Journal of Applied Botany and Food Quality 90, 330 - 338 (2017), DOI:10.5073/JABFQ.2017.090.041 1Department of Morphology and Systematic of Plants, Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Serbia 2Department of Plant Physiology, Institute for Biological Research “Siniša Stanković“, University of Belgrade, Serbia 3Institute for Medicinal Plant Research “Dr. Josif Pančić”, Belgrade, Serbia 4,5Department of Biology, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, Institute for Biology and Macedonian Academy of Sciences and Arts, Skopje, Macedonia Laserpitium ochridanum: antioxidant, antimicrobial and anti-quorum sensing activities against Pseudomonas aeruginosa Ksenija S. Mileski1*, Ana D. Ćirić2, Jovana D. Petrović2, Mihailo S. Ristić3, Vlado S. Matevski4,5, Petar D. Marin1, Ana M. Džamić1 (Received June 6, 2016; Accepted August 23, 2017) Summary This study shows Laserpitium ochridanum essential oil composition, its antifungal potency, and antioxidant, antimicrobial and antiquorum sensing activities of different extracts. Monoterpene hydrocarbons (40.9%) were the most abundant group of constituents in the oil. Sabinene (22.8%), viridiflorol (14.7%) and α-pinene (11.40%) were the main components of the oil. The ethanolic extract had the highest antioxidant capacity in DPPH and ABTS assays and it was the richest in phenolic contents. Microdilution method revealed the strongest antibacterial activity of ethanolic extracts in comparison to other tested extracts and streptomycin. Essential oil of L. ochridanum evidenced the best antifungal potential against used micromycetes. Results of an anti-quorum sensing activity assay indicated high affection of aqueous extract in reduction of PAO1 pyocyanin production (18.07%). Used samples possessed slight reduction of twitching and swimming motility. This study shows for the first time anti-quorum sensing activity of L. ochridanum against Pseudomonas aeruginosa PAO1, as well as its significant antioxidant potential. Introduction The genus Laserpitium L. (Apiaceae) comprises about 30 species, mostly biennial and perennial plants, widely distributed from the Canary Islands to Siberia and Iran (Nikolić, 1973). It is characterized by ternate or several times pinnate leaves, white, yellow or pinkish petals (TuTin, 1968). Laserpitium ochridanum Micevski is rare and an endemic perennial plant, up to 40-60 cm with white colour of the petals which can be found only at the National park Mt. Galičica (FYROM), at 1600-2000 m a.s.l. (Micevski, 2005). Different parts of some widely distributed Laserpitium species (e.g. L. siler, L. latifolium) have been used as traditional herbal medicines in Europe. They are usually used as tonics for strengthening and refreshing, for treating toothache, as diuretics, for treating gastrointestinal disorders, heart and liver dysfunctions, pulmonary tuberculosis, rheumatism and topically in pruritic dermatomycoses, as well as for sleep disorder and major depression in Taiwan (PoPović et al., 2013; Yi-Lin chen et al., 2015). In earlier studies, sesquiterpene lactones were found as the main secondary metabolites in Laserpitium extracts (Appendino et al., 1987, 1993; ĐermaNović et al., 1996). Recently, it was published that sesquiterpene lactones mainly belong to the class of guajanolides (PoPović et al., 2013). However, monoterpene hydrocarbons were predominant compounds in the essential oil (EO) of Laserpitium species (BAser and duMAn, 1997; chizzoLA et al., 1999; chizzoLA, 2007; Petrović et al., 2009; TiriLLini et al., 2009; PoPović et al., 2010, 2013, 2014). Litarature data showed that different Laserpitium species possessed antibac* Corresponding author terial, antifungal, cytotoxic, anticancer, antinociceptive and antiedematous activities (Petrović et al., 2009; TiriLLini et al., 2009; PoPović et al., 2010, 2013, 2014). Lately, the interest in studying different pathogens is rapidly incrising, because of their resistance towards synthetic antibiotics or antimycotics. It is known that the pathogenic, gram-negative bacillus Pseudomonas aeruginosa is a major cause of nosocomial infections, bronchopneumonia, septic shock and wound infections. This opportunistic bacterium forms populations with distinctive density-dependant behavour. By antiquorum sensing (anti-QS) agents, growth of P. aeruginosa can be weaken and some of its pathologically significant virulence factors, such as production of biofilm, swarming motility, pigment and antibiotic production, can be reduced (Soković et al., 2014; sepAhi et al., 2015). Some medical plants possess anti-QS activity and can be considered as potential anti-quorum agents (AL-hussAini and MAhAsneh, 2009; koh et al., 2013; sepAhi et al., 2015). The aim of this study was to define the chemical composition of L. ochridanum essential oil and to determined antioxidant, antimicrobial and anti-QS activities of its extracts. To the best of our knowledge L. ochridanum crude extracts were assayed for the first time for their antioxidant potency combined with total phenolic and flavonoid contents. Also, no anti-QS activity of this species has been reported to date. Material and methods Solvents and chemical reagents Solvents and chemicals that were provided for performing the experiments were of analytical grade. Organic solvents were procured from Zorka pharma, Šabac, Serbia. Gallic acid, 3-tert-butyl4-hydroxyanisole (BHA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu phenol reagent, potassium acetate and aluminum trinitrate nonahydrate were obtained from Sigma-Aldrich Co., St Louis, MO, USA. Sodium carbonate anhydrous was purchased from Centrohem d.o.o, Stara Pazova, Serbia. Potassium peroxide sulphate and L(+)-ascorbic acid were obtained from Fisher Scientific UK Ltd., Loughborough, Leicestershire, UK. 