Cent. Eur. J. Biol. • 7(6) • 2012 • 1116-1122 DOI: 10.2478/s11535-012-0094-4

Central European Journal of Biology

Total phenolic content, flavonoid concentration, antioxidant and antimicrobial activity of methanol extracts from three Seseli L. taxa

Research Article Jelena S. Matejić1, Ana M. Džamić2, Tatjana Mihajilov-Krstev3, Vladimir N. Ranđelović3, Zoran Đ. Krivošej4, Petar D. Marin2,*

1University of Niš, Faculty of Medicine, 18000 Niš, Serbia

2University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, 11000 Belgrade, Serbia

3University of Niš, Faculty of Science and Mathematics, 18000 Niš, Serbia

4University of Pristina, Department of Biology, Faculty of Natural Sciences, 38220 Kosovska Mitrovica, Serbia

Received 19 April 2012; Accepted 14 August 2012

Abstract: Thepresentstudydescribesthetotalphenoliccontent,concentrationsofflavonoidsandin vitroantioxidantandantimicrobialactivityof methanol extracts from Seseli pallasii Besser, S. libanotis (L.) Koch ssp. libanotis and S. libanotis (L.) Koch ssp. intermedium (Rupr.) P. W. Ball, growing wild in Serbia. The total phenolic content in the extracts was determined using Folin-Ciocalteu reagent and their amounts rangedbetween84.04to87.52mgGA(gallicacid)/g.Theconcentrationsofflavonoidsintheextractsvariedfrom4.75to19.37mg

Qu(quercetin)/g.AntioxidantactivitywasanalyzedusingDPPHreagent.Antioxidantactivityrangedfrom0.46to4.63IC50 (mg/ml) and from1.98to2.19mgVitC(vitaminC)/gwhentestedwiththeDPPHandABTSreagents,respectively,usingBHAandVitCascontrols. Theantimicrobialactivityoftheextractswasinvestigatedusingamicro-welldilutionassayforthemostcommonhumangastrointestinal pathogenic bacterial strains: Escherichia coli ATCC25922,Pseudomonas aeruginosaATCC9027,Salmonella enteritidisATCC13076, Bacillus cereusATCC10876,Listeria monocytogenesATCC15313,Staphylococcus aureusATCC25923andCandida albicans ATCC 10231.ThisfindingsuggeststhatSeseli species may be considered as a natural source of antioxidants and antimicrobial agents.

Keywords: Seseli pallasii • S. libanotis • Apiaceae • Antioxidant and antimicrobial activity • DPPH, ABTS • Phenols • Flavonoids ©VersitaSp.zo.o.

1. Introduction S. libanotis (L.) Koch ssp. libanotis has lower leaves that are 2- to 3-pinnate, lobes linear, oblong or The genus Seseli belongs to the family Apiaceae lanceolate, often falcate, acute. This taxon is distributed (=Umbelliferae) and contributes 10 species to the flora in western, central and parts of south Europe, extending of Serbia [1]. Seseli pallasii Besser. (incl. S. varium northwards to southern Sweden [2]. Trev.) - moon carrot, is a glabrous biennial or perennial Seseli libanotis (L.) Koch ssp. intermedium (Rupr.) plant that reaches a height of 30-120 cm. Its leaves vary P. W. Ball is a plant with lower leaves 1- to 2-pinnate, from 2- to 4-pinnate linear to almost filiform. Its petals lobes ovate, coarsely toothed or pinnatifid, obtuse or are white and glabrous, and its fruit is ellipsoid or oblong, subobtuse. This taxon is distributed in eastern and glabrous or slightly tuberculate-verrucose. This taxon is central Europe [2]. distributed in the north of Italy, the Czech Republic and A screening of S. pallasii extracts for chronic Slovakia eastwards to central Ukraine [2]. toxicity and larval inhibition on Spodoptera littoralis

* E-mail: [email protected] 1116 J.S. Matejić et al.

