Abstract: Hydro-distilled essential oils from the aerial parts of Satureja sahendica Bornm. and their antioxidant effects were investigated, mainly by a combination of GC/MS. S. sahendica is one of the endemic species of Satureja in Iran. The plant materials separated to two different parts, 1-inflorescence (S1) 2- leaf and stem (S2). Thirty to forty gram of each samples were subjected to hydro-distillation for 4 h using a clevenger-type apparatus to produce oils. Hydro distillation was repeated thrice for each sample and average was calculated. The oils were found to be yellow liquid and were obtained in yields of 1.66 for S1 and 1.5 for S2 based on v/w. Twenty nine compounds were identified in the oil of S1 that Thymol (32.5%), γ-Terpinen (29.33%) and P-Cymene (23.48) are main constituents and 23 compounds were characterized in the oil of S2 that main constituents were P-Cymene (44.88%), Thymol (28.22%) and γ-Terpinen (10.07%). The antioxidant effect of S1 and S2 compounds were assessed by DPPH assay and RC50 values were and mg mL-1, respectively.
INTRODUCTION
Satureja (Labiatae) species are present in mountainous areas in Iran, mainly in Western and Northern Parts. Satureja sahendica Bornm. is an endemic species of Iran distributed in Western and Northwestern and existence were reported from Zanjan, East Azarbayejan and Kordestan provinces.
It is a late flowering species (late summer and fall), growing on rockwalls and rocky slopes.Foliage (1-2 and 8-12 mm), Flowers (6 mm long) white and attractive for bees (Mozaffarian, 1993).Due to various usages of Satureja species or their oils the components of essential oil of plants identified in Iran and overall the world (Tampieri et al., 2005).
The investigations on the composition of essential oils of S. mutica, S. macrantha and S. intermedia in Iran show that the main constituents in the S. mutica essential oil were P-Cymene (25.8%) and Limonene (16.3%) and in the essential oil of S. intermedia, Thymole (32.3%) and γ-Terpinene (29.3%) are main components.In the essential oil of S. macrantha main component are P-Cymene (25.8%) and Limonene (16.3%) (Sefidkon and Ahmadi, 2000).
Comparison the constituents of essential oil of S. boliviana and S. parvifolia from Argentina demonstrate that main constituents in first plant are γ-Terpinen (15.4%), β-Caryophyllene (10.2%), Germacrene (8.9%), Bicyclo germacrene (8.3%) and 1,8-Cineol (7.4%), whereas in second plant, main component is Piperitenone oxide (69.8%) (Viturro et al., 2000).
The essential oil of S. bachtiarica from Iran mainly have Thymol (44.5%), γ-Terpinen (23.9%), P-Cymene (7.3%), β-Caryophyllene (95.3%) and Borneol (4.2%) and in S. khuzistanica from Iran the main constituents are P-Cymene (39.8%), Carvacrol (29.6%), γ-Terpinene (18.9%) (Sefidkon and Ahmadi, 2000).
The essential oil composition of S. viminea in Castarica was investigated and the main constituents are P-Menth-3-en-8-ol (40%), Polegone (35.3%), P-Mentha-3,8-di-en (18.9%) (Vila et al., 2000).
The various species of Satureja genus are used as a culinary herbs and green leaves and herbaceous sections of stem are used fresh and dried as flavouring agents in seasoning, stews, meat dishes, poultry, sausages and vegetables and as a medicinal plant has been traditionally used as a stimulant, stomachic, carminative, expectorant, antidiarrheic and aphrodisiac (Baser, 2002).
Also researches indicate that the Satureja plants have antimicrobial, antifungal and antibacterial effects (Skocibusic et al., 2004). On the other hand the essential oil of this genus plants are natural antioxidants and have great antioxidative effects (Delazar et al., 2005a, b; Radonic and Milos, 2003; Tomohiro et al., 1994).
The main characteristic of an antioxidant is ability to trap free radicals. Highly reactive free radicals and oxygen species are present in biological systems from a wide variety of sources.These free radicals may oxidize nucleic acids, proteins, lipids or DNA and initiate degenerative disease.
The highly susceptibility to oxidation of the fat and oil polyunsaturated fatty acids used in shuman foods or animal feed requires the application of antioxidants.
The antioxidants can be either synthetic or natural products with scavenge oxygen free radicals, thereby inhibiting or delaying oxidation.
Interest in natural antioxidants has increased dramatically in recent times due to concerns regarding the safety of the chronic consumption of antioxidants (Butylated hydroxyl toluene and Butylated hydroxyl lanisole) the antioxidative efficacy of a variety phytochemicals. The consensus that foods rich in certain phytochemicals can affect the aetiology and pathology of Chronic disease and aging process and the publics conceived belief that natural compounds are innately safer than synthetic compounds and are thus more commercially acceptable.
MATERIALS AND METHODS
Plant materials: The aerial parts of Satureja sahendica Bornm. were collected in flowering stage (late summer), from Mazarnashib, near the Sahand hillside at East Azarbayejan (N: 37° 43– 52.07½, E: 46° 10´ 3.6– with 2048 heigh), herbarium specimens were deposited at Azarbayejan botanical garden in Tabriz Forest and Rangelands Research institute (the herbarium code was: 5395, Satureja sahendica, Sahand-Sufichai-1700 m). The experiments were conducted at the autumn of 2006 in Medicinal Plants Lab, Drug Applied Research Center, Tabriz University of Medical Science.