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and quercetin hydrate were purchased from TCI Europe NV, Binnenveldsweg, Belgium. Malt-broth (MB), tryptic soy broth (TSB), Mueller-Hinton agar (MH), Luria-Bertani medium (LB) (1% w/v NaCl, 1% w/v tryptone, 0.5% w/v yeast extract) and malt agar (MA) were obtained from the Institute of Immunology and Virology, Torlak (Belgrade, Serbia). Streptomycin (Sigma-Aldrich S6501 St. Louis, MQ, USA), ampicillin (Sigma-Aldrich A9393 St. Louis, MQ, USA) and dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MQ, USA) were used in these study. Antimicotic diflucan (containing 50 mg fluconazole) was obtained from Pfizer PGM, Pocesur-Cisse, France. Biological activities of Laserpitium ochridanum Plant material Plant material was collected during the flowering stage, at Mt. Galičica, the national park in Republic of Macedonia (FYROM) in July, 2013 (GPS: N 40°56́30̋, E 20°49́34̋). It was determined as Laserpitium ochridanum Micevski by one of the authors (prof. Vlado S. Matevski). A voucher specimen (BU16778) is deposited at the herbarium of the Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Serbia. Isolation of the essential oil The dark blue EO of L. ochridanum was obtained from 200 g of dry aerial parts by 3 h of hydrodistilation using a Clevenger type apparatus. The yield of the oil was 0.11% for herbal parts (w/w-dry bases). The essential oil obtained was preserved in sealed vials at 4 ºC prior to further analysis. Preparation of plant extracts Dried, ground plant material (10 g) was treated with 200 mL of methanol, ethanol and distilled water to obtain different extracts. The ultrasonic extraction procedure was performed during 24 h in the dark; the extracts were exposed to ultrasound for the first and the last hour of extraction and subsequently filtered through a Whatman filter paper No 1. Methanolic and ethanolic extracts were subjected to solvent evaporation under reduced pressure at maximum temperature of 40 ºC. The frozen aqueous extracts were lyophilized, reduced to a fine dried powder and mixed to obtain homogenous samples. The dried and crude extracts were measured, packed in glass bottles, and stored at 4 ºC until subjection to subsequent analysis. Obtained yields of L. ochridanum extracts were 0.597 g for methanolic (ME), 1.323 g for ethanolic (EE) and 0.983 g for aqueous extracts (AE). Gas chromatography–flame ionization detector (GC–FID) and gas chromatography–mass spectrometry (GC–MS) Qualitative and quantitative analysis of the essential oil was performed using GC and GC-MS methods. The GC analysis of the oil was carried out on a GChP-5890 II apparatus, equipped with splitsplit less injector, attached to an HP-5 column (25 m × 0.32 mm, 0.52 μm film thickness) and fitted to FID. Carrier gas flow rate (H2) was 1 mL/min, split ratio 1:30, injector temperature was 250 °C, detector temperature 300 °C, while column temperature was linearly programmed from 40 to 240 °C (at rate of 4 °/min.). The same analytical conditions were employed for GC-MS analysis, where a 1800C Series II GCD system equipped with HP-5MS column (30 m × 0.25 mm, 0.25 μm film thickness) was used. The transfer line was heated at 260 °C. Mass spectra were acquired in EI mode (70 eV), in m/z range 40-400. The identification of the individual EO components was accomplished by comparison of retention times with standard substances and by matching mass spectral data with those of the Wiley 275 mass spectra library. Confirmation was performed using AMDIS software and literature (AdAMs, 2007). Quantitative analyses were based on area percents obtained by FID. Analyses of total phenols and total flavonoids Total phenolic content (TPC) The spectrophotometric method described by singLeTon et al. (1999) with some modifications was applied for recording total TPCs of all tested L. ochridanum extracts, using Folin-Ciocalteu reagent and GA as a standard. After preparing a 10% Folin-Ciocalteau reagent, the mixtures of 1000 μL of this solution and 200 μL of extracts solutions (1 mg/mL) were left to react for 6 min. After short incubation, 800 μL of 7.5% sodium carbonate solution was added 331 and thus prepared solution was allowed to stand for 2 h at room temperature in the dark. The absorbance was measured at 736 nm versus a blank sample. Total phenols were calculated from the GA calibration curve (10-100 mg/L). Data were expressed as milligrams of GA equivalents per gram of dry plant extract. The values were presented as means of triplicate analysis. Total flavonoid content (TFC) Measurements of TFCs of L. ochridanum extracts were based on the method described by pArk et al. (1997) with slight modification. An aliquot of each extract solution (1 mL) was mixed with 80% ethanol, 10% aluminium nitrate nonahydrate and 1 M potassium acetate. Absorption readings at 415 nm using a spectrophotometer were taken after 40 min. against a blank sample consisting of a 0.5 mL 96% ethanol instead of the tested sample. The TFCs were determined from the QE standard curve (10-100 mg/L). Results were expressed as mg of QE equivalents/g of dry extract. Generally, All measurements were done in triplicates. Antioxidant capacity DPPH assay Series of EO and extracts solutions in appropriate solvents, with concentrations of 0.25-2 μL/mL for EO and 0.025-0.2 mg/mL for extracts were subjected for examination of free radical scavenging activity by DPPH assay. This spectrophotometric procedure described by BLois (1958), was performed to evaluate the quantity of tested solutions needed to reduce 50% of the initial DPPH radical concentration. 