larval growth was recently reported. Lethal doses does not occur with the ABTS assay, especially when and the effect of LD50 doses on growth inhibition and the absorbance is measured at 734 nm [11]. antifeedant were estimated in order to determine the In this study, the antioxidant and antimicrobial potency of the selected extracts based on the mortality activity of Seseli pallasii, S. libanostis ssp. libanotis, and results [3]. S. libanotis subsp. intermedium methanol extracts is In the Eastern part of Turkey, S. libanotis is a reported. As far as we know this is the first report of the common plant used both as a cheese preservative antioxidant activity of these taxa. and to provide aroma. Besides S. libanotis, 25 different species can be used to make herb-flavoured cheese. These herbs, e.g. Allium spp., Thymus spp., Falcaria 2. Experimental Procedures spp. etc., can be added singly or as a mixture to cheese [4]. Leaves of S. libanotis (kelemkesir or kelemenkesir 2.1 Chemicals in Turkish) are consumed as a vegetable in eastern Organic solvents were purchased from “Zorka Turkey [5]. The methanol extract of this plant had a pharma” Sabac, Serbia. Gallic acid, 3-tert-butyl- broad-spectrum antibacterial activity against Bacillus 4-hydroxyanisole (BHA) and 2,2-dyphenyl-1- cereus, B. dipsauri, B. lentimorbus, B. sphaericus, picrylhydrazyl (DPPH) were obtained from Sigma B. subtilis, Corynebacterium ammoniagenes, Kocuria Chemicals Co., St Louis, MO, USA. Folin-Ciocalteu rosea, Neisseria subflava and Micrococcus lylae, which phenol reagent was purchased from Merck, is of interest in relation to the prevention of microbial Darmstadl, Germany. Sodium carbonate anhydrous contamination in foods [6]. (Na2CO3), potassium acetate (C2H3KO2), potassium

Recently, the results of essential oil analyses and peroxidisulphate (K2O8S2) and L(+)- Ascorbic acid antimicrobial effects of different Seseli species were (Vitamin C) were purchased from AnalaR Normapur, published. The essential oil from the aerial parts of VWR, Geldenaaksebaan, Leuven Belgium. Aluminium

S. annuum, wild-growing in Serbia, was characterized nitrate nonahydrate (Al(NO3)3x9H2O) was purchased as containing germacrene-D, sabinene, Z-β-ocimene from Fluka Chemie AG, Buchs, Switzerland. ABTS and limonene. This oil showed antifungal activity against and quercetin hydrate were obtained from TCI Europe fifteen fungi with MICs between 12.5 to 50 7 μl/ml[ ]. NV, Boerenveldsweg, Belgium. All other solvents and Also, essential oil from aerial parts of S. globiferum Vis. chemicals were of analytical grade. which contains sabinene, α-pinene and β-phellandrene showed activity against Pseudomonas aeruginosa, 2.2 Plant material Micrococcus flavus, Lysteria monocytigenes and Aerial plant parts of S. pallasii were collected in July 2003 Escherichia coli, and all investigated micromycetes from Soko Grad (Soko Banja) and parts of S. libanotis [8]. The essential oil isolated from S. montanum ssp. ssp. libanotis were collected in July 2003 from the tommasinii (Reichenb. fil.) Arcangeli was also analyzed Kopaonik mountains. Aerial plant parts S. libanotis and found to possess moderate to strong antimicrobial ssp. intermedium were collected in July 2009 and activity, which can be explained by presence of the fruits were collected in September 2009 from the Stara β-pinene, germacrene-D, sabinene, α-pinene and planina mountains. Voucher specimens for S. pallasii, limonene [9]. The essential oil of S. rigidum which S. libanotis ssp. libanotis (LM 60313), S. libanotis ssp. contains α-pinene, camphene, β-pinene, and limonene intermedium (SL 16433), have been deposited at the was also found to have antimicrobial and potential Herbarium of the Institute of Botany and Botanical antioxidant activity [10]. Garden “Jevremovac”, Faculty of Biology, University of Several methods are available to evaluate Belgrade. antioxidant activities of natural compounds in foods or biological systems. Two methods commonly used 2.3 Preparation of plant extracts in antioxidant activity assays are the 2,2-dyphenyl- Plant material was air dried in the dark and ground to 1-picrylhydrazyl (DPPH) and 2,2’-azinobis(3- a powder. The aerial plant parts (10 g) were powdered ethylbenzthiazoline-6-sulfonic acid) (ABTS) tests. ABTS and extracted with 100 ml methanol. The mixture was is soluble in both aqueous and organic solvents, and exposed to ultrasound for 30 min and after 24 h standing it reacts relatively rapidly compared to DPPH, which in the dark was filtered. The methanol solvent was normally takes several hours for the reaction to be removed by evaporation under reduced pressure, at a completed. Color interference of the DPPH assay with maximum temperature of 40ºC. After evaporation of the samples that contain anthocyanins leads to under- solvent, the crude extract was subjected to subsequent estimation of antioxidant activity. However, this problem analysis.

