Some biological compounds , radical scavenging capacities and antimicrobial activities in the seeds of Nepeta italica L . and Sideritis montana L . subsp . montana from Turkey By I ·

Este estudio ha determinado algunos compuestos biológicos (ácidos grasos, vitaminas liposolubles, esteroles y flavonoides), capacidad atrapadora de radicales libres, y actividades antimicrobianas de las semillas de Nepeta italica L. y Sideritis montana L. subsp. montana. Se encontró que el ácido palmítico (C16:0; 8.54±0.13-3.05±0.04%), ácido oleico (C18:1 n9, 22.41±0.8-18.83±0.1%) y α-linolénico (C18:3 n 3;39.56±0.67-77.04±2.07%) eran mayoritarios en ambas semillas de Nepeta italica L. y Sideritis montana L. subsp. montana. Además, se determinó que tanto Nepeta italica L. como Sideritis montana L. subsp. montana contenían estigmasterol (630.07±1.81μg/g; 80,74±0.71μg/g, respectivamente) y ergosterol (11.01±0.14 μg/g; 161.32±0.63 μg/g respectivamente) y esta última además beta-sitosterol (2.93±0.03μg/g). Además, los resultados muestran que Nepeta italica L. contiene morin (37.79±1.09μg/g), catequina (124.39±2.23μg/g) y naringina (475.96±3.57μg/g) y Sideritis montana L. subsp. montana contiene morin (188.41±2.53μg/g), catequina (64.14±1.86μg/g), naringenina (38.34±1.78 μg/g), como principales flavonoides. También, se determinó que los extractos metanólicos de Nepeta itálica L. y Sideritis montana L. subsp. montana fueron eficaces contra los radicales DPPH. Los resultados obtenidos en el presente estudio mostraron que las vitaminas, los flavonoides y los ácidos grasos de los extractos en las semillas de Nepeta itálica L. y Sideritis montana L. subsp. montana impidieron el crecimiento de los microorganismos utilizados en el ensayo, en diferentes proporciones.


INTRODUCTION
Recently, considerable attention has been devoted to medicinal plants because they contain chemical constituents which exhibit antioxidant properties (Miliauskas et al., 2004;Grzegorczyk et al., 2007;Mohamad et al., 2010).Antioxidants are viewed as compounds that protect cells against oxidative stress, which might cause cell damage (Giao et al., 2007).It is reported that medicinal plants contain a wide range of natural compounds such as phenolic acids, flavonoids, terpenoids, vitamins and tannins, which show antioxidant activity (Ai-li and Chang-Hai, 2006;Bouayed et al., 2007).In particular, members of the Lamiaceae family have been extensively studied for their antioxidant activity and radical scavenging activity (Erdemog ˘lu et al., 2006;Barros et al., 2009).
Nepeta and Sideritis, which are members of the Lamiaceae family, have significant antioxidant capacities (Nakibog ˘lu et al., 2007;Tepe et al., 2007;Güvenç et al., 2010).The genus Nepeta (Nepetoideae subfamily) comprises about 400 species, most of SoME BIoLoGICAL CoMPoUNDS, RADICAL SCAvENGING CAPACITIES AND ANTIMICRoBIAL ACTIvITIES IN THE SEEDS oF… 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.The solvent was then removed on a rotary evaporator at 40°C.The extracted lipids were stored at-25°C until further analysis.The experiment was repeated three times.

Flavonoid analyses and Radical scavenging capacity
2 g seed material was homogenized in 5 ml 80% methanol.Homogenates were centrifuged at 5000 rpm at +4ºC.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 Analyses
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 a 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.

