THE INFLUENCE OF STERILIZATION ON FREE-RADICAL GENERATION, DISCOLORATION AND THE ANTIOXIDANT PROPERTIES OF CERTAIN SPICE HERBS

Abstract

The aim of this work was to assess the influence of steam sterilization on free-radical generation in herbs and on changes in their chromatic parameters, as well as in their antioxidant activity and the content of polyphenols and flavonoids. The research conducted showed that the Hight Temperature Short Time (HTST) sterilization method decreased the number of free radicals in the herbs tested. At the same time, the antioxidant activity as well as the polyphenol and flavonoid contents increased. Changes in chromaticity in sterilized herbs were determined; darkening and shift towards red and yellow hues were observed. The antioxidant properties of herbs before and after sterilization decreased in the following sequence: oregano>thyme>marjoram>basil. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

The aim of this work was to assess the influence of steam sterilization on free-radical generation in herbs and on changes in their chromatic parameters, as well as in their antioxidant activity and the content of polyphenols and flavonoids.

The research conducted showed that the Hight Temperature Short Time (HTST) sterilization method decreased the number of free radicals in the herbs tested. At the same time, the antioxidant activity as well as the polyphenol and flavonoid contents increased. Changes in chromaticity in sterilized herbs were determined; darkening and shift towards red and yellow hues were observed. The antioxidant properties of herbs before and after sterilization decreased in the following sequence: oregano>thyme>marjoram>basil.

- Keywords: EPR, CIELab, antioxidant activity, spices, steam sterilization -

INTRODUCTION

Herb spices have been used for many centuries to imbue foods with desired flavor and aroma characteristics. Among these herbs are basil, [OcímumbasüicumL·), orégano [Origanum vulgare L.), marjoram [Origanum mqjorana L.) and thyme [Thymus vulgaris L.). These plants belong to the mint family, or Labiatae. They contain essential oils of varied composition, organic acids and flavonoids. Thus, they are characterized by specific sensory properties and may positively influence, among others, digestion processes, as they have an antispasmolytic effect and stimulate the excretion of gastric acid. The components in these herbs give them anti-inflammatory properties (OCAÑA-FUENTES et al, 2010), as well as bactericidal and fungicidal properties, which are used in the treatment of various infections, mainly of the upper and lower respiratory tract and the skin (TOHIDPOUR et al, 20 10; ROTA et al., 2008; DE SOUZA et al., 2010). They also stimulate the excretion of mucus from the bronchi and allow for easier expulsion (GROSSO et al., 2010), in addition to having a tranquilizing effect and countering depression (NGUYEN et al, 2010). The herbs are also used as an auxiliary in treating colic and diarrhea. Tests have also shown that they have cholesterol-lowering properties in the blood (BRAVO et al., 2008) and inhibit blood-platelet aggregation (YAZDANPARAST and SHAHRIYARY, 2008).

Apart from their well-known medicinal properties, the herbs and spices are primarily an abundant source of antioxidant compounds, which play an important role in the prevention of lifestyle diseases (YU et al., 2010; LEE et al., 2005). These characteristics are due to the presence of polyphenol compounds in the herbs, such as phenolic diterpen, phenolic acids and flavonoids (HOSSAIN et al., 2011; SELLAMI et al., 2009), as well as vitamin C, tocopherols and carotenoids (SUHAJ, 2006; SGHERRI et al, 2010).