1117 Total phenolic content, flavonoid concentration, antioxidant and antimicrobial activity of methanol extracts from three Seseli L. taxa

2.4 Determination of total phenolic content Sample concentrations for each compound and The total phenolic content of extracts was determined standard antioxidant, which decrease absorption of

spectrophotometrically by the Folin-Ciocalteu method DPPH solution to 50% (IC50), were obtained from according to the procedure reported by Singleton et al. the absorption of curves DPPH solution at 517 nm. with some modifications [12]. Briefly, 300 µl of methanol Calculations were performed with Origin 7.0 software. extracts solution and 1500 µl of 1:10 Folin-Ciocalteau reagent were mixed and after 6 minutes in the dark 2.7 Evaluation of ABTS radical scavenging 1200 µl of sodium carbonate (7.5%) was added. After activity 2 h of incubation in the dark at room temperature, the The ABTS radical-scavenging activity, was determined absorbance at 740 nm was measured (Shimadzu, following the method of Miller and Rice-Evans with UV-Visible PC 1650 spectrophotometer). The total some modifications [15]. The ABTS·+ solution was phenolic concentration was calculated from a gallic prepared by mixing 19.2 mg of ABTS with 5 ml of acid (GA) calibration curve (10-100 mg/L). Data were potassium persulfate (2.46 mM). The solution was expressed as gallic acid equivalents (GA)/g of extract held at room temperature in the dark for 12-16 h averaged from 3 measurements. before use. The ABTS·+ solution (1 ml) was diluted with 100-110 ml water, in order to obtain an absorbance 2.5 Determination of flavonoid content 0.7±0.02 at 734 nm. A fresh ABTS·+ solution was The total flavonoid content was evaluated using prepared for each analysis. Antioxidant or standard aluminium nitrate nonahydrate according to the solutions, 75 μl, were mixed with 3 ml of diluted ABTS·+ procedure reported by Woisky and Salatino with some solution and incubated at 30ºC for 30’. The absorbance modifications [13]. The sample for determination was at 734 nm was measured (Shimadzu, UV-Visible PC prepared by mixing a 600 µl of methanol extracts 1650 spectrophotometer). Water was used as a blank.

solution and 2580 µl of mixture (80% C2H5OH, 10% ABTS radical scavenging activity in methanol extracts

Al(NO3)3 x 9 H2O and 1M C2H3KO2). After 40 min of was calculated from the Vitamin C (VitC) calibration incubation at room temperature, the absorbance at curve (0-2 mg/L) and expressed as Vitamin C (VitC)/g 415 nm was measured using a Shimadzu, UV-Visible of dry extract. All experimental measurements were PC 1650 spectrophotometer. The total flavonoid carried out in triplicate and were expressed as average concentration in methanol extracts was calculated of three analyses ± standard deviation. from a quercetin hydrate (Qu) calibration curve (10-100 mg/L) and expressed as quercetin equivalents 2.8 Antimicrobial activity (Qu)/g of dry extract. Measurements were done in 2.8.1 Microbial cultures triplicates. The antimicrobial activity of all tested samples was evaluated using laboratory control strains obtained 2.6 Evaluation of DPPH scavenging activity from the American Type Culture Collection: Gram (-) The antioxidant activity of the extracts was evaluated bacteria - Escherichia coli ATCC 25922, Pseudomonas by means of the 2,2-diphenyl-1-picrylhydrazil (DPPH) aeruginosa ATCC 9027, Salmonella enteritidis ATCC radical scavenging method. This spectrophotometer 13076; Gram (+) bacteria: Bacillus cereus ATCC 10876, assay uses stable radical DPPH as a reagent [14]. Listeria monocytogenes ATCC15313, Staphylococcus A methanolic solution of investigated extracts (300 μl; the aureus ATCC 25923 and yeast Candida albicans ATCC extracts’ concentrations between 63.3 and 6000 μg/ml) 10231. was added to 2700 μl methanolic solution of DPPH radical (concentration of 0.04 mg/ml) and after shaking, 2.8.2 Micro-well Dilution Assay the reaction mixture was left to react in the dark for The inocula of the microbial strains were prepared 30 minutes at room temperature. from the overnight broth cultures and suspensions Absorbance of the remaining DPPH radical was were adjusted to 0.5 McFarland standard turbidity then measured at 517 nm (A1) on a Shimadzu, (corresponding to 107-108 CFU/ml, depending on UV-Visible PC 1650 spectrophotometer. Each sample genera - consensus standard by the NCCLS) [16]. and the Vitamin C and BHA standards were measured Serial dilutions of the tested samples (methanol in triplicate against a methanol blank (A0). The decrease exstracts from three Seseli taxa - 100 mg/ml of absorption of the DPPH solution was calculated using in 30% ethanol) were prepared in a 96-well the following equation: microtiter plate over the range of 50.0–0.1 mg/ml in Percentage of absorption decrease (517 nm) = inoculated Mueller-Hinton broth. The final volume (A0-A1)x100/A0 was 100 μl and the final microbial concentration was

