Effect of Gamma Irradiation on Total Phenolic Contents and Antioxidant Activities of Satureja Hortensis, Thymus Vulgaris, and Thymbra Spicata from Turkey

Abstract

In this study, the effect of gamma irradiation on the total phenolic content and antioxidant activity of Satureja hortensis, Thymus vulgaris, and Thymbra spicata was evaluated. Plants irradiated in a ⁶⁰Co irradiator to 0, 1.2, 3.0, and 5.1 kGy at 25°C. Control and irradiated samples were extracted with methanol. The antioxidant activity of methanol extracts was determined using phosphomolybdenum assay and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method. The total phenolic content in three spices was found to decrease with irradiation except at 5.1 kGy for S. hortensis. The DPPH radical scavenging activity of the extracts decreased after irradiation.

Keywords:

• Satureja hortensis
• Thymus vulgaris
• Thymbra spicata
• Antioxidant activity
• Gamma irradiation

INTRODUCTION

Satureja hortensis L., Thymus vulgaris L., and Thymbra spicata L. belonging to the Lamiaceae family are most widely used as spices. The leafy parts of S. hortensis (summer savory), T. vulgaris (thyme), and T. spicata (black thyme) have been added to meat, fish, and food products for years. In addition to improving flavour, certain spices and essential oils prolong the storage life of foods by an antimicrobial activity.[1] These spices have many biological activities, such as antibacterial,[1–3] antifungal,[4] and antioxidant.[5–7]

Plants contain a diverse group of phenolic compounds.[8] Many plant phenolic compounds have been reported to possess potent antioxidant activity.[9] The antioxidant activity of phenolic compounds is mainly due to the presence of the conjugated ring structures and hydroxyl groups, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers. In addition, they have a metal chelating potential. The antioxidant effect of plant phenolics has been studied in relation to the prevention of coronary diseases and cancer, as well as age-related degenerative brain disorders.[10,11]

Gamma radiation is a highly effective means of inhibiting the growth of undesired microbes. This is substantiated by the fact that an increasing number of countries have adopted irradiation as a way to ensure the hygienic quality of dehydrated foods. It is allowed up to 10 kGy as the international safe dose, though some countries, including Argentina, have increased this level to 30 kGy without any harmful effects being observed.[12]

There is growing scientific interest on the influence of irradiation processes on antioxidant activity of herb and spices.[12–15] However, the influence of gamma radiation on the antioxidant activity and total phenolic content of S. hortensis, T. vulgaris, and T. spicata has not been studied as yet. Therefore, the present study was planned to determine the effect of gamma irradiation on total phenolic content and antioxidant activity of S. hortensis, T. vulgaris, and T. spicata extracts.

MATERIALS AND METHODS

Samples

Satureja hortensis L. (Lamiaceae), Thymbra spicata L. (Lamiaceae), and Thymus vulgaris L. (Lamiaceae) were collected from Mersin (Southern Turkey). These plants were identified and authenticated by a plant taxonomist. The aerial parts (flower and leaf) of these plants were used in the study.

Irradiation

The samples of dry plant leaf powder were placed in plastic bottles and irradiated at 20°C with the doses of 1, 3, and 5 kGy at the Gammapak Company (Cerkezkoy, Tekirdag, Turkey). The irradiation process was carried out in a ⁶⁰Co gamma irradiator (MDS, Nordion, Ottawa, ON, Canada) at the average of the actual measured absorbed doses of 1.2, 3.0, and 5.1 kGy. Horwell (Harwell, UK) Amber Perspex Dosimeters (Batch R, Type 3042, Range: 1–30 kGy) had been used as reference dosimeters in order to measure the irradiation doses that were absorbed by irradiated dry plant samples. The control and irradiated samples were kept in a desiccator in the dark at 25°C until used for experiments.