Herbs and spices can become contaminated with microorganisms that pose a serious threat to human health. The contamination can be primary, as in the case of soil, dust, bird feces and fertilizer pollution, or secondary, as caused by incorrect gathering, drying, storage, packaging or transportation of plants (KNEIFEL et al., 2002; SCHWEIGGERT et al, 2007). Various methods of removing microorganisms from food products exist. The first method, currently forbidden in European countries, was sterilizing with ethylene oxide gas (TATEO and BONONI, 2006). Food is also sterilized by means of radiation (FARKAS, 2006; POLOVKAand SUHAJ, 2010). Because the influence of radiation on sterilized products is not fully known, the method of irradiation is not very popular in many countries. When using radiation in the sterilization process, special information must be included on the product packaging. The most popular method used in the herband spice-processing industry involves steaming (ALMELA et al., 2002). Modern steam sterilization methods are ecologically benign and ensure microbiological purity. One such method is Natural Microbiological Clear (NMC®) steam sterilization, which employs the High-Temperature, Short-Time (HTST) principle, as well as a special Nauta® mixer. Sterilization is performed using saturated steam, brought in under a pressure of 10 bar at a temperature of 1 10°-140°C in a relatively short time (30-180 seconds). The product is constantly stirred during sterilization. Steam vapor together with essential oils pass through the condenser, where the oils liquefy and are introduced back into the cooled product. Thanks to this, the loss of essential oils is marginal. The humidity of the products obtained does not exceed 2-10%.

Oregano, thyme, basil and marjoram are usually added to dishes at the beginning of the culinary process and are subject to prolonged heat exposure. They also might be added at the end of cooking and thus heated for a short duration.

Considering the possibility of changes in the composition and the quality of herb material exposed to high temperature (BAGDATLIOGLU and ORMAN, 2010), the aim of this work was to assess the influence of steam sterilization on the appearance of free radicals and the changes both in chromaticity parameters in dried herbs and in the antioxidant activity and the content of polyphenols and flavonoids in their water extracts.

MATERIALS AND METHODS

Materials

The plant material for this study were unsterilized (NST) and sterilized (ST) dried herbs: basil, [Ocimum basilicum L.), orégano [Origanum vulgare L.), marjoram [Origanum mqjoranaL.), and thyme [Thymus vulgaris L.).

The material for the study was provided by K.P. P.S. Interjarek company (Goluchów, Poland) where the herbs and spices were sterilized by means of the NMC® steam sterilization method.

Sample preparation and conditions of EPR spectra measurements

Powdered samples in their initial state as well as samples of the sterilized leaves were placed in thin-walled glass test tubes with an outer diameter of 3 mm. Empty tubes gave no electron paramagnetic resonance (EPR) signal at the maximum line amplification and maximum microwave power (70 mW).

EPR spectra of the samples tested were registered at room temperature using an X-band frequency spectrometer (9.3 GHz) with a magnetic field modulation of 100 kHz (RADIOPAN company, Poznan, Poland). The frequency of microwave radiation was registered by means of an MCM 101 meter (EPRAD company, Poznan, Poland).

The samples in the test tubes were placed between the electromagnetic poles, and EPR spectra were registered by computer in the form of the first derivative of absorption. Numerical acquisition of data (JAGMAR company, Krakow, Poland) was used, together with the LabView program and spectroscopic analysis software.

Measuring the concentration of free radicals in the samples

The integral intensity of the EPR line registered as the first derivative of absorption was measured by integration of the area under the curve of absorption resonance performed twice. The integral intensity of the EPR line was used in calculating the concentration of free radicals in the sample (WEIL et al, 1994; EATON et ed., 1998).

Ultramarine (sodium aluminosilicate containing sulphur) was used as the pattern of concentration because the paramagnetic centers present in it are quite stable. To increase the accuracy of the measurement, a ruby crystal (A1202: Cr3*) was used as the so-called 'internal pattern'. For each sample analyzed and the ultramarine, the ruby EPR signal was also registered. The amplitude of the EPR line of the ruby was measured.

The concentration of free radicals in the samples was measured according to the following equation:

N = n^sub u^ (I /WA) / (I^sub u^ / W^sub u^A^sub u^)

where:

n^sub u^ - the number of paramagnetic centers in ultramarine (the reference) (n^sub u^ = 1.2x10^sup 19^spin);

W, W^sub u^ - the receiver gains for sample and ultramarine, respectively;

A, A^sub u^ - the amplitudes of the ruby signal for the sample and ultramarine, respectively;

I, I^sub u^ - the integral intensities for the sample and ultramarine, respectively;

m - the mass of the sample.