1118 J.S. Matejić et al.

106 CFU/ml in each well. The plate was incubated 3.2 Flavonoid concentrations for 24 h at 37ºC. All experiments were performed in The quantities of flavonoids identified in the tested triplicate. Two controls were included - medium with extracts are summarised in Table 1. The concentration 30% ethanol (negative control) and medium with of flavonoids in methanolic extracts of aerial parts Streptomycin, Chloramphenicol and Nystatin (positive S. pallasii and S. libanotis ssp. libanotis samples as control). Microbial growth was determined by adding well as for aerial parts and fruits of S. libanotis ssp. 20 μl of 0.5% triphenyl tetrazolium chloride (TTC) intermedium were determined spectrophotometric aqueous solution [17]. Minimal inhibitory concentration ally with aluminium nitrate nonahydrate. The flavonoid (MIC) was defined as the lowest concentration of the content was expressed as quercetin hydrate equivalents samples inhibiting visible growth (red colored pellet (mg of Qu per gram of dry extract). The concentrations on the bottom of the wells after the addition of TTC). of flavonoids in plant extracts ranged from 4.75 to 19.37 To determine MBC/MFC, the broth was taken from mg Qu/g. The highest flavonoid content was identified in each well without visible growth and inoculated in S. pallasii (with 3 mg/ml extract concentration) and the Mueller-Hinton agar (MHA) for 24 h at 37ºC. Minimal lowest was in fruits of S. libanotis ssp. intermedium (with bactericidal/fungicidal concentration (MBC/MFC) was 2 mg/ml extract concentration). The methanol extract of defined as the lowest samples concentration killing S. libanotis ssp. libanotis had a relatively high flavonoid 99.9% of bacterial/fungal cells. content (12.42 mg Qu/g). Because of their common presence in plants, flavonoids are important components of human and 3. Results and Discussion animal diet. Due to the different biological activities of plant secondary metabolites, their regular consumption 3.1 Total phenolic content may have both positive and negative impact on human Total phenolic and flavonoid content, and antioxidant health [18]. activity (DPPH and ABTS assay) in vitro was determined for methanol extracts of the aerial parts of S. pallasii and 3.3 DPPH scavenging activity S. libanotis ssp. libanotis, as well as for the aerial parts DPPH is a very stable free radical. The effect of an and fruits of S. libanotis ssp. intermedium, separately. antioxidant on DPPH radical scavenging is due to their The results of the total phenolic content determination hydrogen donating ability or their radical scavenging of the examined plant extracts are presented in Table 1. activity. When a solution of DPPH is mixed with that of The total phenols in the methanol extracts, expressed a substance that can donate a hydrogen atom, it gives as gallic acid equivalents (GA) per gram of dry extract, rise to the reduced form diphenylpicrylhydrazine with ranged from 84.04 to 87.53 mg GA/g. The highest the loss of its violet color [19]. phenolic content was found in aerial parts of S. libanotis Free radical scavenging capacities of the tested ssp. intermedium (with 2 mg/ml extract concentration) extracts was measured by DPPH assay and results and the lowest in fruits of S. libanotis ssp. intermedium are shown in Table 2. According to the results obtained, (with 2 mg/ml extract concentration). The methanol methanol extracts were found to be active with an extract of S. libanotis ssp. libanotis (with 1 mg/ml extract IC50 value for S. pallasii of 4.632 mg/ml of solution, for concentration) had a relatively high phenolic content S. libanotis ssp. libanotis 0.460 mg/ml of solution, for the (85.03 mg Qu/g). aerial parts of S. libanotis ssp. intermedium. 3.013 mg/ml