0.2 mL of each dilution was mixed with 1.8 mL of DPPH methanol solution (0.04 mg/mL). The absorbance was recorded at 517 nm after 30 min. of dark incubation at room temperature. BHA and ascorbic acid were used as reference standards and methanol as a blank. The corresponding percentage of inhibitions of each sample was calculated from obtained absorbance values by using following equation: Percentage (%) of inhibition = (Ac-As)/Ac × 100 Tested concentrations of EO and extracts which decrease absorption of DPPH solution for 50% (IC50) were obtained from the curve dependence of absorption of DPPH solution on 517 nm from concentration for each tested solution and used standards. ABTS assay The procedure of MiLLer and rice-evAns (1997) with slightly modifications was followed for determination of in vitro ABTS radicalscavenging potency. Before usage, 5 mL of the mixture of 2.46 mM potassium persulphate and 19.2 mg of ABTS was allowed to react in the dark for 12-16 h at room temperature to obtain ABTS+ solution. 100-110 mL of distilled water was added to 1 mL of ABTS+ solution to adjust an absorbance of 0.7 ± 0.02 units at 734 nm. The mixtures of 2 mL of diluted ABTS·+ solution and 50 μL of each tested extract solution were incubated for 30 min. at 30 ºC and the absorbance was determined spectrophotometrically at 734 nm, using water as a blank. For every experiment a fresh ABTS+ solution was prepared. The results were expressed from an ascorbic acid calibration curve (0-2 mg/L) in mg of ascorbic acid equivalents/g of dry extract. Tests were carried out in triplicate and all measurements were expressed as average of three analyses ± standard deviation. Evaluation of antimicrobial properties Microorganisms and culture conditions The antimicrobial activity of all investigated samples was tested using pure control strains obtained from the mycological laboratory, Department of Plant Physiology, Institute for Biologycal Research 332 K.S. Mileski, A.D. Ćirić, J.D. Petrović, M.S. Ristić, V.S. Matevski, P.D. Marin, A.M. Džamić “Siniša Stanković”, Belgrade, Serbia. The microorganisms included following bacterial strains: Bacillus cereus (food isolate), Listeria monocytogenes (NCTC 7973), Micrococcus flavus (ATCC 10240) and Staphylococcus aureus (ATCC 6538), Enterobacter cloacae (human isolate), Escherichia coli (ATCC 35210), Pseudomonas aeruginosa (ATCC 27853), and Salmonella typhimurium (ATCC 13311). The following micromicetes were used: Aspergillus fumigatus (ATCC 9197), Aspergillus niger (ATCC6275) Aspergillus ochraceus (ATCC 12066), Aspergillus versicolor (ATCC 11730), Candida albicans (ATCC 10231), Penicillium funiculosum (ATCC 10509), Penicillium ochrochloron (ATCC 9112) and Trichoderma viride (IAM 5061). Dilutions of bacterial inocula were cultured on solid MH medium, while micromycetes were maintained on solid MA medium. The cultures were subcultured once a month and stored at +4 °C for further usage (BooTh, 1971). Microdilution method For determination of antimicrobial activity of L. ochridanum oil and extracts, the modified microdilution technique described by hAneL and rAeTher (1998) was applied. The assay was performed by sterile 96-well microtiter plates, by adding pure EO or dilutions of tested extracts (in 5% DMSO) into corresponding medium - TSB and MA for bacteria and fungi, respectively. To achieve the concentration of 1.0 × 108 colony forming units (CFU)/mL for bacterial strains, 100 μL of overnight cultures were mixed with 900 μL of medium in eppendorf. Fungal inocula were prepared by washing spores with sterile 0.85% saline solution (containing 0.1% Tween 80 (v/v)). The microbial cell suspensions were adjusted with sterile saline to a concentration of approximately 1.0 × 106 CFU/mL for bacteria and 1.0 × 105 CFU/mL for fungi in a final volume of 100 μL per well. The microplates were incubated for 24 h at 37 °C for bacteria and for 72 h at 28 °C for fungi. The lowest concentrations of tested samples completely inhibiting the growth of used pathogens were defined as minimum inhibitory concentrations (MICs). The minimum bactericidal/fungicidal concentrations (MBCs, MFCs) were determined as the lowest concentrations with no visible growth after serial subcultivation, indicating 99.5% killing of the original inoculums (hAneL and rAeTher, 1998). In addition, bacterial growth was determined by a colorimetric microbial viability assay, based on reduction of an 0.2% p-iodonitrotetrazolium violet color (INT) aqueous solution (I 8377-Sigma Aldrich, St. Louis, MQ, USA) and compared with positive control for each bacterial strain (cLsi, 2009; TsukATAni et al., 2012). Two replicates were done for each sample. The solution of synthetic standard streptomycin with concentration of 1 mg/mL 5% DMSO was used as positive control for bacteria, while the fluconazole solution (antimicotic diflucan containing 50 mg fluconazole) at concentration of 2 mg/mL 5% DMSO was included for fungi. Sterilized distilled water containing 0.02% Tween 80 and 5% DMSO was used as negative control. Biofilm formation Considering the results obtained in antimicrobial assay and low yields of isolated EO and ME, further anti-QS analyzes were continued with ethanolic and aqueous extracts of L. ochridanum. The samples (0.5, 0.25, 0.125 of MICs, respectively) were tested on biofilm forming ability on polystyrene flat-bottomed microtitre 96 well plates as described by spoering and Lewis (2001); drenkArd and AusuBeL (2002), with some modifications. In brief, 100 μL of overnight culture of P. aeruginosa (1.0 × 108 CFU/mL) was added to each well of the plates in the presence of 100 μL subinhibitory concentrations (subMIC) of L. ochridanum samples (0.5, 0.25 and 0.125 MIC) or 100 mL medium (control). After incubation for 24 h at 37 ºC, each well was washed twice with sterile PBS (pH 7.4), dried, stained for 10 min with 0.1% crystal violet in order to determine the biofilm mass. After drying, 200 μL of 95% ethanol (v/v) was added to solubilise the dye that had stained the biofilm cells. The excess stain was washed off with distilled water. After 10 min, the content of the wells was homogenized and the absorbance at λ = 620 nm was