Chromatic parameter measurement

Chromatic parameters of unsterilized and sterilized herbs were measured using a CM-5 spectrophotometer (Konica, Minolta). The samples were placed on a Petri dish and measured in reflected light with the mirror- surface reflection component attenuated. A 0 30 mm aperture was used as well as a standard colorimetrie observer with a 10° field of vision and D65 illuminant. Before each measurement, the device underwent white tile calibration. Chromatic coordinates were measured according to the conventional CIELab colorimetry system. Differences in the parameters of color brightness were measured in terms of the L* coordinate and the tone of color as coordinates a* and b*. Additionally, the absolute difference of color DE*ab was measured (NSONZI and RAMASWAMY, 1998):

ΔE*ab = [(AL*)^sup 2^ + (ΔL*)^sup 2^ + (Δb*)^sup 2^]^sup 1/2^

where ΔL*, Aa*, and Δb* are indicators of chromatic difference between sterilized and unsterilized herbs.

Preparation of water extracts

Water extracts of the herbs were used in the study, prepared in the form of infusions and decoctions.

For the infusions, 0.5 g of the material was measured, soaked in 5 mL of deionized boiling water and cooked for 15 min in a lid-covered vessel. The infusion was set aside for another 15 minutes and then filtered and filled with water to achieve a final volume of 10 mL.

For the decoctions, 0.5 g of the material was measured, soaked in 5 mL of deionized roomtemperature water and cooked for 30 min in a lid-covered vessel, then filtered and filled with water to achieve capacity final volume of 10 mL.

Measurement of TAS, polyphenol and the flavonoid contents

In the extracts obtained and after having diluted the sample, the Total Antioxidant Status (TAS) was measured, as well as the polyphenol content, using colorimetrie techniques. The measurement of TAS was performed using Randox Laboratories Ltd., United Kingdom. The method of measuring TAS consists of the inhibition of the appearance of ABTS,+ cation radicals proportional to the concentration of antioxidants contained in the sample. The total antioxidant activity was expressed as the Trolox antioxidant activity or TEAC (Trolox Equivalent Antioxidant Capacity).

The technique of measuring polyphenols (SINGLETON and ROSSI, 1965) is based on a chromatic reaction with the use of the Folin-Ciocalteau reagent. The total polyphenol concentration was calculated as mg gallic acid equivalents (GAE). The flavonoid content was measured using the Woisky and Salatino method (WOISKY and SALATINO, 1998) based on the production of chromatic complex compounds with A1C13. The flavonoid concentration was expressed in mg quercetin equivalents (QE).

Statistical analysis

All data were expressed as the means ± standard deviation of five measurements. ANOVA analyses and the STATISTICA PL v. 8.0 software were used to evaluate the results of the experiments. A value of ? < 0.05 was regarded as significant.

RESULTS AND DISCUSSION

The sterilization process, which aims at removing microorganisms from stored products, employs steam at a very high temperature and pressure. Herbs owe their medicinal properties mainly to the high content of antioxidants and essential oils. The latter in the form of fatty acid esters might undergo hydrolysis and oxidation resulting in the generation of superoxide radicals if exposed to conditions favorable to such changes. EPR results showed that HTST steam sterilization does not cause a significant increase in the amount of free radicals. These tests demonstrated that both the initial samples and the sterilized samples were paramagnetic. EPR spectra were registered for samples of all the herbs tested, both the sterilized herbs and those in their initial state. EPR spectra were obtained at low microwave power and at 1 5 dB of attenuation. The EPR spectral parameters used for testing the herbs are shown in Table 1 . The amplitude (A) of the EPR line increases with the number of free radicals present in the sample, and the width (?? ) of the EPR line depends on magnetic interactions between unpaired electrons in the sample. The amplitude (A) of the EPR line decreases following sterilization in all herbs tested, and the width of the EPR line (?? J of the sterilized herbs is greater than the width of the EPR line of the initial samples (Table 1). This means that in the sterilized samples more powerful dipole interactions take place between free radicals. The EPR lines of both the initial samples and the sterilized herbs are characterized by a large width, which indicates powerful dipole interactions between the unpaired electrons of free radicals, and thus the distances between them are short. The relationship between the amplitude and width of the EPR lines and the microwave power are characteristic of a homogenous distribution of free radicals in the sample. In the case of sterilized herbs, these results are especially important because they indicate that the sterilization process influences the samples in a homogenous manner, and spin islands do not appear.