Methanol extracts Extracts concentration (mg/ml) Total phenolic content Total flavonoid content ABTS assay

*S. pallasii 3 84.65±0.004 19.38±0.005 2.19±0.009

*S. libanotis subsp. libanotis 1 85.03±0.004 12.42±0.002 1.98±0.019

*S. libanotis subsp. intermedium 2 87.53±0.005 16.39±0.002 2.04±0.006

**S. libanotis subsp. intermedium 2 84.04±0.052 4.76±0.001 2.06±0.008

BHA 0.1 63.31±0.001 _ 2.66±0.005

Vitamin C 0.1 40.91±0.002 _ _

Table 1. Total phenolic/flavonoid contents and antioxidant capacities by ABTS assay of Seseli sp. extracts.

Each value in the table was obtained by calculating the average of three analysis ± standard deviation. *Aerial parts of plants, **Fruits

1119 Total phenolic content, flavonoid concentration, antioxidant and antimicrobial activity of methanol extracts from three Seseli L. taxa

of solution and the IC50 value for fruits of S. libanotis ssp. 3.5 Antimicrobial activity

intermedium was 4.428 mg/ml. The IC50 values for the Methanol extracts of three Seseli L. taxa showed synthetic antioxidant BHA was 0.093 mg/ml and Vitamin antimicrobial activity against all tested microbial C 0.054 mg/ml were determined in parallel experiments. strains (Table 3) using the antibiotics Streptomycin,

A lower IC50 value indicates higher antioxidant activity. Chloramphenicol and Nystatin for comparison. Methanol When compared to other taxa, the extract of S. libanotis extracts were dissolved in 30% aqueous ethanol. This ssp. libanotis possessed the strongest antioxidant solvent did not show any antimicrobial activity (negative activities. control). Tested extracts had an inhibitory effect on Gram 3.4 ABTS scavenging activity (-) bacteria at concentrations of 0.20-25.0 mg/ml, The results from the ABTS assay are shown in Table 1. but most of them did not show bactericidal effect at The amount ranged from 1.94 to 2.20 mg VitC/g of concentrations of 50.0 mg/ml. All extracts showed good Seseli taxa extracts. The highest activity was identified inhibitory effect on Pseudomonas aeruginosa strain at in S. pallasii (with 3 mg/ml extract concentration) and concentrations of 0.20-1.56 mg/ml which may be of great the lowest in S. libanotis subsp. libanotis (with 1 mg/ml extract concentration). The aerial parts and fruits of Methanol extracts IC (mg/ml) S. libanotis subsp. intermedium (with 2 mg/ml extract 50 concentration) had aproximately the same values. *S. pallasii 4.632 The methanol extract of S. libanotis ssp. libanotis *S. libanotis subsp. libanotis 0.46 showed high antioxidant activity in accordance with *S. libanotis subsp. intermedium 3.013 their high concentration of total phenols and flavonoids. Based on the results of this study, the extracts with the **S. libanotis subsp. intermedium 4.428 highest antioxidant activity had the highest concentration BHA 0.093 of phenols. Phenolic compounds are very important Vitamin C 0.054 plant constituents because of their scavenging ability on free radicals due to their hydroxyl groups. Therefore, the Table 2. Antioxidant activity of Seseli sp. methanol extracts using phenolic content of plants may contribute directly to their DPPH scavenging method.

antioxidant action. *Aerial plant parts, **Fruits

Methanol extracts (MIC/MBC(MFC) in mg/ml) Referent antibiotics

*S.libanotis subsp. *S. libanotis subsp. **S.libanotis subsp. MIC/MBC (MFC) *S.pallasii libanotis intermedium intermedium in μg/ml

