Antioxidant and antimicrobial activity in the seeds of Origanum vulgare L. subsp. gracile (C. Koch) Ietswaart and Origanum acutidens (Hand.-Mazz.) Ietswaart from Turkey

The present study determined some biological compounds (fatty acids, vitamins, sterols and flavonoids), radical scavenging capacity and antimicrobial activity of two Origanum L. species of Origanum vulgare L. subsp. gracile (C. Koch) Ietswaart and Origanum acutidens (Hand.-Mazz.). Ietswaart. Linolenic acid was found to be the main fatty acid in both species, which was followed by palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1 n9), linoleic acid (C18:2 n6) and stearidonic acid (C18:4). In addition, the two Origanum species were recorded as containing high levels of stigmasterol. It was also found that Origanum vulgare L. subsp. gracile (C. Koch) Ietswaart had a high β-sitosterol content (152.8±2.6 µg/g); high amounts of D3 (33.1±0.8 µg/g), K1 (29.4±0.8 µg/g), K2 (26.0±0.8 µg/g) vitamins; and low amounts of α-tocopherol (7.8±0.2 µg/g) and D2 (1.8±0.1 µg/g), while Origanum acutidens (Hand.-Mazz.) Ietswaart contained high amounts of D2 (42.9±1.5 µg/g) vitamin; and low amounts of D3 (2.9±0.1 µg/g), α-tocopherol (3.1±0.1 µg/g), r-tocopherol (3.5±0.2 µg/g), K2 (1.3±0.1 µg/g), retinol (1.3±0.2 µg/g) and retinol acetate (1.2±0.1 µg/g) vitamins. The present study showed that Origanum vulgare L. subsp. gracile (C. Koch) Ietswaart and Origanum acutidens (Hand.-Mazz.) Ietswaart contained the lowest amount of flavonoid. Furthermore, methanol extracts were recorded to be the most effective agent against the DPPH radical in both Origanum L. species examined. Experimental results showed that the vitamins, flavonoids and fatty acid extracts of both Origanum L. species were effective in the inhibition of the growth of the tested microorganisms at varying levels.


INTRODUCTION
Reactive oxygen radicals have been detected to play a significant role in the occurrence and progression of various diseases such as cancer, cardiovascular diseases, atherosclerosis and inflammatory injuries (Verma et al., 2009).Many medicinal plants, especially members of the Lamiaceae-such as oregano, sage and thyme contain large amounts of antioxidants such as polyphenols, ascorbic acid and carotenoids, all of which can play an important role in the absorption and neutralization of the reactive oxygen radicals, quenching singlet and triplet oxygen, or in the decomposition of peroxides (Capecka et al., 2005;Djeridane et al., 2006).
Origanum species are distributed widely around the Mediterranean Region, more than 80% of which is concentrated exclusively in the East Mediterranean Region (Skoula et al., 2008).Turkey is the world's largest producer of oregano and grows the largest number of oregano varieties (Cetingul et al., 2007).The leaves and dried herbs of oregano have been used for medical purposes for centuries (Chun et al., 2005).Recent research has shown that oregano extracts, among all aromatic herbs, have the most effective antioxidant activity and antimicrobial properties (Milos et al., 2000;Kouri et al., 2007) and that oregano is used as an antidiabetic, carminative, tonic, stimulant, diuretic and to treat asthma (nakiboglu et al., 2007).Recently, there is an increasing interest in finding natural antimicrobials from different extracts obtained from plant materials.Phenolic compounds, flavonoids or unsaponifiable matter are among the groups of compounds able to inhibit the growth of specific microorganisms (Dib et al., 2010;Mohamed et al., 2010).
In this scope, the aim of the present study was to determine (i) the fatty acid compositions, vitamin and sterol contents; (ii) flavonoid contents and radical scavenging properties; and (iii) antimicrobial activities of vitamin and sterol, fatty acid and flavonoid contents of the seeds of Origanum vulgare subsp.gracile and Origanum acutidens.

Chemical agents
All chemicals and reagents were purchased from Sigma-Aldrich.

Extraction of seeds
Extracts of fatty acid, sterols and lipid soluble vitamins 2 g of seed materials for fatty acid, sterol and vitamin analyses were finely ground in a mill and were then extracted with hexane/isopropanol (3:2 v/v) (Hara and Radin, 1978).The lipid extracts were centrifuged at 10.000 g for 5 minutes and filtered, and the solvent was then removed on a rotary evaporator at 40°C.The extracted lipids were stored at 225°C until further analysis.The experiment was repeated three times.