Chromatographic analysis and quantification of lipid soluble vitamins and sterols
Lipid-soluble vitamins and phytosterols were extracted from the lipid fraction according to the method of Sánchez-Machado ( 2002) with minor modifications.The extracted lipids of seed material were dissolved in acetonitrile/methanol (75/25 v/v) and 50 mL were injected into the HPLC instrument (Shimadzu, Kyota Japan).The column used was a Supelcosil TM LC18 (250 x 4.6 mm, 5 mm, Sigma, USA).The mobile phase was acetonitrile/methanol (75/25, v/v) and the elution was performed at a flowrate of 1 ml /min.The temperature of the analytical column was kept at 40°C.Detection was performed 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 were performed by frequent comparison with authentic which grow in Central and Southern Europe, North Africa and Central and Southern Asia (Miceli et al., 2005).In Turkey, the genus Nepeta is represented by 44 species, 22 of which are endemic (Davis, 1982;Akpınar et al., 2008).The antispasmodic, diuretic, antiseptic, antitussive, antiasthmatic and febrifuge activities of Nepeta species have been reported in the literature (Tepe et al., 2007).The other genus, Sideritis L. (Lamioideae subfamily), (Lamiaceae) includes approximately 150 species of annual and perennial plants, distributed mainly in the Mediterranean region (Güvenç et al., 2010).Sideritis is represented by 45 species and 53 taxa, of which 39 species are endemic in Turkey (Davis, 1982;Nakibog ˘lu et al., 2007).The endemism ratio of the Sideritis species is very high (78%) for Turkey among other Lamiaceae species (Sagdic et al., 2008).Sideritis species are used in folk medicine as anti-inflammatory, anti-ulcer, cytostatic, antimicrobial, flu vaccine and stimulant circulatory agents (Basile et al., 2006).The objectives of the present study are: i) to determine fatty acid compositions, vitamin and sterol contents; ii) to evaluate the flavonoid contents and radical scavenging properties of Nepeta italica and Sideritis montana subsp.montana seeds.In addition, the antimicrobial activities of both species were investigated.

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

Extraction of plant materials
Fatty acid, sterol and vitamin analyses 2 g seed materials for fatty acid, sterol and vitamin analyses were finely ground in a mill and were then I • RFAN EMRE, MURAT KURS ¸AT, ÖKKES ¸ YILMAz AND PINAR ERECEvIT 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 flavonoids, vitamins and fatty acids in plants were introduced directly into the well.Steril petri dishes (9cm diameter) were placed 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).Wells injected with methanol and hegzan served as negative controls.The experimental studies were carried out three times.

Chromatographic Conditions for Flavonoids
The chromatographic analysis was carried out using a PREvAIL C18 reversed-phase column (15x4.6mm,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, 306 nm for RES, 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 the 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 were mixed with 25 and 50 μL of extract in DMSo.The reaction mixture was left in the dark at room temperature for 30 minutes.Absorption of the 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 (%) = [(Abs control -Abs sample)]/ (Abs control)] x 100 where Abs control is the absorbance of DPPH radical + methanol; Abs sample is the absorbance of DPPH radical + sample extract /standard.

Antimicrobial activity
Antimicrobial tests were carried out by 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 McFarland standard, inoculated into Mueller Hinton Agar (Difco), Malt  et al. (2007) found that the linoleic acid content of five Nepeta species was 10.9-23.5%, with the linoleic acid content of S. montana being the lowest (0.13±0.02%).However, Ertan et al. (2001) determined that the linoleic acid composition of Sideritis species was 45.4-64.0%.
It was indicated that the genera under the subfamily Nepetoideae have been characterized by unsaturated fatty acids and the α-linolenic acid composition was reported as generally over 40% (Cantino and Sanders, 1986;Azcan et al., 2004).In the present study, linolenic acid was the major unsaturated fatty acid in N. italica (39.56±0.67%).Akpınar et al. (2008) determined that the linolenic acid composition of five Nepeta species including N. italica (59.51%) was 48.20-61.62%.Another study, by Kiliç et al. (2007) determined that the linolenic acid content of Nepeta species was 49.8-58.5%.However, the fatty acid composition of S. montana showed a difference in the percentage of linolenic acid.It was reported that in the subfamily Lamioideae, the ratio of 18:3 fatty acid is less than 30%, and, in most species, it is less than 5% (Ertan et al., 2001).However, the present study found that the 18:3 n3 fatty acid content for S. montana subsp.montana was 77.04±2.07%.In contrast to these results, Ertan et al. (2001) found that Sideritis species have lowest linolenic acid content (0.8-2.0%).
The linoleic acid composition of N. italica was found to be 22.8±0.14% in this study.A study Previous studies reported a high correlation between the antioxidant capacity and phenolic constituents of methanol extracts from medicinal plants (Miceli et al., 2005;Maisuthisakul et al., 2007;Li et al., 2008;Sagdic et al., 2008).The present results show that N. italica and S. montana exhibit antioxidant properties.

