Research Article Received: 21 June 2014
Revised: 25 September 2014
Accepted article published: 15 October 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6959
Antioxidant activity and essential oil composition of Satureja hortensis L. as influenced by sulfur fertilizer Sharareh Najafiana* and Maryam Zahedifarb Abstract BACKGROUND: The quantity and quality of essential oils in plants can be influenced by various factors, including genetic traits, agricultural practices and environmental conditions such as nutrient availability. Macronutrients such as sulfur (S) are among the major factors influencing plant growth and development. Therefore this study was carried out to determine the effect of S fertilization at three levels (0, 0.05 and 0.1 g S kg−1 soil) on the essential oil composition and antioxidant activity of Satureja hortensis L. RESULTS: Application of 0.05 g S kg−1 soil increased the levels of 𝜶-terpinene, p-cymene, myrcene, 𝜶-thujene and 𝜶-pinene but decreased the level of 𝜸-terpinene in the essential oil. This S application was most suitable for obtaining higher proportions of 𝜶-terpinene, p-cymene, myrcene, 𝜶-thujene and 𝜶-pinene, while application of 0.1 g S kg−1 soil was preferable for carvacrol. 𝜸-Terpinene was most abundant in the control. All extracts showed a considerable DPPH-inhibitory effect, with IC50 ranging from 0.720 g L−1 in the control to 0.363 g L−1 with application of 0.1 g S kg−1 soil. The maximum antioxidant effect was observed with application of 0.1 g S kg−1 soil. CONCLUSION: The results revealed that the use of chemical fertilizers such as S could improve the antioxidant activity of plant extracts significantly. Studying the secondary plant metabolites, mainly essential oils, is an interesting research area, so further studies are recommended to determine the effect of chemical fertilizers on the composition and antioxidant activity of essential oils of other aromatic plants. © 2014 Society of Chemical Industry Keywords: chemical fertilizer; DPPH; antioxidant activity; essential oil
INTRODUCTION Antioxidant compounds are vital health-protecting factors in foods and have been shown to reduce the risk of chronic ailments such as heart disease and cancer.1 Antioxidants delay autoxidation by preventing the formation and/or interrupting the propagation of free radicals. Phenolic antioxidant compounds can be divided into several general groups, e.g. flavonoids, volatile oils, phenolic acids and diterpenes.2 Compounds in these groups exert their antioxidant effect in different ways. For example, generally, phenolic acids trap free radicals, while flavonoids scavenge free radicals or chelate metals.3 Phenolic compounds that are a major constituent of human nutrition are a large group of secondary metabolites widely distributed in plants. Several approaches have been used to monitor and compare the antioxidant activity of foods. Measurement of 2,2-diphenyl1-picrylhydrazyl (DPPH) free radical-scavenging ability is a simple, rapid and inexpensive method for determining the antioxidant activity of foods. This method is generally used to assess the ability of individual compounds to act as free radical scavengers or hydrogen donors in order to assess the antioxidant activity of foods. Recently, it has been used to evaluate the antioxidant activity in complex biological systems. It can also be applied for the assessment of overall antioxidant capacity in both liquid and solid materials as a non-selective approach for any specific J Sci Food Agric (2014)
component. The overall antioxidant capacity of foods can be useful to determine their functional properties.4 Essential oils are natural aromatic compounds found in the flowers, seeds, stems, bark, roots or other parts of plants. They are often used as medicinal oils for skin treatments to remediate cancer and can be extracted using the following approaches: solvent extraction, distillation or steam expression. The quantity and quality of essential oils in plants can be influenced by various factors such as genetic traits,5 environmental conditions (e.g. light, temperature, nutrient and water availability)6,7 and agricultural practices (e.g. harvest date, postharvest conditions).8 Macronutrients are among the major factors influencing plant growth and development. Sulfur (S) is one of the macronutrients with important functions in plants. It is found in cysteine, cystine
∗
Correspondence to: Sharareh Najafian, Department of Agriculture, Payame Noor University, PO Box 19395-3697, Tehran, Iran. E-mail: sh.najafi[email protected]
a Department of Agriculture, Payame Noor University, PO Box 19395-3697, Tehran, Iran b Department of Rangeland and Watershed Management, College of Agriculture and Natural Resources, Fasa University, Fasa, Iran
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www.soci.org and methionine, amino acids that make up proteins. Furthermore, it activates certain enzyme (hydrogenase) systems9 and is a main part of nutritional vitamins such as vitamin B1 (thiamine) and vitamin H (biotin).10 Sulfur fertilization has also been shown to increase the oil content of crop seeds.11 It was also found that S application as SO4 2− increased the yield of crops such as corn, wheat and oilseed rape.12 On the other hand, decreased growth, dry matter yield and SPAD chlorophyll meter reading of barley leaves were reported be Astolfi et al.13 Satureja hortensis L. (Lamiaceae), summer savory, is an annual, herbaceous aromatic and medicinal plant native to southern Europe and naturalized in parts of North America.14 The aerial parts of some Satureja plants are widely used in foods as a flavor component, in herbal teas and in folk and traditional medicines to treat ailments such as nausea, cramp, indigestion, muscle pain, diarrhea and infectious diseases.15 Alizadeh et al.16 reported that application of complete fertilizer caused a slight but non-significant change in the essential oil composition of S. hortensis, with the levels of some components such as carvacrol, 𝛿-terpinene and 𝛼-terpinene being affected. They stated that application of 1500 mg of fertilizer per plant decreased the amount of carvacrol significantly but increased 𝛿-terpinene and 𝛼-terpinene. Zheljazkov et al.12 concluded that the oil yield of sweet basil was maximized by application of 80 kg S ha−1 . Khan and Hussain17 showed that the highest seed and oil yields of mustard were obtained with application of 20 kg S ha−1 . It is reported that nutrients and antioxidants together may reduce levels of reactive oxygen species more effectively than single dietary antioxidants, because they can act in synergy.18 Since there appears to be no previous research on the subject, the aim of this study was to investigate the effect of S fertilizer on the essential oil composition and antioxidant activity of summer savory.