Extracts of flavonoids
2 g of seed materials were homogenized in 5 ml of 80% methanol.Homogenates were centrifuged at 5000 rpm at 4°C.After centrifugation, the supernatant was concentrated by reduced-pressure rotary evaporation.Each extract was re-suspended in dimethyl sulphoxide (DMSO) to produce a stock solution.The experiment was repeated three times.

Fatty Acids
Fatty acids in the lipid extracts were converted into methyl esters by means of 2% sulphuric acid (v/v) in methanol (Christie, 1990).The fatty acid methyl esters were extracted with n-hexane.The methyl esters were then separated and quantified by gas chromatography and flame-ionization detection (Shimadzu GC 17 Ver.3)coupled to a Glass GC 10 computer software.Chromatography was performed with a capillary column (25 m in length and 0.25 mm in diameter, Permabound 25, Macherey-nagel, Germany) using nitrogen as carrier gas (flow rate 0.8 ml/min.).The temperatures of the column, detector and injection valve were 130-220, 240, and 280°C, respectively.Identification of the individual methyl esters was performed by frequent comparison with authentic standard mixtures that were analyzed under the same conditions.

Lipid soluble vitamins and sterols
Lipide-soluble vitamins and phytosterols were extracted from the lipid fraction by the method of Sânchez-Machado ( 2002) with minor modifications.The HPLC analyses were performed using a Shimadzu instrument (Shimadzu, Kyota, Japan) equipped with a UV detector.Seperations were performed on a Supelcosil TM LC18 (250  4.6 mm, 5 M. KURS ¸AT, I • .EMRE, Ö.yILMAz AnD P.ERECEVIT mm, Sigma, USA).The mobile phase was acetonitrile/ methanol (75/25 v/v), at a flow rate of 1.0 ml/min.The temperature of the analytical column was kept at 40°C; the injection volume of each sample was 50 µl.Chromatograms were recorded at at 320 nm for retinol (vitamin A) and retinol acetate, and 215 nm for d-tocopherol, vitamin D, α-tocopherol, α-tocopherol acetate, 202 nm for phytosterols, 265 nm for vitamin K1.Identification of the individual vitamins and phytosterols was performed by frequent comparison with authentic external standard mixtures analyzed under the same conditions (Lôpez-Cervantes et al., 2006).Class Vp 6.1 software assisted in the interpretation of the data.The results of analyses were expressed as µg/g for each sample.

Flavonoids
A chromatographic analysis was carried out using a PREVAIL C18 reversed-phase column (154.6mm,5µm, USA); the mobile phase was methanol/water/ acetonitrile (46/46/8, v/v/v) containing 1.0% acetic acid (zu et al., 2006).This mobile phase was filtered through a 0.45 µm membrane filter (Millipore), then de-aerated ultrasonically prior to use.Catechin (CA), naringin (nA), rutin (RU), resveratrol (RES), myricetin (MyR), morin (MOR), naringenin (nAR), quercetin (QU) and kaempferol (KA) were quantified by DAD separation at 280 nm for CA and nA, 254 nm for RU, MyR, MOR and QU, and 265 nm for KA.Flow rate and injection volume were 1.0 ml/min and l0 µL, respectively.The chromatographic peaks of the extracts were confirmed by comparing their retention times with those of the reference standards.Quantification was carried out by the integration of the peak using the external standard method.All chromatographic operations were carried out at a temperature of 25°C.

Antioxidant assay by DPPH radical scavenging activity
The free radical scavenging effect of seed extracts was assessed by the decoloration of a methanolic solution of DPPH • according to the method of Liyana-Pathiranan and Shahidi (2005).A solution of 25 mg / L DPPH in methanol was prepared and 4.0 ml of this solution was mixed with 25 and 50 µL of extract in DMSO.The reaction mixture was left in the dark at room temperature for 30 minutes.The absorption of a blank sample containing the same amount of methanol and DPPH solution was prepared and measured daily.The absorbance of the mixture was measured spectrophotometrically at 517 nm. 1 µM quercetin was used as a reference.
The ability to scavenge DPPH radicals was calculated by the following equation: DPPH radical scavenging activity (%) 5 [(Abs control 2 Abs sample)]/ (Abs control)]  100 where Abs control is the absorbance of DPPH radical 1 methanol; Abs sample is the absorbance of DPPH radical 1 sample extract /standard.