Preparation of Extracts

The aerial parts of the plant materials were dried in the shade at room temperature. Dried aerial parts of plants were ground to fine powder using a blender. Then the powdered plant material (10 g) was extracted using a Soxhlet type extractor with 100 ml methanol (MeOH) at 60°C for 6 h. Thereafter, the extract was filtered through Whatman No. 1 paper (Maidstone, England) in a Buchner funnel (Coors, USA). Methanol was evaporated under reduced pressure in a rotary evaporator (Rotavapor, Buchi, Flawil, Switzerland; T < 40°C). After determining the yield, the extract was dissolved in methanol for further study.[16]

Determination of Total Phenolic Compounds

The amount of total phenolic compounds in the methanol extracts of S. hortensis, T. vulgaris, and T. spicata was estimated according to the Folin-Ciocalteu method, using gallic acid as the reference compound.[17] Briefly, 1 mg of dry extract was dissolved in 1 ml of methanol. This solution (40 μl) was mixed with 2.4 ml of distilled water, 200 μl of Folin-Ciocalteu reagent, and 600 μl of 20% sodium carbonate. Then, the volume was made up to 4.0 ml with distilled water. After incubation at room temperature for 2 h, the absorbance of the reaction mixture was measured at 765 nm. The data are presented as the average of triplicate analyses. Results were expressed as mg of gallic acid equivalents/g extract.

Phosphomolybdenum Assay

The antioxidant activity of the extracts was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al.[18] The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate/Mo (V) complex at acid pH. 0.4 ml of the methanolic extract (1 mg/ml) was combined with 4 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95°C for 90 min. Then, the absorbance of the solution was measured at 695 nm against blank after cooling to room temperature. In the case of the blank, 0.4 ml of methanol was used in place of sample. The mean of three readings was used and the antioxidant capacity of the extracts was expressed as mg of ascorbic acid equivalents /g extract.

Scavenging Activity on the 2,2-diphenyl-1-picrylhydrazyl Radical (DPPH Test)

The free radical scavenging activity of the extracts, based on the scavenging activity of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, was determined by the method described by Lee et al.[19] A 50-μl aliquot of the proper methanolic extract dilution in the concentration range of 0.1–2 mg/ml was added to 1 ml of the methanolic DPPH solution (0.1 mM). Absorbance at 517 nm was determined after 30 min, and the percentage inhibition was calculated from [(A ₀ – A ₁)/A ₀] × 100, where A ₀ is the absorbance of the control and A ₁ is the absorbance of the extract/standard. The same procedure was repeated with butylated hydroxytoluene as the positive control. IC₅₀ value is the extraction concentration at which DPPH radicals are reduced by 50%. IC₅₀ values of the methanolic extracts were calculated from linear regression analysis. The measurements were performed in triplicate and the results were averaged.

Table 1 The percent yield, total phenolic content, and total antioxidant activity of S. hortensi s, T. vulgaris, and T. spicata.

Plants	Treatment	Yield (%)	Gallic acid equivalent (mg /g)	Ascorbic acid equivalent (mg/g)
Satureja hortensis	Control	11.00	107.65 ± 1.2^b, *	232.98 ± 5.0^a, *
	1.2kGy	14.00	81.28 ± 1.2^d	191.32 ± 0.7^b
	3.0kGy	16.00	92.43 ± 1.3^c	195.90 ± 0.8^b
	5.1kGy	15.00	142.81 ± 1.8^a	233.05 ± 4.8^a
Thymus vulgaris	Control	14.00	35.30 ± 1.0^a	172.85 ± 0.6^a
	1.2kGy	14.00	23.46 ± 0.6^b	152.18 ± 0.7^d
	3.0kGy	12.00	21.77 ± 0.6^c	153.47 ± 0.0^c
	5.1kGy	12.00	21.77 ± 0.6^c	154.62 ± 0.7^b
Thymbra spicata	Control	13.00	75.19 ± 1.3^a	216.80 ± 0.0^b
	1.2kGy	14.00	57.27 ± 1.5^b	205.21 ± 0.0^c
	3.0kGy	13.00	75.19 ± 1.5^a	263.65 ± 3.3^a
	5.1kGy	13.00	55.58 ± 1.0^b	210.32 ± 0.1^bc
*Means followed by the same letter in a column are not significantly different at p = 0.05.
Values expressed are mean ± standard deviation of three experiments.