MATERIALS AND METHODS To determine the effect of S fertilizer on the essential oil composition and antioxidant activity of S. hortensis L., a greenhouse experiment was carried out on a loamy calcareous soil (Table 1). Some physical and chemical properties of the studied soil were measured using standard methods: sodium bicarbonateextractable phosphorus (P),19 diethylenetriaminepentaacetic acid (DTPA)-extractable iron (Fe), manganese (Mn), zinc (Zn) and copper (Cu),20 calcium carbonate equivalent,21 total nitrogen (N),22 organic matter (wet oxidation method), pH in saturated paste (pH meter) and electrical conductivity in saturated extract (electrical conductivity meter). The experiment was a randomized complete design with six replicates. Treatments consisted of soil application of three elemental S levels (0, 0.05 and 0.1 g S kg−1 soil). Each plastic pot contained 3 kg of soil. Pots were watered with distilled water to near field capacity and maintained at this moisture level by adding water to a constant weight. To prevent any possible deficiency of nutrients other than S, all pots received uniform application of 0.02 g P kg−1 soil as Ca(H2 PO4 )2 ·H2 O, 0.15 g N kg−1 soil as CO(NH2 )2 (one half was added at planting and the other half was shoot dressed 3 weeks after emergence) and 0.01, 0.01 and 0.005 g Mn, Zn and Cu kg−1 soil respectively as their sulfates in aqueous form. All pots received uniform application of 0.01 g Fe kg−1 soil as Fe chelate of ethylenediaminedihydroxyphenylacetic acid (FeEDDHA) in aqueous form. Twenty S. hortensis seeds were planted about 1 cm deep and thinned to ten uniform stands 2 weeks after emergence. Plants were harvested 12 weeks after
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S Najafian, M Zahedifar
Table 1. Some physical and chemical properties of studied soil
ECe Soil (dS property pH m−1 ) Value
7.8
0.4
CCE (g kg−1 ) 450
OM (g kg−1 ) 15
Olsen P (mg kg−1 ) 4.5
DTPAextractable (mg kg−1 ) Fe 2.3
Mn
Zn
Cu
3.7 0.96 1
ECe , electrical conductivity, CCE, calcium carbonate equivalent; OM, organic matter.
planting. Plant samples were dried in shade and prepared for analysis. Preparation of plant extracts Dried plant samples (20 g) were soaked in 200 mL of methanol/water (90:10 v/v) for 2 days, with a change of solvent after 1 day. The extracts were filtered and concentrated in a rotary evaporator for up to 10 min. The powders were weighed and their yields determined. Before use, the powders were kept at −20 ∘ C. Immediately before each measurement, the desired concentration of powder in methanol was prepared and its antioxidant activity and total phenol content were measured.23 Using DPPH for determination of antioxidant activity Scavenging of the stable DPPH free radical was used to determine the antioxidant activity of plant extracts and standard antioxidant gallic acid. In a modified assay,24 200 μL of 100 mmol L−1 DPPH radical solution in methanol was mixed with 20 μL of 12.5–3200 μg mL−1 methanol extracts or gallic acid. The solutions were maintained at room temperature for about 30 min. DPPH radical inhibition was determined using an ELx808 microplate reader (BioTek Instruments Inc., Winooski, VT, USA) at 515 nm. IC50 (concentration (mg L−1 ) required to inhibit DPPH radical formation by 50%) values of samples were calculated from the nonlinear regression between log concentration (μg mL−1 ) of test extracts and mean % radical-scavenging activity using MATLAB (The MathWorks Inc., Natick, MA, USA). The blank subtracted from all measurements was methanol extract without DPPH. Antioxidant activity was determined using the equation [( ) ] antioxidant activity = 100 – Asample –Ablank ∕Acontrol × 100 where A denotes absorbance. DPPH (without plant extract) and methanol were used as control and blank respectively. Headspace volatile extraction Dried plant samples (3 g) were crushed and placed in 20 mL headspace vials. The vials were immediately sealed with silicone rubber septa and aluminum caps and then transferred to a headspace tray. Headspace extraction was performed using a CombiPAL system (CTC Analytics AG, Zwingen, Switzerland) comprising a heater, an agitator and a headspace autosampler. The vials were heated to 80 ∘ C and held for 20 min while being agitated; the temperature of the sampling needle and transmission lines was 85 ∘ C.25 Identification of oil constituents using gas chromatography/mass spectrometry (GC/MS) GC/MS analysis was carried out using an Agilent 7890 GC/MS system (Agilent Technologies Inc., Shanghai, China) operating at