read on a Sunrise™ - TecanELISA reader. The experiment was done in triplicate and repeated two times and values were presented as a mean values ± SE. Twitching and flagella motility After growth in the presence or absence of subMICs of L. ochridanum ethanolic and aqueous extracts, streptomycin and ampicillin, the cells of P. aeruginosa PAO1 were washed twice with sterile PBS and re-suspended in PBS at 1.0 × 108 CFU/mL (OD of 0.1 at 660 nm). In brief, the cells were stabbed into a nutrient agar plate with a sterile toothpick and incubated overnight at 37 °C. The plates were then removed from the incubator and incubated at room temperature for two more days. Colony edges and the zone of motility were measured with a light microscope (o’TooLe and koLTer, 1998a, b). SubMICs of extracts (0.5 MICs) were mixed into 10 mL of molten LB medium and poured immediately over the surface of a solidified LB plate as an overlay. The plate was point inoculated with an overnight culture of PAO1 once the overlaid agar had solidified and incubated at 37 °C for 3 days. The extent of swimming was determined by measuring the area of the colony (sAndY and FoongYee, 2012). The experiment was done in triplicate and repeated two times. The colony diameters were measured three times in different direction and values were presented as a mean values ± SE. Preparation of stock solutions of plant extracts for antimicrobial tests Different quantities of stock solutions of L. ochridanum extracts, dissolved in 5% DMSO (20 mg/mL) were tested against various pathogenic microorganisms. Inhibition of synthesis of P. aeruginosa PAO1 pyocyanin The flask assay was used to quantify the inhibitory activity of the L. ochridanum against P. aeruginosa pyocyanin production. Overnight culture of the bacillus PAO1 was diluted to OD600 nm 0.2. Then, 0.5 MICs of tested extracts dissolved in 5% of DMSO (1.25 mg/mL for EE and 12.50 mg/mL for AE), were added to the bacteria (5 mL) and incubated at 37 °C for 24 h. The treated culture was extracted with chloroform (3 mL), followed by mixing the chloroform layer with 0.2 M HCl (1 mL). Absorbance of the extracted organic layer was measured at 520 nm using a Shimadzu UV1601 spectrophotometer (Kyoto, Japan) (sAndY and Foong-Yee 2012). The experiment was done in triplicate and repeated two times. The values were expressed as ratio (OD520/OD600) × 100. Anti-quorum sensing activity of extracts Bacterial strains, growth media and culture conditions In this study, Pseudomonas aeruginosa PAO1 from the Institute for Biological Research “Siniša Stanković”, Belgrade, Serbia, was used. Bacteria were routinely grown in Luria-Bertani (LB) medium with shaking (220 rpm) and cultured at 37 °C. Statistical analysis Three samples were used and all the assays were carried out in triplicates. The results are expressed as mean values and standard deviation (SD). The results were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD Test with a = 0.05. This analysis was carried out using SPSS v. 18.0 program. Biological activities of Laserpitium ochridanum Results Essential oil composition Referring to the results presented in Tab. 1, fifty nine components were identified in L. ochridanum EO. Monoterpene hydrocarbons were the most abundant group of compounds (40.87%), followed by oxygenated sesquiterpenes (24.14%), oxygenated monoterpenes (15.27%) and sesquiterpene hydrocarbons (13.17%). The dominant compounds of the EO were sabinene (22.8%), viridiflorol (14.7%) and α-pinene (11.4%) (Tab. 1). Tab. 1: Chemical composition of EO of L. ochridanum aerial parts. Compounds KIE KIL % 919.1 924 0.32 α-Pinene 924.8 932 11.36 Thuja-2,4(10)-diene 944.9 953 0.17 Sabinene 965.6 969 22.76 0.80 α-Thujene β-Pinene 974.7 974 Myrcene 985.7 988 0.69 n-Octanal 997.2 998 0.39 α-Terpinene 1009.4 1014 1.02 p-Cymene 1018.0 1020 0.35 β-Phellandrene 1020.9 1025 0.96 γ-Terpinene 1051.3 1054 1.93 cis-Sabinene hydrate 1057.3 1065 0.56 n-Octanol 1069.3 1063 3.86 Terpinolene 1080.7 1086 0.51 6-Camphenone 1088.1 1095 0.59 trans-Sabinene hydrate 1097.0 1098 0.29 6-Camphenol 1113.5 1111 0.43 α-Campholenal 1119.3 1122 0.56 trans-Pinocarveol 1132.4 1135 0.85 trans-Sabinol 1135.5 1137 0.40 trans-Verbenol 1139.6 1140 1.79 Terpinen-4-ol 1171.0 1174 3.86 Thuj-3-en-10-al 1178.9 1181 0.31 α-Terpineol 1186.8 1186 0.28 Myrtenal 1188.8 1193 0.37 Myrtenol 1192.1 1194 0.54 Verbenone 1204.0 1204 0.19 Octanyl acetate 1207.5 1211 1.26 0.50 Isobornyl acetate 1277.8 1283 α-Terpinyl acetate 1343.0 1346 0.21 Cyclosativene 1366.2 1369 0.27 α-Copaene 1370.2 1374 1.12 Daucene 1376.8 1380 0.69 β-Cubebene 1381.1 1387 0.23 β-Elemene 1383.4 1389 0.43 (E)-Caryophyllene 1409.0 1417 1.62 trans-α-Bergamotene 1427.0 1432 0.18 α-humulene 1443.5 1452 0.66 (E)-β-Farnesene 1450.2 1454 0.19 cis-Muurola-4(14),5-diene 1460.6 1465 0.20 γ-Himachalene 1467.9 1468 0.81 Dauca-5,8-diene 1471.4 1471 1.96 ar-Curcumene 1475.5 1479 0.19 β-Selinene 1487.0 1489 1.73 cis-Eudesma-6,11-diene Bicyclogermacrene β-Bisabolene δ-Cadinene α-Calacorene Spathulenol Caryophyllene oxide Viridiflorol 1-epi-Cubenol β-Cedren-9-one β-Eudesmol α-Bisabolol Chamazulene Neophytadiene (isomer II) Incensole acetate Total Number of constituents Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others 333 1488.0 1494.9 1500.6 1514.3 1546.1 1570.3 1573.4 1588.9 1627.5 1631.6 1649.4 1679.3 1721.9 1829.5 2181.9 1489 1500 1505 1522 1544 1577 1582 1592 1627 1630 1649 1685 1730 1830 2184 0.83 0.44 0.12 0.70 0.80 2.65 1.16 14.71 0.54 2.44 0.44 2.20 3.04 1.02 0.52 100.00 59 40.87% 15.27% 13.17% 24.14% 6.55% KIE = Kovats (retention) index experimentally determined (AMDIS) KIL = Kovats (retention) index - literature data (ADAMS, 2007) Total phenolic contents According to the results obtained for TPC in L. ochridanum extracts (Tab. 2), phenols were present from 111.28 to 141.30 mg GA/g of dry extract for methanolic and