Both in the initial samples and in the sterilized samples, free radicals are present. Their concentrations are similar and amount to 1018 spin/g (Table 1). It has been determined that in all the herbs tested the concentration of free radicals decreases following sterilization (Fig. 1).

The highest concentration of free radicals was observed in basil, both sterilized and unsterilized, while the lowest concentration was found in orégano and marjoram. The decrease in the number of free radicals might be connected with the increased antioxidant activity observed in the herbs following sterilization (Fig. 1). In the case of orégano, which has the highest antioxidant value, the smallest number of free radicals was observed. Conversely in the case of basil, the greatest number of free radicals was accompanied by the lowest antioxidant activity among the herbs tested.

During sterilization, a change in the color of the plant products took place, which was demonstrated by changes in the CIELab values in (Fig. 2). In sterilized herbs, a decrease in the L* chromaticiry coordinate was observed in comparison with the unsterilized samples. This decrease means that less light was reflected by the surface of the material, which is most likely due to a loss of water during sterilization and greater porosity of the dried herb surface. This change results in dried herbs being darker and the L* coordinate shifting towards black. The value of the L* parameter, that is, the brightness of the material, also depends among other things on the number of compounds of brown hue. During the process of drying, enzymatic and nonenzymatic reactions of browning take place in the plant tissue, causing an increase in the content of brown pigments and thus a darkening of the dry material (PERERA, 2005). The greatest change in the parameter (AL* = -3.5) was observed in basil and the least in thyme (AL* + -1.05) and orégano (AL* + -1.16).

The value of the a* chromaticity coordinate responsible for red (+) and green (+) hues increased in the sterilized herbs, that is, following the shift towards red, which is confirmed by other reports in the literature (ALIBAS, 2007; ALIBAS et ed., 2007). In the unsterHized herbs, the value of the a* coordinate is characterized by a greater amount of green hue. Evidently, sterilization causes a decrease in the share of green color, which might be caused by degradation of chlorophyll a and b during the high-temperature process. The changes in Da* are similar for all herbs tested and amount to between 1.41 and 2.0 (Fig. 2). The positive value of the b* parameter is responsible for a yellow hue. As a result of sterilization, the value of this parameter increased b*=1.09 in orégano to ??*=3.63 in thyme (Fig. 2). The increase in the share of yellow might caused by the process of browning as well as the depletion of chlorophyll, which often conthe carotenoids of yellow, orange and red present in the leaves. Similar changes in b* chromaticity parameter were observed in and parsley leaves (THERDTHAI and ZHOU, DOYMAZ et ed., 2006).

The value of the total chromaticity difference (AE*ab) was determined based on the bright(L*), red color (a*) and yellow color (b*) of final product. The more distant these values from the model - the unsterilized herbs - greater was the total chromaticity difference.

Assessing the total chromaticity difference (AE*ab) between the sterilized and the unsterilized herbs, it may be said that the orégano sample is characterized by a color that was altered the least by sterilization (??*=2. 13). The remaining herbs displayed higher values of ??* (from 4.25 in thyme to 4.58 in marjoram) (Fig. 2).

Higher values were found for all parameters analyzed, i.e., the antioxidant activity, as well as the polyphenol and flavonoid concentrations in the extracts of the sterilized herbs in comparison with the extracts of the unsterilized ones. The influence of temperature on the antioxidant activity of plant products is not single-valued and depends, among other things, on the temperature value and the time of exposure (KATSUBE et al., 2009; TOMAINO et al., 2005). The increase in the antioxidant activity observed as a result of exposure to high temperatures might be caused by many factors. As a result of heating, increased pressure or enzymatic hydrolysis, damage to cell walls occurs and an increase in the bioaccessibiliry of the antioxidant substances might result (MCINERNEY et al., 2007). Furthermore, during thermal processing new antioxidant compounds might be created (JEONG et al, 2004). What seems the most important consideration, however, is that thermal processing, which, on the one hand, is detrimental to many components, inactivates enzymes responsible for enzymatic oxidation of natural antioxidants on the other hand, contributing to the retention of antioxidant activity (GONÇALVES et al, 2010). An example of this effect might be fruits and vegetables subjected to so-called blanching, which retain their antioxidant activity during storage to a greater extent than non-blanched fruits and vegetables (PATRAS et al., 2010). An important factor influencing the antioxidant content is not only the increased temperature itself but also the environment in which it acts. Heating plant products in water causes relatively fast penetration of heat to the inside of the tissues, which causes longer exposure of the whole mass of the processed product to the agent and high antioxidant-compound loss.