Statistical Analysis

Data from the experiments were subjected to analysis of variance (ANOVA) using SPSS 10.0 on Windows (Chicago, IL, USA). Means were separated at the 5% significance level by the least significant difference test. Bivariate correlations were analysed by Pearson's test using SPSS 10.0 on Windows.[20]

RESULTS AND DISCUSSION

Extraction Yields

The extraction yields of S. hortensis, T. vulgaris, and T. spicata in methanol were determined and are shown in Table 1. The methanol extracts of S. hortensis showed increased yields after exposure to radiation. The extract of T. vulgaris showed a linear decrease in the dry weight with an increase of the gamma radiation dose (3.0 and 5.1 kGy) while the dry weight yield of the extract of T. vulgaris samples irradiated at 1.2 kGy, was found to be the same as that of the control. T. spicata irradiated at 1.2 kGy had higher yields than the control and samples irradiated at higher doses.

The increase in the dry weights of extracts following irradiation might be due to degradation of some high molecular weight components, thus changing these components from insoluble to soluble ones in the test solvents.[14] A similar increase in extraction yields of Agaricus blazei with radiation treatment has also been reported by Huang and Mau.[21] Khattak et al.[14] reported an increase in the methanol extract of Nigella sativa after exposure to radiation. Kim et al.[22] found an increase in the extraction yields of Korean medicinal herbs after treating with gamma irradiation. The difference in extraction yields, as compared to that reported in the literature may be due to different chemical composition of the plants.

Total Phenolic Content

The total phenolic content of control and irradiated samples of S. hortensis, T. vulgaris, and T. spicata in methanol was determined using Folin-Ciocalteau's reagent. The results are expressed as mg equivalents of gallic acid/g dry extract and are given in Table 1. For the control samples, S. hortensis had the highest total phenolic content (107.65 ± 1.2 mg/g) while T. vulgaris had the least total phenolic content (35.30 ± 1.0 mg/g). Gamma irradiation significantly affected the total phenolic content of S. hortensis, T. vulgaris, and T. spicata (p < 0.05).

A significant decrease in phenolic content was observed in the methanolic extracts of T. vulgaris following 1.2, 3.0, and 5.1 kGy irradiation (p < 0.05). The phenolic content of the control sample was found to be 35.30 mg/g. The phenolic content decreased to 21.77 mg/g for T. vulgaris irradiated with a dose of 5.1 kGy. Gamma irradiation decreased the total phenolic content in T. vulgaris extracts by about 38.32% at 5.1 kGy compared to the control. Significant change in the total phenolic content of T. spicata was observed following irradiation except at 3 kGy radiation (p < 0.05). The phenolic content decreased to 57.27 and 55.58 mg/g for T. spicata irradiated with a dose of 1.2 and 5.1 kGy, respectively. Gamma irradiation decreased the total phenolic content in T. spicata extract by about 26.08% at 5.1 kGy compared to the control.

The effect of gamma irradiation on the phenolic content of S. hortensis, T. vulgaris, and T. spicata has not been investigated previously, but similar observations on other plants and spices were reported. Research on the effect of gamma irradiation on almond skin showed that the almond skin extract had increased phenolic content with an irradiation dose of 4 kGy and above.[13] Variyar et al.[23] found increased amounts of phenolic acids in irradiated cloves and nutmeg. In contrast, Koseki et al.[24] determined a decrease, with respect to control, of the amount of total phenolic compounds in dehydrated rosemary after irradiation doses of between 10 and 30 kGy. Mishra et al.[25] found no significant effect on total phenolics in radiation processed tea leaves. Perez et al.[12] showed that total phenolic content remained the same as that of the control in the methanol extract of Rosmarinus officinalis. For cumin seeds, Kim et al.[15] found a non significant increase in the phenolic content in the irradiated cumin. As similar to our present study, De-Toledo et al.[26] reported that the dose of 8 kGy promoted an increase in the content of total phenolic compounds in soybean while total phenolic content decreased at doses of 2 and 4 kGy.