Antimicrobial activity
Antimicrobial tests were carried out according to the well agar method using 100 µL of suspension containing 10 6 cells / mL of bacteria, 10 4 cells / mL yeast and cells / mL dermatophyta fungi as per the McFarland standard, inoculated into Mueller Hinton Agar (Difco), Malt Extract Agar (Difco), and Sabouroud Dextrose Agar (Oxoid), respectively.Wells were prepared in the plates with the help of a cork-borer (0. 85 cm). 10 µl of the flavonoid, vitamin and fatty acid extracts were introduced directly into the wells one at a time.The wells were also injected with methanol and hexane.Steril petri dishes (9 cm diameter) were kept at 4°C for 2h.Then, the inoculated plates were incubated at 370.1°C for 24 h for bacterial strains and also at 250.1°C for 72 h for yeast and dermatophyta fungi.Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms (Collins and Lyne, 1987).The standard antibiotics nystatin and streptomysin were used as a positive control (respectively; for yeasts and bacteria) and methanol and hexzane were used as negative control in this study.The experimental studies were repeated three times.

Flavonoid contents
Due to their naturally high phenolic antioxidant contents, the use of Lamiaceae extracts (which includes oregano, rosemary, thyme and spearmint) can be important in the scope of the antioxidant applications (Chun et al., 2005).Many previous studies have demonstrated the antioxidant and free radical scavenging activities of various polyphenols (Kukiç et al., 2006;nakiboglu et al., 2007).

Radical scavenging activity by DPPH method
The methanol extracts of the two Origanum species examined in this study were found to be the most effective extracts against the DPPH radical (Table 3, Figure 1).While the radical scavenging activity of the methanol extracts in the seeds of O. vulgare varied in the range of 87.41.2%(for 25 µl) and 89.30.4% (for 50 µl) , in the seeds of O. acutidens it varied from 89.60.6% (for 25 µl) to 90.50.5% (for 50 µl).
Several previous literature studies have reported findings similar to those of the present study (Kulisica et al., 2004;Ivanova et al., 2005;Kouri et al., 2007).O. vulgare was reported to have lower .OH scavenging activity and DPPH radical scavenging capacity when compared to Origanum sipyleum, and Sideritis sipylea (nakiboglu et al., 2007).Ünver et al. (2009) found that the water extracts of O. vulgare had a free radical scavenging activity higher than that of Salvia officinalis but lower than that of mentha piperita.In contrast, an oregano study by Capecka et al. (2005) recorded a lower rate of DPPH scavenging accompanied by a significantly high inhibition of linoleic acid peroxidation.

Antimicrobial activity
Table 4 lists the antimicrobial activities of vitamins, flavonoids and fatty acids in two Origanum species, negative control groups and standard antibiotics.no previous literature study reported the antimicrobial activity of the extracts of vitamins, flavonoids and fatty acids of the two species examined in the present study.Therefore, this is the first study which demonstrates the antimicrobial activity of biological compounds contained in the seeds of these plants.Results of the present study show that the vitamin, flavonoid and fatty acid extracts of the two species inhibited the growth of microorganisms used in the test at varying levels.However, some extracts were observed to have no effect on the tested microorganisms and the flavonoids and vitamin extracts to have a generally reduced effect compared to the antibiotic group.
It can be seen in Table 4 that the antimicrobial activities of the vitamin extracts of O. vulgare subsp.gracile, except for C. glabrata, had an inhibition zone in the 8.30.2-23.60.3 mm range.However, the flavonoid extracts of O. vulgare subsp.gracile did not have any activity against gram-negative bacteria but had an antimicrobial effect on the  .Some literature studies showed that the extracts of O. vulgare inhibited the growth of bacteria and fungi and the synthesis of microbial metabolites (Oliveira et al., 2009).According to Kursat and Erecevit (2009), the plant extracts of O. vulgare had less antimicrobial activity against S. aureus (9 mm) and B. megaterium (9 mm) but more activity against K. pneumoniae (17 mm) and Epidermophyton spp.(11 mm).Dikba7 et al. (2009) showed that the essential oils of O. acutidens had a strong antibacterial effect against both grampositive and gram-negative bacteria.yi≈it and Kandemir (2002) recorded antimicrobial activity of the ethanol extracts of O. acutidens against grampositive bacteria but no such activity against gramnegative bacteria or the yeast species Candida albicans.A previous study by Kordalı et al. (2008) reported the inhibitory effects of the essential oils isolated from O. acutidens on the mycelial growth of 17 phytopathogenic fungal species (Kordalı et al., 2008).

Antioxidant and antimicrobial activity in the seeds of Origanum vulgare L. subsp. gracile (C. Koch) Ietswaart and Origanum acutidens (Hand.-Mazz.) Ietswaart from Turkey
AnTIOxIDAnT AnD AnTIMICROBIAL ACTIVITy In THE SEEDS OF ORigAnum VuLgARE L. SUBSP.gRACiLE (C.KOCH)…