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Influence of S fertilizer on Satureja hortensis essential oil 70 eV ionization energy, equipped with an HP-5 MS capillary column (Chrom Tech, Apple Valley, MN, USA) (phenyl methyl siloxane, 30 m × 0.25 mm i.d., 25 μm) with helium as carrier gas and a split ratio of 1:50. Retention indices (RIs) were determined using retention times of n-alkanes that were injected after the essential oil under the same chromatographic conditions. RIs of all components were determined based on an approach using n-alkanes as standards. Compounds were identified by comparison of their RIs (HP-5) with those reported by others in the literature and also by comparison of their mass spectra with published mass spectral data in the Adams Library, Wiley GC/MS Library and MassFinder 2.1 Library.26 – 28
RESULTS AND DISCUSSION Chemical composition of essential oil Eighteen essential oil compounds were identified in the control, whereas 15 and 17 compounds were obtained with application of 0.05 and 0.1 g S kg−1 soil respectively. The identified constituents with their respective levels and RIs are summarized in Table 2. The main oil constituents in the control were 𝛾-terpinene (674 g kg−1 ), 𝛼-terpinene (108 g kg−1 ), myrcene (44 g kg−1 ), 𝛼-thujene (42 g kg−1 ), p-cymene (38 g kg−1 ), 𝛼-pinene (28 g kg−1 ) and carvacrol (21 g kg−1 ). Application of 0.05 g S kg−1 soil changed these components to 𝛾-terpinene (639 g kg−1 ), 𝛼-terpinene (125 g kg−1 ), myrcene (52 g kg−1 ), 𝛼-thujene (49 g kg−1 ), p-cymene (42 g kg−1 ), 𝛼-pinene (32 g kg−1 ) and carvacrol (15 g kg−1 ). Finally, the composition was 𝛾-terpinene (664 g kg−1 ), 𝛼-terpinene (90 g kg−1 ), carvacrol (72 g kg−1 ), p-cymene (37 g kg−1 ), myrcene (43 g kg−1 ), 𝛼-thujene (31 g kg−1 ) and 𝛼-pinene (22 g kg−1 ) with application of 0.1 g S kg−1 soil. The most important result of this study was the increasing trend in levels of 𝛼-terpinene, p-cymene, myrcene, 𝛼-thujene and 𝛼-pinene with application of 0.05 g S kg−1 soil (Table 2). The 𝛼-terpinene concentration was 108 g kg−1 without S fertilization but reached 125 g kg−1 after application of 0.05 g S kg−1 soil, i.e. a 15.7% increase. Myrcene (18.2% increase), 𝛼-thujene (16.7% increase) and 𝛼-pinene (14.3% increase) exhibited a similar trend. Another component showing the same trend was p-cymene, which is a monoterpene. Monoterpenes are the primary components of plant essential oils, and the effects of many medicinal herbs have been attributed to them.29,30 The p-cymene concentration was 38 g kg−1 without S fertilization but reached 42 g kg−1 after application of 0.05 g S kg−1 soil, i.e. a 10.5% increase. In contrast, the concentration of 𝛾-terpinene with lower molecular weight decreased slightly (by 5.2%) with application of 0.05 g S kg−1 soil. This phenomenon could be due to evaporation, oxidation or other unwanted changes in essential oil components with application of 0.05 g S kg−1 soil. The genus Satureja presents great variability in the concentrations of major components of its essential oil owing to the existence of different species and subspecies but also to many other factors, mainly environmental and climatic conditions.16 In previous studies, thymol and carvacrol in particular were found to be the principal constituents of the oils isolated from several Croatian Satureja species.31,32 It is interesting that various isolates of winter savory from Croatia and Bosnia and Herzegovina have carvacrol (up to 841.9 g kg−1 ) as the main constituent.33 A review of the published literature reveals that the essential oil of winter savory shows large variations in the relative concentrations of major components such as carvacrol (50–690 g kg−1 ), linalool (10–620 g kg−1 ), 𝛾-terpinene (10–310 g kg−1 ) and J Sci Food Agric (2014)
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Table 2. Effect of S application on concentrations of chemical compounds (g kg−1 ) identified in essential oil of Satureja hortensis, and their retention indices Applied S fertilizer (g S kg−1 soil)
No. Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
𝛼-Thujene 𝛼-Pinene Camphene Sabinene 𝛽-Pinene Myrcene 𝛼-Phellandrene 𝛿-3-Carene 𝛼-Terpinene p-Cymene Limonene 𝛽-Phellandrene (E)-𝛽-Ocimene 𝛾-Terpinene p-Cymen-9-ol (Z)-Ocimenone Carvacrol Caryophyllene 𝛽-Bisabolene
Retention index
0 (control)
0.05
923 42 ± 2.1b 49 ± 2.3a 930 28 ± 1.6a 32 ± 2a 945 2 ± 0.2a 2 ± 0.2a 969 3 ± 0.3a 4 ± 0.35a 973 11 ± 1.2a 13 ± 1.1a 987 44 ± 2.3b 52 ± 2.3a 1003 9 ± 0.8b 11 ± 1.3a 1008 1 ± 0.08b 2 ± 0.25a 1014 108 ± 5.5b 125 ± 6a 1021 38 ± 1.9b 42 ± 2.2a 1025 9 ± 0.95a 1 ± 0.1c 1026 2 ± 0.25b 3 ± 0.4a 1043 1 ± 0.1b 1 ± 0.1b 1059 674 ± 21a 639 ± 22b 1211 3 ± 0.35 – 1224 1 ± 0.08 – 1299 21 ± 1.5b 15 ± 1.8b 1415 – – 1504 1 ± 0.08b –
0.1 31 ± 1.9c 22 ± 1.4b 2 ± 0.3a 3 ± 0.3a 11 ± 1.2a 43 ± 2.4b 8 ± 0.9b 1 ± 0.1b 90 ± 4c 37 ± 1.9b 7 ± 0.8b 3 ± 0.4a 2 ± 0.2a 664 ± 25a – – 72 ± 3.2a 1 ± 0.1 3 ± 0.5a
Data are mean ± standard deviation of six replications. Means in a row followed by the same letter are not significantly (P < 0.05) different by Duncan’s multiple range test.