ethanolic extract, respectively. In tested extracts, TFC ranged from 21.38 to 67.69 mg QE/of dry extract for aqueous and ethanolic extract, respectively. In general, greater variation in tested extracts was recorded in flavonoid contents. The highest phenolic and flavonoid concentrations were measured in EE of L. ochridanum (Tab. 2). Antioxidant activity The results of obtained antioxidant activity for L. ochridanim are listed in Tab. 2. In DPPH test, used extracts exhibited similar antioxidant activity, stronger than BHA, but lower activity compared to ascorbic acid. Still, the strongest radical scavenging activity was recorded for EE (0.113 ± 0.002 mg/mL), which was in accordance with the highest measured total phenolic and flavonoid contents. EO of L. ochridanum showed the lowest antioxidant potency compared to all other samples. Results obtained by the ABTS test showed that the AE was the most effective agent in concentration of 2.172 ± 0.005 mg ascorbic acid/g of dry extract. According to the obtained results this sample had slightly lower antioxidant capacity than standard QE (2.749 ± 0.004 mg ascorbic acid/g of dry extract). Antimicrobial properties Antibacterial activity The results presented in Tab. 3 indicate that L. ochridanum extracts exhibited moderate antibacterial activity. The EE was the strongest in bactericidal activity (MBCs = 1.00-5.00 mg/mL), while the lowest potency had AE (MBCs = 11.00-14.00 mg/mL). Both, methanolic and ethanolic extracts were more effective compared to streptomycin, but all extracts, including aqueous, showed stronger inhibitory activity on L. monocytogenes and E. cloacae than used antibiotic. K.S. Mileski, A.D. Ćirić, J.D. Petrović, M.S. Ristić, V.S. Matevski, P.D. Marin, A.M. Džamić 334 Tab. 2: TPC, TFC and antioxidant activity of L. ochridanum extracts and EO (means ± SD). L. ochridanum extracts/ EO Total phenolic contents Antioxidant activity TPC 1 mg/mL (mg GA/g of DE) TFC 1 mg/mL (mg QE/g of DE) DPPH (IC50 = mg/mL) ABTS 1 mg/mL (mg ascorbic acid/g of DE) ME 111.28 ± 0.005c 31.31 ± 0.010b 0.12 ± 0.011b 1.63 ± 0.009b EE 141.30 ± 0.013a 67.69 ± 0.018a 0.11 ± 0.002b 1.56 ± 0.004b AE 125.30 ± 0.010b 0.004c 0.000b 2.17 ± 0.005a EO / Standards 21.38 ± 0.12 ± 1.88 ± 0.009c / / / ±0.012b BHA 0.13 ascorbic acid 0.03 ± 0.008a / QE 2.75 ± 0.004a Indicated letters mean significant difference (p < 0.05) B. cereus and S. aureus (MBCs = 1.00-11.00 mg/mL) were the most sensitive bacteria, while E. coli and M. flavus (MBCs = 5.00->14.00 mg/mL) proved to be the most resistant strains. Antifungal activity The results obtained for the antifungal activity of investigated samples are presented in Tab. 4. The EO of this species had the strongest activity in inhibition of micromycetes growth (MFCs = 0.55-2.20 mg/mL) and it was similar to the activity of applied fluconazole (MFCs = 0.03-1.50 mg/mL). Among the investigated extracts, the EE showed the highest activity (MFCs = 5.00 mg/mL) on all used fungal strains, exept for A. niger (MFCs =18.00 mg/mL) (Tab. 4). The most resistant micromycetes were A. niger and A. fumigatus, while the most sensitive strains were A. versicolor, P. ochrochloron and P. funiculosum. Fungi T. viride and P. ochrocloron were more sensitive to L. ochridanum oil (MFC = 1.10 mg/mL), than to fluconazole (MFC = 1.50 mg/mL) (Tab. 4). Tab. 3: Antibacterial activity of L. ochridanum extracts in mg/mL (means ± SD). ME L. ochridanum/ Bacteria MIC EE MBC 0.06a MIC 0.03a AE MBC 0.02a 0.00a MBC 0.02a B. cereus 0.40 ± M. flavus 4.00 ± 0.02c 5.00 ± 0.02b 2.00 ± 0.01ab 5.00 ± 0.00b 10.00 ± 0.01b 0.04c 0.07b 0.00a 0.10a 0.02a 1.00 ± 0.50 ± 1.00 ± Streptomycin MIC 6.00 ± 11.00 ± MIC 0.01a 2.50 ± 0.06a 2.50 ± 0.00a 5.00 ± 0.00b 0.10c 20.00 ± 0.02c 1.50 ± >14.00 ± 0.03b 0.01a MBC 0.01a L. monocytogenes 3.00 ± P. aeruginosa 3.00 ± 0.00c 4.00 ± 0.05b 0.50 ± 0.02a 2.00 ± 0.01a 5.00 ± 0.00a 11.00 ± 0.02a 2.50 ± 0.07a 5.00 ± 0.01b 0.01c 0.03c 0.02b 0.01b 0.00b 0.01b 0.04a 5.00 ± 0.03b 10.00 ± 0.02b 20.00 ± 0.00c 4.00 ± 0.50 ± 2.00 ± 5.00 ± E. coli 4.00 ± E. cloacae 2.00 ± 0.01b 4.00 ± 0.01b 1.00 ± 0.10a 4.00 ± 0.02b 5.00 ± 0.10a 0.00b 0.05b 0.06a 0·05a 0.01b S. typhymurium 2.00 ± S. aureus 0.40 ± 0.10a 8.00 ± 3.00 ± 4.00 ± 1.00 ± 1.00 ± 0.07b 1.00 ± 0.05a 5.00 ± 2.00 ± 10.00 ± 10.00 ± 2.00 ± 0.03a 5.00 ± 0.07a 11.00 ± >14.00 ± 15.00 ± 2.50 ± 11.00 ± 0.01a >14.00 ± 0.02b 0.01a 5.00 ± 0.01b 2.50 ± 0.05a 5.00 ± 0.00b 2.50 ± 11.00 ± 0.00a Indicated letters mean significant difference (p < 0.05) Tab. 4: Antifungal activity of L. ochridanum extracts and EO in mg/mL (means ± SD). L. ochridanum/ Fungi ME MIC EE MFC MIC AE MFC MIC Fluconazole EO MFC MIC MFC MIC MFC C. albicans 8.00 ± 0.02c 12.00 ± 0.01b 6.00 ± 0.10c 12.00 ± 0.05b 12.00 ± 0.01b 14.00 ± 0.03a 0.55 ± 0.00b 1.10 ± 0.02b 0.02 ± 0.05a 0.03 ± 0.03a T. viride 3.00 ± 0.00a 8.00 ± 0.02a 1.10± 0.03b 1.00 ± 0.02d 1.50± 0.02d 0.01b 0.00d 1.50 ± 0.02d P. ochrochloron 6.00 ± P. funiculosum 0.03b 10.00 ±0.10a 3.00 ± 0.01a 8.00 ± 0.05a 0.00b 0.01b A. fumigatus 6.00 ± A. versicolor 6.00 ± 0.03b 8.00 ± 0.20a 0.02b 14.00 ± 0.03c 5.00 ± 0.05b 14.00 ± 0.07b 10.00 ± 0.01a 12.00 ± 0.02a 0.55 ± 0.07b 4.00 ± 0.02a 5.00 ± 0.05a 10.00 ± 0.00a 14.00 ± 0.00a 0.28 ± 0.05a 4.00 ± 0.00a 5.00 ± 0.00a 12.00 ± 0.07b 14.00 ± 0.02a 0.55 ± 0.05b 6.00 ± 0.10c 14.00 ± 0.20b 12.00 ± 0.10b 14.00 ± 0.02a 0.55 ± 0.10b 4.00 ± 0.01a 5.00 ± 0.03a 10.00 ± 0.20a 12.00 ± 0.07a 0.28 ± 0.02a 0.03a 0.02a 0.00a 0.05a 0.10b A. ochraceus 6.00 ± A. niger 8·00 ± 0.01c 19.00 ± 0.05c 6.00 ± 0.00c 18.00 ± 0.05c 12.00 ± 0.01b 18.00 ±0.10b 1.10 ±0.07c 19.00 ± 4.00 ± Indicated letters mean significant difference (p < 0.05) 5.00 ± 10.00 ± 14.00 ± 0.55 ± 1.10 ± 1.00 ± 2.20 ± 0.01c 0.25 ± 0.01b 0.50 ± 0.00b 2.20 ± 0.07c 0.50 ± 0.02c 1.00 ± 0.00c 0.55± 0.00a 0.13 ± 0.00a 0.50 ± 0.01b 0.02b 0.50 ± 0.05c 1.00 ± 0.03c 1.10 ± 2.20 ± 0.00c 0.25 ± 0.03b 1.00 ± 