectra noise level. The peak-to-peak ESR signal intensity of this line were found to decrease very rapidly in the 10 days of storage, then the rate of decrease became slowly. The decay curve was fitted by first, second order, sum of two first order and second order decay functions as well as by the sum of first and second order decay functions. The decrease of signal intensity during the 33 days can be well described by a sum of first and second order kinetic functions (I=I01
exp(-k1t)+I02/(I02k2t+1)). In this function, I and I01,2 represent signal intensity at any time and at
zero, respectively, and k1,2 represent reaction rate constants. The reaction rate constants,
calculated from this fitting procedure, were k1 = 0.15 per day and k2 = 0.0007 per day. This
proves that the ESR signal decay at room temperature followed two reaction steps having different kinetic. The first reaction (k1) has a high and the second one (k2) has a slow reaction
rate constant. In other words, the radical species responsible from the first reaction are unstable compared to the species responsible from second reaction at laboratory conditions.
The calculated decay data by using these constant values determined after the fitting procedure was also given in Fig. 3 as dashed lines.
Although the fading study was ended after 33 days from irradiation treatment, we investigated the ESR spectrum of sage tea after about 9 months from irradiation process. During this long storage period, the sample was stored in the same normal laboratory conditions (in the dark). The two satellites lines, the unambiguous evidence of irradiation (EN 1787, 2000), still were observed. At the end of this long storage period, the left satellite signal intensity was at least three times that of the unirradiated spectra noise level (this trend was not shown in Fig. 3.). The stability of the radiation-induced cellulosic radical signals determines the potential of the ESR technique for detection of irradiated foods. So, EN 1787 standard can be applied up to at least 9 months after irradiation treatment for sage tea sample. The other spectral parameters of the left satellite signal such as g-factor and peak-to-peak linewidth did not change at normal laboratory conditions during 9 months storage period.
Radical species decay faster at high temperature due to increase in the molecular motions. To test this idea and to determine kinetic features and activation energies of the contributing radical species at high temperatures, annealing studies were performed in water bath at normal laboratory conditions. To investigate the effects of annealing temperatures on the decay characteristics of cellulose-derived radical signals, the sage tea samples irradiated at 7 kGy were annealed at four different temperatures ( 293, 313, 333 and 353 K) for predetermined times between 2.5 and 90 min. Although the samples were annealed at predetermined temperatures, all the spectra were recorded at room temperature after cooling the samples down to room temperature. The peak-to-peak amplitude of the ESR signal at g = 2.0265 was determined at each annealing time for each annealing temperature and it was plotted versus the annealing time. The results are shown in Fig. 4. As seen from Fig. 4, the intensity of the signal decays at all temperatures, however, these decays are faster at higher temperatures. Besides signal intensity, other features such as peak-to-peak line width (ΔHpp)
and g-factor of the signal at g = 2.0265 were also studied, but no significant changes have been observed in the whole annealing temperatures and time ranges within our experimental error limits (Δg = ± 0.0001 and Δ(ΔHpp = ±0.02 mT).The experimental decay data at each
annealing temperature were fitted by first, second order, sum of two first order and second order decay functions as well as by the sum of first and second order decay functions, as in the case of long-term decay at room temperature. A sum of first and second order decay functions was found to be the best approximation to fit the thermal decay of the g = 2.0265 signal. The reaction rate constants (k1, k2) of the contributing radical species were calculated
from the fitting procedure for each isotherm. Theoretical decay data calculated by using these reaction rate constant values were also given in Fig. 4 as dashed lines. As it is seen from this figure, the agreement between experimental and theoretical decay data is fairly well. This result also confirm that the ESR signal decay at room and high temperatures followed two reaction steps of cellulose-derivative radicals having different kinetics.
The rate constant k is expected to exhibit an exponential dependence on the temperature of the type [ k(T) = k0 exp(-E/RT)], where E is the activation energy, R the gas constant, k0 the
frequency factor and T the absolute temperature. If so, the ln(k) − 1/T plot should give a straight line whose slope is proportional to the activation energy. The reaction activation energy values of E1 = 23.2 ± 1.6 (for k1) and E2 = 32.0 ± 4.1 (for k2) kJmol-1 were calculated
from these plots (Fig.5) for the two cellulose-derivative radical signal components, responsible for the left satellite ESR line (near g = 2.02) of irradiated sage tea samples. This result shows that the free radicals responsible from the ESR signals of irradiated black and rooibos tea with the reaction activation energies of 33.8 ± 3.1 kJmol-1 and 46.0 ± 3.5 kJmol-1
are more stable than free radicals of irradiated sage tea (Polat and Korkmaz, 2008). In other words, the radiation-induced free radicals (near g = 2.02) of irradiated sage tea are less resistant to the temperature than free radicals of black and rooibos tea.
4. Conclusion
Unirradiated sage tea sample exhibits a asymmetric ESR singlet. Irradiation at ambient conditions caused a weakly increase in signal intensity of this central line without any changes in g-factor. Two satellite lines at left and right of central line was observed at g = 2.0265 and g = 1.9910 with a separation of 6.03 mT after irradiation at a dose of 4 kGy. Variation of the cellulose-derived radical signal (g= 2.0265) intensity with absorbed dose follows a polynomially increasing function in the dose range of 0.5-10 kGy. At room temperature, the post-irradiation decrease in intensity is about 46 % during the 33 days. The thermal decay at room and high temperatures of the signal at g = 2.0265 from sage tea can be described best by a sum of first and second order decay functions. This may support the existence of at least two possible decay mechanisms of the g = 2.0265 ESR signal in sage tea sample. High radiation yield at the permitted food irradiation dose range (1-10 kGy) and stability of the induced free radicals (near g = 2.02) at room temperature (Figs. 2 and 3) indicate that ESR technique can be used in identification of irradiated sage tea even at the end of a storage period of ~ 9 months. That is to say, EN 1787 standard can be applied to sage tea during the ~9 months after the irradiation process.
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