Gram (-) bacteria Streptomycin

Escherichia coli 12.5/>50.0 25.0/>50.0 12.5/>50.0 0.78/>50.0 16.0/16.0 ATCC 25922 Pseudomonas aeruginosa 0.39/>50.0 0.78/>50.0 1.56/50.0 0.20/>50.0 8.0/8.0 ATCC 9027 Salmonella enteritidis 6.25/>50.0 0.78/50.0 6.25/50.0 0.39/>50.0 4.0/4.0 ATCC 13076

Gram (+) bacteria Chloramphenicol

Bacillus cereus 12.5/50.0 3.125/25.0 0.78/25.0 0.78/25.0 4.0/16.0 ATCC 10876 Listeria monocytogenes 6.25/12.5 3.125/12.5 1.56/6.25 0.78/12.5 8.0/16.0 ATCC15313 Staphylococcus aureus 3.125/25.0 3.125/12.5 0.78/25.0 0.78/25.0 1.0/8.0 ATCC 25923

Fungal strain Nystatin

Candida albicans 12.5/>50.0 6.25/50.0 3.125/>50.0 0.78/>50.0 16.0/16.0 ATCC 10231

Table 3. Antimicrobial activity of methanol extracts against pathogenic microbial strains using Micro- well Dilution Assay

*Aerial plant parts, **Fruits

1120 J.S. Matejić et al.

importance, since strains of this genus are the most libanotis (MIC/MBC=0.78-3.125/12.5-25.0 mg/ml) frequent causes of food spoilage in the fridge or after methanol extracts, as Bacillus cereus (MIC/MBC=0.78- thawing. When it comes to Gram (+) bacteria, inhibitory 3.125/25.0-50.0 mg/ml) was reported previously [6]. values of extracts were detected over the range of These preliminary results, obtained using a micro-well 0.78-12.5 mg/ml, while bactericidal concentrations dilution assay, show that Seseli methanol extracts are a ranging from 6.25 to 50.0 mg/ml. Prominent inihibitor potential source of numerous antimicrobial compounds activity was obtained with extracts from the aerial active against microbial strains responsibile for different parts S. libanotis subsp. intermedium on the Listeria gastrointestinal disorders and food spoilage. monocytogenes strain (MIC/MBC=1.56/6.25 mg/ml). Extracts showed high anticandidal activity (MIC=0.78/12.5 mg/ml), but fungicidal activity was 4. Conclusions not shown even at the highest tested concentrations, except when aerial parts S. libanotis subsp. libanotis All extracts evaluated from the different Seseli taxa (MFC=50.0 mg/ml) extracts were used. contained protective agents against oxidative stress More prominent antimicrobial inhibitory activity was provoked by DPPH and hydroxyl radicals. They also obtained when S. libanotis extracts (especially with fruit exhibited good antioxidant activity when compared with extracts from S. libanotis subsp intermedium) were the synthetic antioxidants BHA and Vitamin C. All tested used, compared to S. pallasii extract. extracts inhibited the growth of a number of human In a recent study, using disk diffusion, it was pathogens. These microorganisms cause food spoilage shown that the n-hexane extracts some Seseli and isolates of the tested plants may be developed as species contained no antimicrobial activity. Decreased natural food preservatives to prevent gastrointestinal inhibitory zones (10-12 mm) were only obtained when disorders. S. resinosum and S. hartvigii extracts were used on Staphylococcus aureus strain [20], compared to the reference antibiotics. This strain was sensitive to the Acknowledgements essential oil from Seseli libanotis [21] and to seeds obtained from this species which showed great activity The authors are grateful to the Ministry of Education and against enteropathogens [22]. Our results confirm Science of the Republic of Serbia for financial support that Staphylococcus aureus is sensitive to Seseli (Grants No. 173029 and 173030).