0.10c Biological activities of Laserpitium ochridanum Anti-QS activity evaluation Biofilm formation Tab. 5 presents the effects of L. ochridanum extracts on P. aeruginosa PAO1 biofilm formation. The samples were tested at 0.5, 0.25 and 0.125 of MIC values. L. ochridanum extracts showed significant difference in terms of anti-biofilm formation activity. L. ochridanum EE showed dose dependant inhibitory activity, reducing from 8.63% to 63.88% of biofilm formation, where the best result was obtained in the presence of 0.5 MIC of the extract. Results revealed Tab. 5: Effects of L. ochridanum extracts on biofilm formation of P. aeruginosa PAO1 (%). L. ochridanum/ Standards Biofilm formation* 0.5 MIC (% ± SE) 0.25 MIC (%± SE) 0.125 MIC (% ± SE) EE 36.12 ± 1.73 60.82 ± 1.05 91.37 ± 0.42 AE n.d. n.d. n.d. Ampicillin 69.16 ± 0.65 56.46 ± 0.46 92.16 ± 0.37 Streptomycin 49.40 ± 0.46 70.97 ± 0.36 88.36 ± 0.42 *Biofilm formation values were calculated as: ((mean A620 control wellmean A620 treated well)/mean A620 control well) × 100. − Values are expressed as means ± SE. − n.d. not determinate 335 that L. ochridanum EE reduced biofilm formation more effectively than both antibiotics allowing formation of PAO1 in the range from 36.12% to 91.37%. Contrary, the tested subMIC concentrations of AE did not show any suppression of P. aeruginosa biofilm formation (Tab. 5). In the presence of commercial antibiotics streptomycin and ampicillin, biofilm formation occurred in narrower range, with slightly stronger biofilm inhibition recorded for streptomycin. Twitching and flagella motility In addition, the ethanolic and aqueous extracts of L. ochridanum reduced the twitching and flagella motility activity of P. aeruginosa (Tab. 6 and Fig. 1). As presented in Tab. 6, the color of the colony ranged from white, through light green to green. Used extracts changed the color and diameter of treated colonies to a certain extent. In the presence of extracts, colonies where white and larger (14.00 mm and 17.67 mm for EE and AE, respectively) in comparison to colonies treated with antibiotics (Tab. 6). The green colony of P. aeruginosa with streptomycin, had minimal growth (11.00 mm) and completely reduces protrusions. Also, the most reduced flagella in size, shape and number were in colony with streptomycin (Fig. 1). Inhibition of synthesis of P. aeruginosa PAO1 pyocyanin SubMICs of L. ochridanum samples were tested for inhibition of P. aeruginosa pigment production and both extracts showed substantial activity in pigment synthesis inhibition. The affection was observed by the reduction of the green pigmentation of the samples, Tab. 6: Effects of L. ochridanum extracts on twitching and flagella motility of P. aeruginosa (PAO1). L. ochridanum extracts/ Standards EE Colony diameter (mm ± SE) Flagella diameter (μm) Colony colour Colony edge 14.00 ± 2.65 40-160 White Rare flagella AE 17.67 ± 5.51 16-80 White Tiny flagella Streptomycin 11.00 ± 1.00 24-56 Green Tiny flagella Ampicillin 13.33 ± 5.03 16-56 Green Regular flagella P. aeruginosa (PAO1) 12.00 ± 1.00 56-80 Light green Regular flagella Fig. 1: Light microscopy of colony edges of P. aeruginosa in twitching motility, grown in the presence or absence of L. ochridanum extracts and commercial antibiotics. The colonies from the bacteria grown with extracts in concentration of 0.5 MIC (A-B). The colony with EE was with moderately reduced protrusions (A); In the presence of AE colony formed almost regular protrusions (B); P. aeruginosa colony in the presence of streptomycin (0.5 MIC) with reduced protrusion (C); P. aeruginosa colony in the presence of ampicillin with regularly formed protrusions (D); P. aeruginosa produced a flat, widely spread, irregularly shaped colony in the absence of extracts and commercial antibiotics (E); Magnification: (A-D) × 100. K.S. Mileski, A.D. Ćirić, J.D. Petrović, M.S. Ristić, V.S. Matevski, P.D. Marin, A.M. Džamić 336 compared to the coloration of the control PAO1 sample (Fig. 2). The strongest inhibition of pigment’s production was detected for L. ochridanum AE. In the presence of tested concentrations of extracts, pyocyanin was less produced (23.46% and 18.07% for ethanolic and aqueous extracts, respectively), than by control strain (141.55%). All extracts were better in prevention of pigment production regarding to applied antibiotics (Fig. 2). Production of pyocianin (%) 160 140 120 100 80 60 40 20 0 L. ochridanum L. ochridanum Streptomycin ethanol aqueous Ampicillin PA01 Fig. 2: Reduction of pyocyanin production of P. aeruginosa PAO1 by L. ochridanum extracts streptomycin and ampicillin tested at subMICs (mg/mL). Discussion The chemical compositions of EOs of different Laserpitium species have been previously reported (BAser and duMAn, 1997; chizzoLA et al., 1999; chizzoLA, 2007; Petrović et al., 2009; TiriLLini et al., 2009; PoPović et al., 2010, 2014). Results obtained for L. ochridanum EO revealed sabinene, viridiflorol and α-pinene as the most dominant compounds (Tab. 1). Recent studies on L. ochridanum herb oil showed sabinene and α-pinene as the major components, which is in accordance with our results. In contrary, limonene, which was the main constituent of fruit oil, was not detected in our sample (PoPović et al., 2014, 2015). Our results revealed that EE of L. ochridanum was the richest in phenols and flavonoids among all extracts. Also, EE had the highest DPPH radical scavenging activity, while AE was the strongest agent in ABTS radical scavenging assay. In general, all extracts possessed comparable activity to synthetic antioxidants. In contrary, EO showed low antioxidant capacity. Results obtained in a microdilution assay, indicated that grampositive and gram-negative bacteria showed similar sensitivity to L. ochridanum extracts. According to the literature data, L. ochridanum chloroform extract of roots and