p-cymene (30–270 g kg−1 ), arising from the existence of different chemotypes.34 The main constituents of S. hortensis (summer savory) essential oil are the phenols thymol (290 g kg−1 ), carvacrol (265 g kg−1 ), 𝛼-terpinene (226 g kg−1 ), p-cymene (93 g kg−1 ) and other terpenoids.12 Finally, it can be concluded that application of 0.05 g S kg−1 soil is the most suitable for obtaining higher proportions of 𝛼-terpinene, p-cymene, myrcene, 𝛼-thujene and 𝛼-pinene, whereas application of 0.1 g S kg−1 soil is preferable for obtaining particular components such as carvacrol. On the other hand, 𝛾-terpinene is mainly produced in the control. Many studies have shown that the most abundant phenolic components in Satureja are carvacrol, 𝛾-terpinene, thymol, p-cymene, 𝛽-caryophyllene, linalool and other terpenoids. The concentrations of these constituents vary between different Satureja species.14 Some investigators have shown that the essential oil of Satureja species has a variety of activities, including antibacterial and antifungal properties, and exhibits strong inhibition of a wide range of bacteria and fungi in human, food and plant pathogens.12 Some plants of the Lamiaceae family are rich in phenolic compounds such as flavonoids, phenolic acids and phenolic diterpenes and have high antioxidant activities.35,36 Flavonoids and phenolic compounds exert multiple biological effects such as antioxidant, free radical-scavenging and anti-inflammatory activities.37,38 Oxidative damage in the human body plays an important causative role in disease initiation and progression.39 IC50 is an appropriate parameter to measure the progress of oxidation in oils and is therefore considered as a good indicator of the effectiveness of antioxidants. All extracts showed a considerable DPPH-inhibitory effect, with IC50 ranging from 0.720 g L−1 in the control to 0.363 g L−1
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IC50(mg L−1)
800 700 600 500 400 300 200 100 0 Gallic acid
Control
0.05 g S kg–1 soil
0.1 g S kg–1 soil
Treatments
Figure 1. Effect of S application on antioxidant activity of Satureja hortensis measured by DPPH assay, and comparison with gallic acid (IC50 is concentration required to inhibit DPPH radical formation by 50%).
with application of 0.1 g S kg−1 soil (Fig. 1). The linear regression equation between IC50 (Y, mg L−1 ) and S fertilization level (X, g kg−1 soil) was determined as Y = −3.575X + 773.01 (R2 = 0.79, P < 0.01) showing that IC50 decreased in response to applied S. Extensive studies have been carried out on the antioxidant activity of many species of the Lamiaceae family.2,36,40 They demonstrated that members of this family had very strong antioxidant capacity. The findings of the present study indicate that savory oils, in addition to other properties, have potential as topical antioxidants. The maximum antioxidant effect was observed with application of 0.1 g S kg−1 soil. In this treatment the concentration of carvacrol increased 2.4-fold compared with the control. The results obtained in our study show good correlation within one species.
CONCLUSION Studying the secondary plant products, especially essential oils, is an interesting research area that requires further studies on other aromatic plant essential oils consisting of various components. Furthermore, the results revealed that the addition of chemical fertilizers such as S could improve the antioxidant activity of plant extracts significantly.
ACKNOWLEDGEMENTS The authors thank Mr Engineer Hamid Reza Satari and Mr Engineer Ahmad Reza Ghasemi, superintendents of Eram Garden, for their support.