References

[1] Nikolić V., Genus Seseli, In: Josifović M. (Ed.), activity of the essential oil of Seseli annuum wild- Flora of Serbia 5, Belgrade, SANU, 1973 growing in Serbia, Fitoterapia., 2007, 78, 319-322 [2] Ball P.W., Genus Seseli L., In: Tutin T.G., Heywood [8] Janaćković P., Soković M., Vujisić L., Vajs V., V.H., Burges N.A., Moore D.M., Valentine D.H., Vucković I., Krivosej Z., et al., Composition and Walters S.M., Webb D.A. (Eds.), Flora Europaea 2, antimicrobial activity of Seseli globiferum essential Cambridge University Press, London, 1968 oil, Nat. Prod. Commun., 2011, 6, 1163-1166 [3] Pavela R., Screening of Eurasian plants for [9] Siljegović J., Glamoclija J., Soković M., Vucković I., insecticidal and growth inhibition activity against Tesević V., Milosavljević S., et al., Composition and Spodoptera littoralis larvae, Afr. J. Agric. Res., antimicrobial activity of Seseli montanum subsp. 2011, 6, 2895-2907 tommasinii essential oil, Nat. Prod. Commun., [4] Ozturk A., Ozturk S., Kartal, S., The characteristics 2011, 6, 263-266 and uses of herbs added to herby cheeses in Van, [10] Stojković S., Petrović S., Kukić J., Dzamić A., Ristić Herb J. Syst. Bot., 2000, 7, 167–181 M., Milenković M., et al., Chemical composition [5] Baytop T., Treatment with Plants in Turkey, Addition, and antimicrobial and antioxidant activity of Seseli 2nd ed., Nobel Medical Publications, Ankara, 1999 rigidum flower essential oil, Chem. Nat. Comp., [6] Ozturk S., Ercisli S., Chemical composition and 2007, 45, 253-256 in vitro antibacterial activity of Seseli libanotis, [11] Arnao M.B., Some methodological problems in World J. Microbiol. Biotechnol., 2006, 22, 261-265 the determination of antioxidant activity using [7] Milosavljević S., Tesević V., Vucković I., Jadranin M., chromogen radicals: A practical case, Trends Food Vajs V., Soković M., et al., Composition and antifungal Sci. Tech., 2000, 11, 419–421

1121 Total phenolic content, flavonoid concentration, antioxidant and antimicrobial activity of methanol extracts from three Seseli L. taxa

[12] Singleton V.L., Orthofer R., Lamuela-Raventos R.M., oils from aromatic plants used in Brazil, Braz. J. Analysis of total phenols and other oxidation substrates Microbiol., 2004, 35, 275–280 and antioxidants by means of Folin–Ciocalteu reagent, [18] Stobiecki M., Kachlicki P., Isolation and identification Meth Enzymol., 1999, 299, 152–178 of flavonoids, In: Grotewold E. (Ed.), The Science [13] Woisky R., Salatino A., Analysis of propolis: some of Flavonoids, 2nd ed., Springer Science and parameters and procedures for chemical quality Business Media, New York, 2006 control, J. Apic. Res., 1998, 37, 99-105 [19] Molyneux P., The use of the stable free radical [14] Blois M.S., Antioxidant determinations by the use of diphenylpicrylhydrazyl (DPPH) for estimating a stable free radical, Nature., 1958, 181, 1199-1200 antioxidant activity, Songklanakarin J. Sci. Technol., [15] Miller N., Rice-Evans C., Factors influencing the 2004, 26, 211-219 antioxidant activity determined by the ABTS radical [20] Tosun A., Ozkal N., Yildiz S., Antimicrobial activity cation assay, Free Radic Res., 1997, 26, 195–199 screening of some Seseli L. species growing in [16] NCCLS – National Committee for Clinical Turkey, J. Fac. Pharm., 2004, 33, 151-155 Laboratory Standards, Performance standards [21] Seyd M., Chaudhary F.M., Bhatty M.K., Antimicrobial for anti-microbial susceptibility testing: eleventh activity of Essential Oils of Umbelliferae Family. informational supplement, Document M100-S11, Part III. Seseli libanotis, Ligusticum stewartii and National Committee for Clinical Laboratory Pycnocycla aucheriana Oils, Pak. J. Sci. Ind. Res., Standard, Wayne, PA, USA, 2003 1989, 32, 316-319 [17] Sartoratto A., Machado A.L.M., Delarmelina C., [22] Meene S., Chaudhary F.M., Bhatty M.K., Figueira G.M., Duarte M.C.T., Rehder V.L.G., Antimicrobial activity of seeds of Seseli libanotis, Composition and antimicrobial activity of essential Pak. J. Sci. Ind. Res., 1989, 32, 316-319

1122