rhizomes exhibited antimicrobial activity against some food spoilage microorganisms (PoPović et al., 2015). In comparison with those results, it can be concluded that methanolic and ethanolic extracts of L. ochridanum in our study showed higher antimicrobial potency. We found that extracts indicated stronger antibacterial than antifungal potential (Tab. 3 and 4). EO of L. ochridanum showed similar inhibitory effect as fluconazole (Tab. 4). Also, L. ochridanum EO was more effective than L. garganicum EO in growth inhibition of the most resistant micromycete A. niger (TiriLLini et al., 2009). Inhibition of bacterial QS offers new strategies for the treatment of bacterial infections. Anti-QS agents can interfere with the bacterial communication system and can disrupt the pathogenicity process of bacteria (sepAhi et al., 2015). Therefore, L. ochridanum extracts were submitted for anti-QS screening for the first time since it is of great importance to discover new potential anti-QS agents. The screening results indicated that EE and AE samples showed some potential of anti-QS activity. L. ochridanum EE demonstrated an inhibition at the initial stage of biofilm formation in the manner of different tested concentrations. That is of great importance since bacteria form biofilms as a protection against host’s immune system and as a factor of antibiotic resistance (Soković et al., 2014). It was clearly indicated that tested concentrations of L. ochridanum extracts were more effective on P. aeruginosa pigment production than those of applied antibiotics. This green, toxic pigment acts as a virulence factor in bacteria, so reduction of its production is crucial for increasing the effectiveness of host defense. While AE notably demonstrated the best activity in suppression of pyocyanin synthesis, it did not show any reduction of colony formation at the tested subMICs. Opposite results obtained for AE in these two assays could be associated with possible different mechanisms responsible for its activity. At this point, there is no sufficient data to highlight the exact method of QS inhibition. A few potential modes of action have been proposed, many of them to interfere with the QS system such as inhibition of biosynthesis of auto-inducer molecules, inactivation or degradation of the auto-inducer, interference with the signal receptor and inhibition of the genetic regulation system. Due to complex phytochemistry of plant extracts, different compounds could be associated with specific effects linked to the QS system, so the difference in the activities of L. ochridanum aqueous sample suggest less polar nature of L. ochridanum active compounds in reduction of P. aeruginosa biofilm formation. The importance of plant extracts preparation should be also considered (Adonizio et al., 2006; Adonizio, 2008: Glamočlija et al., 2015). In the research of Adonizio et al. (2008), where aqueous extract of Schefflera actinophylla, with no anti-QS activity, was used as a negative control, it was concluded that plant extracts differentially affect biofilm formation. Inhibition of swarming and twitching motility of PAO1 by L. ochridanum samples was achieved in moderate extent with better results obtained for EE. Both types of motilities are important in the initial stages of biofilm formation of P. aeruginosa (o’TooLe and koLTer, 1998b). Conclusions This study established the chemical characterization of L. ochridanum EO and provided new data concerning antioxidant and anti-QS activities of crude extracts/EO. The presence of phenolic compounds and previously reported sesquiterpene lactones mostly contribute to biological potency of L. ochridanum extracts. Among all extracts, EE possessed the best potency in evaluated antioxidant and antibacterial activities. The EO revealed strong antifungal potential. L. ochridanum showed promising anti-QS effectiveness that was sufficient for the reduction of biofilm formation and pyocyanin production. To establish the application of this species in various pharmaceutical, dietary or alternative medicine branches, further research is needed especially concerning that the exact mechanistic interactions with the QS system should be resolved. Acknowledgements The authors are grateful to the Ministry of Education, Science and Technological Development of the Republic of Serbia for financial support Grants No. 173029 and 173032. References AdAMs, r.p., 2007: Identification of essential oil components by gas chromatography/mass spectrometry, 4th ed. Carol Stream, IL, USA: Allured Publishing Corp. Adonizio, A.L., downuM, k., BenneTT, B.c., MAThe, k., 2006: Anti- Biological activities of Laserpitium ochridanum quorum sensing activity of medicinal plants in Southern Florida. J. Ethnopharmacol. 105, 427-435. DOI: 10.1016/j.jep.2005.11.025 Adonizio, A.L., 2008: Anti-quorum sensing agents from South Florida medicinal plants and their attenuation of Pseudomonas aeruginosa pathogenicity. FIU Electronic Theses and Dissertations 13, 14-21. A L-hussAini, r., MAhAsneh, A.M., 2009: Microbial growth and quorum sensing antagonist activities of herbal plants extracts. Molecules 14, 3425-3435. DOI: 10.3390/molecules14093425 Appendino, g., crAvoTTo, g., nAno, g.M., 1993: Sesquiterpene lactones from Laserpitium gallicum. Phytochemistry 33, 883-886. Appendino, g., vALLe, g.M., gAriBoLdi, p., 1987: Sesquiterpene lactols from Laserpitium halleri. Phytochemistry 26, 1755-1757. BAser, k.h.c., duMAn, h., 2011: Composition of the essential oil of Laserpitium petrophilum Boiss. et Heldr. J. Essent. Oil Res. 9, 707-708. DOI: 10.1080/10412905.1997.9700818 BLois, M.s., 1958: Antioxidant determinations by the use of a stable free radical. Nature 181, 1199-1200. DOI: 10.1038/1811199a0 BooTh, c., 1971: Fungal culture media. In: Norris, J.R., Ribbons, D.W. (eds), Methods in microbiology, 4th ed. London, UK: Academic Press. chen, Y.L., Lee, c.Y., huAng, k.h., kuAn, Y.h., chen, M., 2015: Prescription patterns of Chinese herbal products for patients with sleep disorder and major depressive disorder in Taiwan. J. Ethnopharmacol. 