However, when heated in the air, the inside of the product remains at a lower temperature than the surface, which causes lesser antioxidant loss (TURKMEN et al, 2005; FRANCISCO et al., 2010). As Munyakahas demonstrated (MUNYAKA et al. , 20 1 0) , blanching by the HTST method (high temperature short time) is the best way of retaining vitamin C while processing broccoli.

The antioxidant activity in herbs both before and after sterilization decreased in the following sequence: oregano>thyme>marjoram>basil (Fig. 3). Decoctions displayed slightly lower values of antioxidant activity than infusions, which might be caused by a longer period of heating and the decomposition of antioxidant substances vulnerable to high temperatures, such as for example ascorbic acid. Changes in the polyphenol concentrations were similar to changes in the antioxidant activity (Fig. 4). The highest polyphenol concentration in the sterilized herbs was measured in orégano (20.67-21.22 mg GAE/ mL), while the lowest concentration was measured in basil (6.13-7.40 mg GAE/mL). Contrary to the antioxidant activity, higher concentrations of polyphenols were measured in the decoctions than in the infusions. This slight increase in polyphenol concentration in decoctions might be caused by longer heating and the aforementioned increase in bioaccessibility. The tendency of the changes in herb flavonoids before and after sterilization is similar to the trend in polyphenols. However, the changes are very minute (Fig. 5). Both sterilization and the way in which the water extracts were prepared had little influence on the flavonoid concentration. Higher flavonoid concentrations were found in the sterilized herbs and their decoctions. The concentrations of flavonoids measured in the herbs decreased in the following sequence: oregano>thyme>marjoram>basil. An attempt at assessing the correlation between the antioxidant activity and the polyphenol concentration in the sterilized (r=0.878, p<0.05) and unsterilized (r=0.875, p<0.05) herb extracts resulted in determining a linear correlation. The correlation between the content of phenolic compounds and antioxidant activity in various plant extracts was proven by MILIAUSKAS et al. (2004). Lower correlations in the sterilized (r=0.839, p<0.05) and unsterilized (r=0.762, p<0.05) herb extracts were found between the antioxidant activity and the flavonoid content, as well as between the polyphenol and flavonoid concentration in the sterilized (r=0.873, p<0.005) and unsterilized (r=0.768. p<0.05) herb extracts.

CONCLUSION

The research conducted showed that the HTST sterilization method decreased the number of free radicals in the herbs tested. At the same time, the antioxidant activity as well as the polyphenol and flavonoid contents increased. Changes in chromaticity in sterilized herbs were observed in the form of darkening and a shift towards red and yellow hues. The antioxidant properties of herbs before and after sterilization decreased in the following sequence: oregano>thyme>marjoram>basil.

References

REFERENCES

Alibas I., Akbudak B., Akbudak N. 2007. Microwave drying characteristics of spinach. J Food Eng. 78:577-583.

Alibas I. 2007. Energy consumption and colour characteristics of nettle leaves during microwave, vacuum and convective drying. Biosyst. Eng. 96:495-502.

Almela L., Nieto-Sandoval J.M. and Fernández-López J.A. 2002. Microbial inactivation of paprika by a high-temperature short-X time treatment. Influence on color properties. J Agrie. Food Chem. 50:1435-1440.

Bagdatlioglu N. and Orman S. 2010. The effect of steam sterilization on antioxidant activities of sage, orégano and basil. Ital. J. Food Sci. 22:343-346.