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Influence of S fertilizer on Satureja hortensis essential oil 31 Milos M, Radonic A, Bezic N and Dunkic V, Localities and seasonal variations in the chemical composition of essential oils of Satureja montana L. and S. cuneifolia Ten. Flav Fragr J 16:157–160 (2001). 32 Mirjana S and Nada B, Chemical composition and antimicrobial variability of Satureja montana L. essential oils produced during ontogenesis. J Essent Oil Res 16:387–391 (2004). 33 Kustrak D, Kuftinec J, Blazvic N and Maffei M, Comparison of the essential oil composition of two subspecies of Satureja montana. J Essent Oil Res 8:7–13 (1996). 34 Cazin C, Jonard R, Alain P and Pellecuer J, L’evolution de la composition des huiles essentielles chez divers chemotypes des Sarriette des montangnes (Satureja montana L.) obtenus par l’isolement in vitro des apex. CR Acad Sci III 6:237–240 (1985). 35 Wong CC, Li HB, Cheng KW and Chen F, A systematic survey of antioxidant activity of 30 Chinese medicinal plants using the
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Revised: 25 September 2014
Accepted article published: 15 October 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6959
Antioxidant activity and essential oil composition of Satureja hortensis L. as influenced by sulfur fertilizer Sharareh Najafiana* and Maryam Zahedifarb Abstract BACKGROUND: The quantity and quality of essential oils in plants can be influenced by various factors, including genetic traits, agricultural practices and environmental conditions such as nutrient availability. Macronutrients such as sulfur (S) are among the major factors influencing plant growth and development. Therefore this study was carried out to determine the effect of S fertilization at three levels (0, 0.05 and 0.1 g S kg−1 soil) on the essential oil composition and antioxidant activity of Satureja hortensis L. RESULTS: Application of 0.05 g S kg−1 soil increased the levels of 𝜶-terpinene, p-cymene, myrcene, 𝜶-thujene and 𝜶-pinene but decreased the level of 𝜸-terpinene in the essential oil. This S application was most suitable for obtaining higher proportions of 𝜶-terpinene, p-cymene, myrcene, 𝜶-thujene and 𝜶-pinene, while application of 0.1 g S kg−1 soil was preferable for carvacrol. 𝜸-Terpinene was most abundant in the control. All extracts showed a considerable DPPH-inhibitory effect, with IC50 ranging from 0.720 g L−1 in the control to 0.363 g L−1 with application of 0.1 g S kg−1 soil. The maximum antioxidant effect was observed with application of 0.1 g S kg−1 soil. CONCLUSION: The results revealed that the use of chemical fertilizers such as S could improve the antioxidant activity of plant extracts significantly. Studying the secondary plant metabolites, mainly essential oils, is an interesting research area, so further studies are recommended to determine the effect of chemical fertilizers on the composition and antioxidant activity of essential oils of other aromatic plants. © 2014 Society of Chemical Industry Keywords: chemical fertilizer; DPPH; antioxidant activity; essential oil
INTRODUCTION Antioxidant compounds are vital health-protecting factors in foods and have been shown to reduce the risk of chronic ailments such as heart disease and cancer.1 Antioxidants delay autoxidation by preventing the formation and/or interrupting the propagation of free radicals. Phenolic antioxidant compounds can be divided into several general groups, e.g. flavonoids, volatile oils, phenolic acids and diterpenes.2 Compounds in these groups exert their antioxidant effect in different ways. For example, generally, phenolic acids trap free radicals, while flavonoids scavenge free radicals or chelate metals.3 Phenolic compounds that are a major constituent of human nutrition are a large group of secondary metabolites widely distributed in plants. Several approaches have been used to monitor and compare the antioxidant activity of foods. Measurement of 2,2-diphenyl1-picrylhydrazyl (DPPH) free radical-scavenging ability is a simple, rapid and inexpensive method for determining the antioxidant activity of foods. This method is generally used to assess the ability of individual compounds to act as free radical scavengers or hydrogen donors in order to assess the antioxidant activity of foods. Recently, it has been used to evaluate the antioxidant activity in complex biological systems. It can also be applied for the assessment of overall antioxidant capacity in both liquid and solid materials as a non-selective approach for any specific J Sci Food Agric (2014)
component. The overall antioxidant capacity of foods can be useful to determine their functional properties.4 Essential oils are natural aromatic compounds found in the flowers, seeds, stems, bark, roots or other parts of plants. They are often used as medicinal oils for skin treatments to remediate cancer and can be extracted using the following approaches: solvent extraction, distillation or steam expression. The quantity and quality of essential oils in plants can be influenced by various factors such as genetic traits,5 environmental conditions (e.g. light, temperature, nutrient and water availability)6,7 and agricultural practices (e.g. harvest date, postharvest conditions).8 Macronutrients are among the major factors influencing plant growth and development. Sulfur (S) is one of the macronutrients with important functions in plants. It is found in cysteine, cystine
∗
Correspondence to: Sharareh Najafian, Department of Agriculture, Payame Noor University, PO Box 19395-3697, Tehran, Iran. E-mail: sh.najafi[email protected]
a Department of Agriculture, Payame Noor University, PO Box 19395-3697, Tehran, Iran b Department of Rangeland and Watershed Management, College of Agriculture and Natural Resources, Fasa University, Fasa, Iran
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www.soci.org and methionine, amino acids that make up proteins. Furthermore, it activates certain enzyme (hydrogenase) systems9 and is a main part of nutritional vitamins such as vitamin B1 (thiamine) and vitamin H (biotin).10 Sulfur fertilization has also been shown to increase the oil content of crop seeds.11 It was also found that S application as SO4 2− increased the yield of crops such as corn, wheat and oilseed rape.12 On the other hand, decreased growth, dry matter yield and SPAD chlorophyll meter reading of barley leaves were reported be Astolfi et al.13 Satureja hortensis L. (Lamiaceae), summer savory, is an annual, herbaceous aromatic and medicinal plant native to southern Europe and naturalized in parts of North America.14 The aerial parts of some Satureja plants are widely used in foods as a flavor component, in herbal teas and in folk and traditional medicines to treat ailments such as nausea, cramp, indigestion, muscle pain, diarrhea and infectious diseases.15 Alizadeh et al.16 reported that application of complete fertilizer caused a slight but non-significant change in the essential oil composition of S. hortensis, with the levels of some components such as carvacrol, 𝛿-terpinene and 𝛼-terpinene being affected. They stated that application of 1500 mg of fertilizer per plant decreased the amount of carvacrol significantly but increased 𝛿-terpinene and 𝛼-terpinene. Zheljazkov et al.12 concluded that the oil yield of sweet basil was maximized by application of 80 kg S ha−1 . Khan and Hussain17 showed that the highest seed and oil yields of mustard were obtained with application of 20 kg S ha−1 . It is reported that nutrients and antioxidants together may reduce levels of reactive oxygen species more effectively than single dietary antioxidants, because they can act in synergy.18 Since there appears to be no previous research on the subject, the aim of this study was to investigate the effect of S fertilizer on the essential oil composition and antioxidant activity of summer savory.