Antimicrobial Activity
The antimicrobial activities of the vitamins, flavonoids and fatty acids in the extracts from the seeds of N. italica, S. montana, negative control group and standard antibiotics are reported in Table 4.The experimental results show that the vitamins, flavonoids and fatty acid extracts of two species inhibited the growth of microorganisms used in the test at different ratios.The vitamin extracts of N. italica and S. montana showed antimicrobial activities against all microorganisms and the vitamin and flavonoid extracts usually have higher effects in comparison to antibiotics.
The vitamin extracts of N. italica and S. montana have the highest antimicrobial efficiency (inhibition zone between 9.220.1-33.2±0.1 mm and 13.3±0.2-35.1±0.3 mm, respectively).As shown in Table 4, the extracts of flavonoids from N. italica showed antibacterial and antifungal activity against the microorganisms tested: E. coli (13.3±0.2 mm), K.

Radical scavenging activity by DPPH method
In the present study, the radical-scavenging activities of the methanol extracts of Nepeta italica and Sideritis montana were determined according to the DPPH radical scavenging test (Table 3).The antioxidant potential of the flavonoid contents of N. italica and S. montana was examined at two different volumes (25 μl and 50 μl).It was determined that the methanol extracts of N. italica and S. montana seeds were most effective against DPPH radicals.The methanol extracts of N. italica and S. montana almost completely inhibited  4).
A previous study reported that the Sideritis species could be used as natural antimicrobial and antioxidant agents in food preservation and human health (Sagdic et al., 2008).Several studies suggested that Nepeta and Sideritis species have antimicrobial and antifungal activities (Diaz et al., 1988;Ezer and Abbasog ˘lu, 1996;Nostro et al., 2001;Basile et al., 2006).Kursat and Erecevit (2009) (Hernandez-Perez and Rabal, 2002;Proestos et al., 2006).However, this is the first study to report that the vitamins, flavonoids and fatty acids of S. montana and N. italica possess antibacterial and antifungal activities.In conclusion, it can be argued that the inhibitory effects of both extracts on the growth of tested microorganisms are a significant finding, in view of their potential applications as natural antimicrobial agents in the development of new drugs for the treatment of infectious diseases involving resistant microorganisms.

Table 1 Fatty acid compositions in the seeds of N. italica and S. montana subsp. montana
SoME BIoLoGICAL CoMPoUNDS, RADICAL SCAvENGING CAPACITIES AND ANTIMICRoBIAL ACTIvITIES IN THE SEEDS oF… by Akpınar et al. (2008) determined that the linoleic acid content of N. italica was 17.67% and the linoleic acid content of another four Nepeta species was 15.66-22.33%.Another study by Kiliç

Table 4 Antimicrobial activities of seed extracts containing vitamins, flavonoids and fatty acids Microorganisms Inhibition zone (mm)
Radical inhibition activities of different volumes of methanol extracts of N. italica and S. montana seeds by DPPH radicals.the plant extract of S. montana did not have antibacterial or antifungal effects on other tested microorganisms: P. aeruginosa, K. pneumonia, C. Glabrata, Epidermophyton sp. and Trichophyton sp.other studies suggested that Nepeta and Sideritis extracts did not have antimicrobial capacity N.i: N. italica, S. m: S. montana subsp.montana.Standard: *:Nystatin (30 μg/disc), **: Streptomysin sulphate (10 μg/disc), Control : methanol and hexane (10 μl), NT: not tested.