The differences in effects were attributed to the different phenolic compounds present in the various plants. The increased phenolic content in irradiated S. hortensis could be attributed to the release of phenolic compounds from a glycosidic component and the degradation of larger phenolic compounds into smaller ones by gamma radiation. Adamo et al.[27] thought that the destructive processes of oxidation and the gamma irradiation were capable of breaking the chemical bonds of polyphenols and thereby releasing soluble phenols of low molecular weights.

Phosphomolybdenum Assay

The total antioxidant activity of control and irradiated samples of S. hortensis, T. vulgaris, and T. spicata in methanol was determined using phosphomolybdenum assay. The results are expressed as mg equivalents of ascorbic acid/g dry extract and are given in Table 1. For the control samples, S. hortensis showed the highest antioxidant activity while T. vulgaris showed the least antioxidant activity. Total antioxidant activity of control and irradiated samples in methanol extract were found to be statistically different (p < 0.05). The total antioxidant activity decreased in methanolic extracts of S. hortensis irradiated with 1.2 and 3 kGy, respectively, while total antioxidant activity of samples irradiated at 5.1 kGy was found to be the same as that of the control. The antioxidant activity of the control sample of T. vulgaris was found to be 172.85 ± 0.6 mg/g. The antioxidant activity decreased to 154.62 ± 0.7 mg/g for T. vulgaris irradiated with a dose of 5.1 kGy. Total antioxidant activity of T. vulgaris methanol extract was decreased by about 10.36% at 5.1 kGy compared to the control. The antioxidant activity decreased to 205.21 ± 0.0 and 210.32 ± 0.1 mg/g in methanolic extracts of T. spicata irradiated with 1.2 and 5.1 kGy compared to that of the control (216.80 ± 0.0 mg/g), respectively, while the antioxidant activity increased (263.65 ± 3.3 mg/g) following irradiation with 3 kGy. Irradiation decreased the total antioxidant activity in T. spicata by about 2.98% at 5.1 kGy compared to the control. There is no information available in the literature on the effect of gamma irradiation on the total antioxidant activity using phosphomolybdenum assay of S. hortensis, T. vulgaris, and T. spicata, as well as other biological materials.

Phenolic substances have been shown to be responsible for the antioxidant activity of plant materials.[28] Gamma irradiation might degrade antioxidant components or decompose some components into antioxidant components. They may change the content and composition of antioxidant components, thereby affecting the antioxidant properties.[21] Bivariate correlations analysed by Pearson's test revealed that there is a correlation between total phenolic contents and total antioxidant activities of the methanolic extracts of control and irradiated S. hortensis (r ² = 0.828). Also, the same correlation was observed for the methanolic extracts of control and irradiated T. vulgaris and T. spicata (r ² = 0.972 and 0.691, respectively).

DPPH Test

The scavenging activity of control and irradiated S. hortensis, T. vulgaris, and T. spicata extracts, expressed as inhibition percentage (% I) of DPPH radical, was analysed in a range of concentrations between 0.25 and 2 mg/ml methanol extract (Fig. 1). DPPH scavenging activities increased as concentrations increased from 0.25 to 2 mg/ml for all the methanol extracts with or without irradiation. However, gamma irradiation decreased the scavenging activity in all the methanol extracts tested. At 2 mg/ml concentration, scavenging activity of S. hortensis decreased about 0.33% after 5 kGy irradiation compared to the control. These decreases were 20.68 and 3.97% for T. vulgaris and T. spicata, respectively.