The air parts of collected plants separated to two parts: inflorescence (S1), leaf and stem (S2) and air dried then storage for 3 month in 2°C at light and air impervious pockets. The plant materials were subjected to hydrodistillation for 4 h using a Clevenger-type apparatus to produce oils.
The oils were dried over Anhydrous sodium chloride and stored in sealed vials at low temperature (2°C) and umbrage before analysis.
Gas chromatography: GC analysis was performed using a Shimadzu GC-17A-Ver. 3. Gas chromatograph equipped with a DB-5 fused column (60 m 0.25 mm i.d., film thickness 1.4 μm).
Oven temperature was held at 50°C for 3 min and then programmed to 250°C at a rate of 3°C min–1.Injector and detector temperature were 260°C, helium was used as carrier gas with a linear velocity of 32 m sec–1.
Gas chromatography-mass spectrometry: GC-MS analysis were carried out on a Shimadzu GC-17A-Ver. 3 equipped with Quadropule (QP5050) MS with DB-5 column (60 m 0.25 mm i.d.,).Oven temperature was 50-250°C at a rate of 3°C min–1, transfer line temperature 260°C, carrier gas helium with a linear velocity of 32 cm sec–1.
Identification of components: The components of the oil were identified by comparison of their mass spectra with those of computer library or with authentic compounds and confirmed by comparison their retention indices, either with those of authentic compounds or with data published in the literature (Adams, 2004).
DPPH assay: DPPH assay is a rapid, simple and inexpensive method to measure antioxidant capacity, involves the use of the free radical 2,2-Diphnyl-1- picrylhydrazyl.
The odd electron in the DPPH free radical gives a strong absorption maximum at 517 nm and purple in color.The color turns from purple to yellow when the odd electron of DPPH radical becomes paired with a hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H.
In this research DPPH was obtained from Sigma Ltd. and stock solutions (1%) of the plant essential oils were papered in CHCl3. Serial dilutions were carried out to obtain concentration of 3.12x10–2, 1.56x10–2, 7.8x10–3, 3.9x10–3 and 1.9x10–3 mg mL–1. Diluted solutions (5 mL each) were mixed with DPPH (5 mL) and allowed to stand for 30 min for any reaction to occur. The UV absorbance was recorded at 517 nm. The experiment was performed in triplicate and the average absorption was noted for each concentration.
RESULTS AND DISCUSSION
The oils isolated by hydrodistillation from two distinct aerial parts
of S. sahendica (S1 ad S2) were found to be yellow liquids were
obtained in yields of 1.66% (v/w) and 1.5% (v/w), respectively. The components
are listed in order of their elution on the DB-5 column (Table
1).
| Table 1: | Percentage composition of essential oils of S1 and S2 samples |
Twenty nine components of the S1 sample oil, representing 98.6% of total oil and 23 components of the S2 sample representing 97.38% were identified. The main components of the S1 samples oil were Thymol (32.57%), γ-Terpinene (29.33%) and P-Cymene (23.48%). The major constituents of the S2 samples oil were P-Cymene (44.8%), Thymole (28.22%) and γ-Terpenine (10.07%).
The DPPH antioxidant assay is based on the ability of 2,2, diphenyl, 1-picrylhydrazyl (DPPH) a stable free radical contains an odd electron which is responsible for the absorbance at 517 nm and also for visible deep purple color. When DPPH accepts an electron donated by an antioxidant compound the DPPH is decolourised which can be quantitatively measured from the changes in absorbance. The S1 and S2 samples displayed significant free radical scavenging activity in the DPPH assay.
Comparing the oil composition of S1 and S2 essential oils showed some
differences (Fig. 1). The first major compound of S1
is Thymol, while the first major compound of S2 is P-Cymene. Some minor
or trace components such as δ-Carene, γ-Elemene, β-Camigrene,
β-Farnesene, γ-Cadinene that was found in S1 was not observed
in the oil of S2 and some minor components such as P-Cymene, Sabinene
hydrate that was found in S1 essential oil was not observed in the oil
of S1. Investigations showed that the oil composition of S. sahendica
is like that of S. bachtiarica, S. spicigera and S.
cuneifolia (Biavati and Ozcan, 2004; Sefidkon and Jamzad, 2000).
Also, the investigations on the antioxidant activity of S1 and S2 samples
and their RC50 values showed that essential oil of aerial parts
of S. sahendica had a great antioxidant effect and due to high
amount of Thymole and P-Cymene and other terpenoids in the oil, it can
be used as folk remedies for many diseases (with bactericidal, carminative,
digestive, expectorants, fungicidal, laxative, antidiuretic, sedative
and antioxidant activities). The essential oil of this plant can be applied
in flavouring condiments, relishes, soups, sausages, canned meats and
in spicy table sauces.
| Fig. 1: | Comparison between some main components of essential oils of samples |