Bravo E., Amrani S., Aziz M., Harnafi H. and Napolitano M. 2008. Ocimum basüicum ethanolic extract decreases cholesterol synthesis and lipid accumulation in human macrophages. Fitoterapia. 79:515-523.

De Souza E.L., de Barros J.C., de Oliveira C.E.V. and da Conceiçâo M.L. 2010. Influence of Origanum vulgare L. essential oil on enterotoxin production, membrane permeability and surface characteristics of Staphylococcus aureus. Int. J. Food Microbiol. 137:308-31 1.

Doymaz I., Tugrul N. and Pala M. 2006. Drying characteristics of dill and parsley leaves. J. Food Eng. 77:559-565.

Eaton G.R., Eaton S.S. and Salikhov K.M. Eds. 1998. Foundations of modern EPR. Singapore: World Scientific.

Farkas J. 2006. Irradiation for better food. Trends Food Sci. Tech. 17:148-152.

Francisco M., Velasco P., Moreno D.A., Garcia-Viguera C. and Cartea M.E. 2010. Cooking methods of Brassica rapa affect the preservation of glucosinolates, phenolics and vitamin C. Food Res. Int. 43:1455-1463.

Gonçalves E.M., Pinheiro J., Abreu M., Brandäo T.R.S. and Silva C.L.M. 2010. Carrot Daucus carota L.) peroxidase inactivation, phenolic content and physical changes kinetics due to blanching. J. Food Eng. 97:574-581.

Grosso C., Figueiredo A.C., Burillo J., Mainar A.M., Urieta J.S., Barroso J.G., Coelho J.A. and Palavra A.M.F. 2010. Composition and antioxidant activity of Thymus vulgaris volatiles: Comparison between supercritical fluid extraction and hydrodistillation. J. Sep. Sci. 33:221 1-2218.

Hossain M.B., Barry-Ryan C, Martin-Diana A.B. and Brunton N.P. 2011. Optimisation of accelerated solvent extraction of antioxidant compounds from rosemary [Rosmarinus officinalis L.J, marjoram Origanum mqjoranaL.) and orégano [Origanum vulgare L.) using response surface methodology. Food Chem. 126:339-346.

Jeong S.M., Kim S.Y., Kim D.H., Jo S.C., Nam K.C., Ann D.U. and Lee S.C. 2004. Effect of heat treatment on the antioxidant activity of extracts from citrus peels. Journal of Agricultural and Food Chem. 52:3389-3393.

Katsube T, Tsurunaga Y., Sugiyama M., Furuno ? and Yamasaki Y 2009. Effect of air-drying temperature on antioxidant capacity and stability of pofyphenolic compounds in mulberry Monis alba L.). Food Chem. 1 13:964-969.

Kneifel W., Czech E. and Kopp ?. 2002. Microbial contamination of medicinal plants. A review. Planta Med. 68:5- 1 5.

Lee S.J., Umano ?., Shibamoto T. and Lee K.G. 2005. Identification of volatile components in basil Ocimum basüicum L.) and thyme leaves [Thymus vulgaris L.) and their antioxidant properties. Food Chem. 91:131-137.

Mclnerney J.K., Seccafien C.A., Stewart CM. and Bird A.R. 2007. Effects of high pressure processing on antioxidant activity, and total carotenoid content and availability, in vegetables. Innovativ Food Sci. Emerg. Technol. 8:543-548.

Miliauskas G., Venskutonis PR. and van Beek TA. 2004. Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chem. 85:231-237.

Munyaka A.W., Oey I., Van Loey A. and Hendrickx M. 20 1 0. Application of thermal inactivation of enzymes during vitamin C analysis to study the influence of acidification, crushing and blanching on vitamin C stability in Broccoli Brassica olerácea L var. italica). Food Chem. 1209:591-598.

Nguyen P.M., Kwee E.M. and Niemeyer E.D. 2010. Potassium rate alters the antioxidant capacity and phenolic concentration of basil Ocimum basüicumL.) leaves. Food Chem. 123:1235-1241.