MATERIALS AND METHODS To determine the effect of S fertilizer on the essential oil composition and antioxidant activity of S. hortensis L., a greenhouse experiment was carried out on a loamy calcareous soil (Table 1). Some physical and chemical properties of the studied soil were measured using standard methods: sodium bicarbonateextractable phosphorus (P),19 diethylenetriaminepentaacetic acid (DTPA)-extractable iron (Fe), manganese (Mn), zinc (Zn) and copper (Cu),20 calcium carbonate equivalent,21 total nitrogen (N),22 organic matter (wet oxidation method), pH in saturated paste (pH meter) and electrical conductivity in saturated extract (electrical conductivity meter). The experiment was a randomized complete design with six replicates. Treatments consisted of soil application of three elemental S levels (0, 0.05 and 0.1 g S kg−1 soil). Each plastic pot contained 3 kg of soil. Pots were watered with distilled water to near field capacity and maintained at this moisture level by adding water to a constant weight. To prevent any possible deficiency of nutrients other than S, all pots received uniform application of 0.02 g P kg−1 soil as Ca(H2 PO4 )2 ·H2 O, 0.15 g N kg−1 soil as CO(NH2 )2 (one half was added at planting and the other half was shoot dressed 3 weeks after emergence) and 0.01, 0.01 and 0.005 g Mn, Zn and Cu kg−1 soil respectively as their sulfates in aqueous form. All pots received uniform application of 0.01 g Fe kg−1 soil as Fe chelate of ethylenediaminedihydroxyphenylacetic acid (FeEDDHA) in aqueous form. Twenty S. hortensis seeds were planted about 1 cm deep and thinned to ten uniform stands 2 weeks after emergence. Plants were harvested 12 weeks after
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S Najafian, M Zahedifar
Table 1. Some physical and chemical properties of studied soil
ECe Soil (dS property pH m−1 ) Value
7.8
0.4
CCE (g kg−1 ) 450
OM (g kg−1 ) 15
Olsen P (mg kg−1 ) 4.5
DTPAextractable (mg kg−1 ) Fe 2.3
Mn
Zn
Cu
3.7 0.96 1
ECe , electrical conductivity, CCE, calcium carbonate equivalent; OM, organic matter.
planting. Plant samples were dried in shade and prepared for analysis. Preparation of plant extracts Dried plant samples (20 g) were soaked in 200 mL of methanol/water (90:10 v/v) for 2 days, with a change of solvent after 1 day. The extracts were filtered and concentrated in a rotary evaporator for up to 10 min. The powders were weighed and their yields determined. Before use, the powders were kept at −20 ∘ C. Immediately before each measurement, the desired concentration of powder in methanol was prepared and its antioxidant activity and total phenol content were measured.23 Using DPPH for determination of antioxidant activity Scavenging of the stable DPPH free radical was used to determine the antioxidant activity of plant extracts and standard antioxidant gallic acid. In a modified assay,24 200 μL of 100 mmol L−1 DPPH radical solution in methanol was mixed with 20 μL of 12.5–3200 μg mL−1 methanol extracts or gallic acid. The solutions were maintained at room temperature for about 30 min. DPPH radical inhibition was determined using an ELx808 microplate reader (BioTek Instruments Inc., Winooski, VT, USA) at 515 nm. IC50 (concentration (mg L−1 ) required to inhibit DPPH radical formation by 50%) values of samples were calculated from the nonlinear regression between log concentration (μg mL−1 ) of test extracts and mean % radical-scavenging activity using MATLAB (The MathWorks Inc., Natick, MA, USA). The blank subtracted from all measurements was methanol extract without DPPH. Antioxidant activity was determined using the equation [( ) ] antioxidant activity = 100 – Asample –Ablank ∕Acontrol × 100 where A denotes absorbance. DPPH (without plant extract) and methanol were used as control and blank respectively. Headspace volatile extraction Dried plant samples (3 g) were crushed and placed in 20 mL headspace vials. The vials were immediately sealed with silicone rubber septa and aluminum caps and then transferred to a headspace tray. Headspace extraction was performed using a CombiPAL system (CTC Analytics AG, Zwingen, Switzerland) comprising a heater, an agitator and a headspace autosampler. The vials were heated to 80 ∘ C and held for 20 min while being agitated; the temperature of the sampling needle and transmission lines was 85 ∘ C.25 Identification of oil constituents using gas chromatography/mass spectrometry (GC/MS) GC/MS analysis was carried out using an Agilent 7890 GC/MS system (Agilent Technologies Inc., Shanghai, China) operating at