Figure 1 Effect of gamma irradiation on the scavenging activity of methanolic extracts of S. hortensis, T. vulgaris, and T. spicata on DPPH radical. Each value is expressed as mean ± standard deviation (n = 3).

For better comparison of the scavenging activity of S. hortensis, T. vulgaris, and T. spicata, the results obtained from the DPPH radical assay were expressed as IC₅₀ values (Fig. 2). For the control samples, a comparison of the three spices showed differences in scavenging activity at all concentrations in the order: S. hortensis > T. spicata > T. vulgaris. Also, IC₅₀ values of S. hortensis, T. vulgaris, and T. spicata extracts were 10.47, 24.04, and 10.52 μg/ml, respectively (Fig. 2). A low IC₅₀ value is indicative of strong antioxidant activity. Gamma irradiation increased the IC₅₀ values in S. hortensis methanol extracts by 29.60, 23.68, and 3.72% at a dose level of 1.2, 3.0, and 5.1 kGy, respectively, compared to the control. Likewise, an increase was observed in T. vulgaris methanol extracts by 16.84, 6.44, and 26.58% at the same radiation doses, respectively. A similar increase was determined in T. spicata methanol extracts by 38.23, 15.68, and 29.94% at the same radiation doses, respectively. This might be due to some conformational changes induced by radiation in the structure of phenolic contents.

Figure 2 IC₅₀ values (μg/ml) for methanolic extracts of S. hortensis, T. vulgaris, and T. spicata irradiated with various doses of gamma irradiation.

Though various researchers have worked on the free radical scavenging activity of herbs and spices,[12,14,15] no data is reported on the effect of gamma irradiation on the free radical scavenging activity of S. hortensis, T. vulgaris, and T. spicata. Many researchers reported different results for effect of gamma irradiation on the free radical scavenging activity of plants. In accordance with our results, it was determined that the DPPH radical scavenging activity reduced after irradiation at 2 kGy.[29] Lampart-Szczapa et al.[30] showed increasing doses of irradiation to lower antioxidant effects of lupin extracts. Suhaj et al.[31] determined that the scavenging activity of methanol extract of black pepper treated with doses of between 5 and 30 kGy tended to decrease immediately after irradiation. On the other hand, Variyar et al.[32] indicated that the scavenging activity of soybean increased with increasing doses from 0.5 to 5 kGy. A previous study on Nigella sativa showed that gamma irradiation enhanced the scavenging activity in acetone and methanol extracts of plant.[14] In contrast, Mishra et al.[25] reported that the free radical scavenging activity of tea was not affected due to radiation treatment within a dose of 10 kGy. For Rosmarinus officinalis, Perez et al.[12] reported that the antioxidant activity of methanol extracts of an irradiated plant remained the same as in the control in a DPPH test. According to another report, irradiation resulted in a non significant increase in the DPPH radical scavenging ability of cumin ethanolic extract at 1, 3, 5, and 10 kGy when compared to the control, but cumin irradiated with 10 kGy showed a non significant decrease when compared to that of cumin irradiated with 3 and 5 kGy.[15] The differences in the effect of gamma irradiation on the free radical scavenging activity of plants may be due to the difference in their chemical composition, solvent used for extraction, and other characteristics.

Conclusion

The results showed that both control and irradiated S. hortensis, T. vulgaris, and T. spicata possessed excellent antioxidant activity and DPPH radical scavenging activity. Gamma radiation decreased total phenolic content on selected plants except 5.1 kGy for S. hortensis. The free radical scavenging activity decreased with gamma irradiation between 1.2 and 5.1 kGy. The decrease in the phenolic content and scavenging activity might be acceptable, when the increasing demand and benefit of the quality, safety as well as shelf life of plant material were considered. Further studies to understand related to the mechanism of gamma irradiation on their antioxidant activity and the effect of gamma irradiation on their different phenolic compounds is necessary.