Nsonzi F. and Ramaswamy H.S. 1998. Quality evaluation of osmo-convective dried blueberries. Dry Technol. 16:705-723.

Ocaña-Fuentes ?., Arranz-Gutiérrez E., Señorans F.J. and Reglero G. 2010. Supercritical fluid extraction of orégano Origanum vulgare] essentials oils: Anti-inflammatory properties based on cytokine response onTHP-1 macrophages. Food Chem. Toxicol. 48:1568-1575.

Patras ?., Tiwari B.K. and Brunton N.P. 2010. Influence of blanching and low temperature preservation strategies on antioxidant activity and phytochemical content of carrots, green beans and broccoli. Food Sci. Technol. 43:1313-1319.

Perera CO. 2005. Selected Quality Attributes of Dried Foods. Dry Technol. 23:717-730.

Polovka M. and Suhaj M. 2010. The effect of irradiation and heat treatment on composition and antioxidant properties of culinary herbs and spices. A Review. Food Rev. Int. 26:138-161.

Rota M.C, Herrera ?., Martinez R.M., Sotomayor J.A. and Jordán M.J. 2008. Antimicrobial activity and chemical composition of Thymus vulgaris. Thymus zygis and Thymus hyemalis essential oils. Food Control. 19:681-687.

Schweiggert U., Reinhold C and Schieber A. 2007. Conventional and alternative processes for spice production - a review. Trends Food Sci. Technol. 18:260-268.

Sellami LH., Maamouri E., Chahed T., Wannes W.A., Kchouk M.E. and Marzouk B. 2009. Effect of growth stage on the content and composition of the essential oil and phenolic fraction of sweet marjoram Origanum mqjorana L.). Ind. Crop Prod. 30:395-402.

Sgherri G, Cecconami S., Pinzino C, Navari-Izzo F. and Izzo R. 2010. Levels of antioxidants and nutraceuticals in basil grown in hydroponics and soil. Food Chem. 123:416-422.

Singleton V.L. and Rossi J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture. 16:144-158.

Suhaj M. 2006. Spice antioxidants isolation and their antiradical activity: a review. J. Food Compos. Anal. 19: 531-537.

Tateo F and Bononi M. 2006. Determination of ethylene chlorohydrin as marker of spice fumigation with ethylene oxide. J. Food Compos. Anal. 19:83-87.

Therdthai N. and Zhou W. 2009. Characterization of microwave vacuum drying and hot air drying of mint leaves (Mentha cordifoUa Opiz ex Fresen). J. Food Eng. 91:482-489.

Tohidpour A, Sattari M., Omidbaigi R., Yadegar A. and Nazemi J. 20 1 0. Antibacterial effect of essential oils from two medicinal plants against Methicillin-resistant Staphylococcus aureus (MRSA) . Phytomedicine. 17:142-145.

Tomaino A., Cimino F., Zimbalatti V, Venuti V, Sulfaro V, De Pasquale A. and Saija A. 2005. Influence of heating on antioxidant activity and the chemical composition of some spice essential oils. Food Chem. 89:549-554.

Turkmen N., Sari FY. and Velioglu S. 2005. The effect of cooking methods on total phenolics and antioxidant activity of selected green vegetables. Food Chem. 93:713-718.

Weil J., Bolton J.R. and WertzJ.E. 1994. Electron Paramagnetic Resonance: Elementary Theory and Practical Applications. New York: John Wiley and Sons Inc.

Woisky R.G. and Salatino A. 1998. Analysis of propolis: Some parameters and procedures for chemical quality control. J. Apicult. Res. 37:99-105.

Yazdanparast R. and Shahriyary L. 2008. Comparative effects of Artemisia dracunculus. Satureja hortensis and Origanum mqjorana on inhibition of blood platelet adhesion, aggregation and secretion. Vase. Pharmacol. 48:32-37.

Yu Y.M., TzengY.W. and ChangW.C. 2010. Origanummajoranah. prevents cardiovascular disease caused by obesity: suppression of leptin-induced proliferation in vascular smooth muscle cells. Atherosclerosis Supp. 11:137.

Paper received September 5, 2011 Accepted February 7, 2012