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Influence of S fertilizer on Satureja hortensis essential oil 70 eV ionization energy, equipped with an HP-5 MS capillary column (Chrom Tech, Apple Valley, MN, USA) (phenyl methyl siloxane, 30 m × 0.25 mm i.d., 25 μm) with helium as carrier gas and a split ratio of 1:50. Retention indices (RIs) were determined using retention times of n-alkanes that were injected after the essential oil under the same chromatographic conditions. RIs of all components were determined based on an approach using n-alkanes as standards. Compounds were identified by comparison of their RIs (HP-5) with those reported by others in the literature and also by comparison of their mass spectra with published mass spectral data in the Adams Library, Wiley GC/MS Library and MassFinder 2.1 Library.26 – 28
RESULTS AND DISCUSSION Chemical composition of essential oil Eighteen essential oil compounds were identified in the control, whereas 15 and 17 compounds were obtained with application of 0.05 and 0.1 g S kg−1 soil respectively. The identified constituents with their respective levels and RIs are summarized in Table 2. The main oil constituents in the control were 𝛾-terpinene (674 g kg−1 ), 𝛼-terpinene (108 g kg−1 ), myrcene (44 g kg−1 ), 𝛼-thujene (42 g kg−1 ), p-cymene (38 g kg−1 ), 𝛼-pinene (28 g kg−1 ) and carvacrol (21 g kg−1 ). Application of 0.05 g S kg−1 soil changed these components to 𝛾-terpinene (639 g kg−1 ), 𝛼-terpinene (125 g kg−1 ), myrcene (52 g kg−1 ), 𝛼-thujene (49 g kg−1 ), p-cymene (42 g kg−1 ), 𝛼-pinene (32 g kg−1 ) and carvacrol (15 g kg−1 ). Finally, the composition was 𝛾-terpinene (664 g kg−1 ), 𝛼-terpinene (90 g kg−1 ), carvacrol (72 g kg−1 ), p-cymene (37 g kg−1 ), myrcene (43 g kg−1 ), 𝛼-thujene (31 g kg−1 ) and 𝛼-pinene (22 g kg−1 ) with application of 0.1 g S kg−1 soil. The most important result of this study was the increasing trend in levels of 𝛼-terpinene, p-cymene, myrcene, 𝛼-thujene and 𝛼-pinene with application of 0.05 g S kg−1 soil (Table 2). The 𝛼-terpinene concentration was 108 g kg−1 without S fertilization but reached 125 g kg−1 after application of 0.05 g S kg−1 soil, i.e. a 15.7% increase. Myrcene (18.2% increase), 𝛼-thujene (16.7% increase) and 𝛼-pinene (14.3% increase) exhibited a similar trend. Another component showing the same trend was p-cymene, which is a monoterpene. Monoterpenes are the primary components of plant essential oils, and the effects of many medicinal herbs have been attributed to them.29,30 The p-cymene concentration was 38 g kg−1 without S fertilization but reached 42 g kg−1 after application of 0.05 g S kg−1 soil, i.e. a 10.5% increase. In contrast, the concentration of 𝛾-terpinene with lower molecular weight decreased slightly (by 5.2%) with application of 0.05 g S kg−1 soil. This phenomenon could be due to evaporation, oxidation or other unwanted changes in essential oil components with application of 0.05 g S kg−1 soil. The genus Satureja presents great variability in the concentrations of major components of its essential oil owing to the existence of different species and subspecies but also to many other factors, mainly environmental and climatic conditions.16 In previous studies, thymol and carvacrol in particular were found to be the principal constituents of the oils isolated from several Croatian Satureja species.31,32 It is interesting that various isolates of winter savory from Croatia and Bosnia and Herzegovina have carvacrol (up to 841.9 g kg−1 ) as the main constituent.33 A review of the published literature reveals that the essential oil of winter savory shows large variations in the relative concentrations of major components such as carvacrol (50–690 g kg−1 ), linalool (10–620 g kg−1 ), 𝛾-terpinene (10–310 g kg−1 ) and J Sci Food Agric (2014)
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Table 2. Effect of S application on concentrations of chemical compounds (g kg−1 ) identified in essential oil of Satureja hortensis, and their retention indices Applied S fertilizer (g S kg−1 soil)
No. Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
𝛼-Thujene 𝛼-Pinene Camphene Sabinene 𝛽-Pinene Myrcene 𝛼-Phellandrene 𝛿-3-Carene 𝛼-Terpinene p-Cymene Limonene 𝛽-Phellandrene (E)-𝛽-Ocimene 𝛾-Terpinene p-Cymen-9-ol (Z)-Ocimenone Carvacrol Caryophyllene 𝛽-Bisabolene
Retention index
0 (control)
0.05
923 42 ± 2.1b 49 ± 2.3a 930 28 ± 1.6a 32 ± 2a 945 2 ± 0.2a 2 ± 0.2a 969 3 ± 0.3a 4 ± 0.35a 973 11 ± 1.2a 13 ± 1.1a 987 44 ± 2.3b 52 ± 2.3a 1003 9 ± 0.8b 11 ± 1.3a 1008 1 ± 0.08b 2 ± 0.25a 1014 108 ± 5.5b 125 ± 6a 1021 38 ± 1.9b 42 ± 2.2a 1025 9 ± 0.95a 1 ± 0.1c 1026 2 ± 0.25b 3 ± 0.4a 1043 1 ± 0.1b 1 ± 0.1b 1059 674 ± 21a 639 ± 22b 1211 3 ± 0.35 – 1224 1 ± 0.08 – 1299 21 ± 1.5b 15 ± 1.8b 1415 – – 1504 1 ± 0.08b –
0.1 31 ± 1.9c 22 ± 1.4b 2 ± 0.3a 3 ± 0.3a 11 ± 1.2a 43 ± 2.4b 8 ± 0.9b 1 ± 0.1b 90 ± 4c 37 ± 1.9b 7 ± 0.8b 3 ± 0.4a 2 ± 0.2a 664 ± 25a – – 72 ± 3.2a 1 ± 0.1 3 ± 0.5a
Data are mean ± standard deviation of six replications. Means in a row followed by the same letter are not significantly (P < 0.05) different by Duncan’s multiple range test.
p-cymene (30–270 g kg−1 ), arising from the existence of different chemotypes.34 The main constituents of S. hortensis (summer savory) essential oil are the phenols thymol (290 g kg−1 ), carvacrol (265 g kg−1 ), 𝛼-terpinene (226 g kg−1 ), p-cymene (93 g kg−1 ) and other terpenoids.12 Finally, it can be concluded that application of 0.05 g S kg−1 soil is the most suitable for obtaining higher proportions of 𝛼-terpinene, p-cymene, myrcene, 𝛼-thujene and 𝛼-pinene, whereas application of 0.1 g S kg−1 soil is preferable for obtaining particular components such as carvacrol. On the other hand, 𝛾-terpinene is mainly produced in the control. Many studies have shown that the most abundant phenolic components in Satureja are carvacrol, 𝛾-terpinene, thymol, p-cymene, 𝛽-caryophyllene, linalool and other terpenoids. The concentrations of these constituents vary between different Satureja species.14 Some investigators have shown that the essential oil of Satureja species has a variety of activities, including antibacterial and antifungal properties, and exhibits strong inhibition of a wide range of bacteria and fungi in human, food and plant pathogens.12 Some plants of the Lamiaceae family are rich in phenolic compounds such as flavonoids, phenolic acids and phenolic diterpenes and have high antioxidant activities.35,36 Flavonoids and phenolic compounds exert multiple biological effects such as antioxidant, free radical-scavenging and anti-inflammatory activities.37,38 Oxidative damage in the human body plays an important causative role in disease initiation and progression.39 IC50 is an appropriate parameter to measure the progress of oxidation in oils and is therefore considered as a good indicator of the effectiveness of antioxidants. All extracts showed a considerable DPPH-inhibitory effect, with IC50 ranging from 0.720 g L−1 in the control to 0.363 g L−1
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IC50(mg L−1)
800 700 600 500 400 300 200 100 0 Gallic acid
Control
0.05 g S kg–1 soil
0.1 g S kg–1 soil
Treatments
Figure 1. Effect of S application on antioxidant activity of Satureja hortensis measured by DPPH assay, and comparison with gallic acid (IC50 is concentration required to inhibit DPPH radical formation by 50%).
with application of 0.1 g S kg−1 soil (Fig. 1). The linear regression equation between IC50 (Y, mg L−1 ) and S fertilization level (X, g kg−1 soil) was determined as Y = −3.575X + 773.01 (R2 = 0.79, P < 0.01) showing that IC50 decreased in response to applied S. Extensive studies have been carried out on the antioxidant activity of many species of the Lamiaceae family.2,36,40 They demonstrated that members of this family had very strong antioxidant capacity. The findings of the present study indicate that savory oils, in addition to other properties, have potential as topical antioxidants. The maximum antioxidant effect was observed with application of 0.1 g S kg−1 soil. In this treatment the concentration of carvacrol increased 2.4-fold compared with the control. The results obtained in our study show good correlation within one species.
CONCLUSION Studying the secondary plant products, especially essential oils, is an interesting research area that requires further studies on other aromatic plant essential oils consisting of various components. Furthermore, the results revealed that the addition of chemical fertilizers such as S could improve the antioxidant activity of plant extracts significantly.
ACKNOWLEDGEMENTS The authors thank Mr Engineer Hamid Reza Satari and Mr Engineer Ahmad Reza Ghasemi, superintendents of Eram Garden, for their support.
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