171, 307-316. DOI: 10.1016/j.jep.2015.05.045 chizzoLA, r., 2007: Composition of the essential oil from Laserpitium gallicum grown in the wild in Southern France. Pharm. Biol. 45, 182-184. DOI: 10.1080/13880200701213054 chizzoLA, r., johaNNeS, N., ChlodwiG, F., 1999: Fruit oil of Laserpitium siler L. grown in France. J. Essent. Oil Res. 11, 197-198. DOI: 10.1080/10412905.1999.9701109 Clinical and laboratory standards institute methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically, 2009: Aproved standards. 8th ed. Clinically and laboratory standards Institute, M07-A8, Wayne: CLSI publication. drenkArd, e., AusuBeL, F.M., 2002: Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416, 740743. DOI :10.1038/416740a Đermanović, m., SteFaNović, m., Đermanović, v., milovaNović, M., 1996: Structure elucidation of the sesquiterpene lactones from plant species Laserpitium latifolium L. J. Herbs Spices Med. Plants. 3, 3-10. DOI: 10.1300/J044v03n02_02 Glamočlija, j., ćirić, a., Nikolić, m., F erNaNdeS, Â., BarroS, l., cALheLh, r.c., F erreirA, i.c., Soković, m., vaN GrieNSveN, l.j., 2015: Chemical characterization and biological activity of Chaga (Inonotus obliquus), a medicinal “mushroom”. J. Ethnopharmacol. 162, 323-332. DOI: 10.1016/j.jep.2014.12.069 hAneL, h., r AeTher, w., 1998: A more sophisticated method of determining the fungicidal effect of water-insoluble preparations with a cell harvester, using miconazole as an example. Mycoses 31, 148-154. DOI: 10.1111/j.1439-0507.1988.tb03718.x koh, c.L., sAM, c.k., Yin, w.F., TAn, L.Y., k rishnAn, T., chong, Y.M., chAn, k.g., 2013: Plant-derived natural products as sources of antiquorum sensing compounds. Sensors 13, 6217-6228. DOI: 10.3390/s130506217 Micevski, k., 2005: Laserpitium L. In: Matevski, V. (ed.), Flora of Republic of Macedonia, 6th ed. Skopje, Macedonian Academy of Sciences and Arts, 1647-1651. MiLLer, n., r ice-evAns, c., 1997: Factors influencing the antioxidant activity determined by the ABTS radical action assay. Free Radic. Res. 26, 195-199. Nikolić, v., 1973: Laserpitium L. In: Josifivić, M. (ed.), Flora of Republic of Serbia, 5th ed. Belgrade, Serbian Academy of Sciences and Arts, 308313. o’TooLe, g.A., koLTer, r., 1998a: Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28, 449-461. 337 DOI: 10.1046/j.1365-2958.1998.00797.x o’TooLe, g.A., koLTer, r., 1998b: Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30, 295-304. DOI: 10.1046/j.1365-2958.1998.01062.x Park, Y.k., koo, m.h., ikeGaki, m., CoNtado, j.l., 1997: Comparison of the flavonoid aglycone contents of Apis mellifera propolis from various regions of Brazil. Arq. Biol. Tecnol. 40, 97-106. P etrović, s., Pavlović, M., PoPović, v., couLAdis, M., TzAkou, o., mileNković, m., vučićević, d., Niketić, m., 2009: Composition of antimicrobial activity of essential oils from flower and leaf of Laserpitium zernyi Hayek. J. Essent. Oil Res. 21, 467-470. DOI: 10.1080/10412905.2009.9700220 PoPović, v., heYeriCk, a., P etrović, S., CaleNBerGh, S.v., k araliC, i., Niketić, m., deForCe, d., 2013: Sesquiterpene lactones from the extracts of two Balkan endemic Laserpitium species and their cytotoxic activity. Phytochemistry 87, 102-111. DOI: 10.1016/j.phytochem.2012.11.011 PoPović, v., heYeriCk, a., P etrović, S., cALenBergh, s.v., k aralić, i., Niketić, M., deForce, d., 2013: Cytotoxic activity of Laserpitium latifolium L. extract and its daucane and phenylpropanoid constituents. Rec. Nat. Prod. 7, 245-249. PoPović, v., P etrović, S., Pavlović, m., mileNković, m., CouladiS, m., tzakou, o., duraki, S., Niketić, m., 2010: Essential oil from the underground parts of Laserpitium zernyi: potential source of alpha-bisabolol and its antimicrobial activity. Nat. Prod. Commun. 5, 307-310. PoPović, v., P etrović, S., tomić, m., StePaNović-P etrović, r., miCov, a., Pavlović-droBaC, m., CouladiS, m., Niketić, m., 2014: Antinociceptive and anti-edematous activities of the essential oils of two Balkan endemic Laserpitium species. Nat. Prod. Commun. 9, 125-128. PoPović, v., P etrović, d.S., mileNković, t.m., droBaC., m., CouladiS, a.m., Niketić, S.m., 2015: Composition and antimicrobial activity of the essential oils of Laserpitium latifolium L. and L. ochridanum Micevski (Apiaceae). Chem. Biodivers. 12, 170-177. DOI: 10.1002/cbdv.201400127 PoPović, v., Stojković, d., Nikolić, m., heYeriCk, a., P etrović, S., Soković, m., Niketić, m., 2015: Extracts of three Laserpitium L. species and their principal components laserpitine and sesquiterpene lactones inhibit microbial growth and biofilm formation by oral Candida isolates. Food Funct. 6, 1205-1211. DOI: 10.1039/c5fo00066a sAndY, s.M., Foong-Yee, T., 2012: Anti-quorum sensing and antimicrobial activities of some traditional chinese medicinal plants commonly used in South-East Asia. Malays. J. Microbiol. 8, 11-20. DOI: 10.21161/mjm.34911 sepAhi, e., TArighi, s., AhMAdi, F.s., BAgheri, A., 2015: Inhibition of quorum sensing in Pseudomonas aeruginosa by two herbal essential oils from Apiaceae family. J. Microbiol. 53, 176-180. DOI: 10.1007/s12275-015-4203-8 singLeTon, v.L., orThoFer, r., LAMueLA-r AvenTos, r.M., 1999: Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Method Enzymol. 299, 152-178. DOI: 10.1016/S0076-6879(99)99017-1 Soković, m., ćirić, a., Glamočlija, j., Nikolić, m., vaN GrieNSveN, j.l.d.l., 2014: Agaricus blazei hot water extract shows anti-quorum sensing activity in the nosocomial human pathogen Pseudomonas aeruginosa. Molecules 19, 4189-4199. DOI: 10.3390/molecules19044189 spoering, A.L., Lewis, k., 2001: Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing of antimicrobials. J. Bacteriol. 183, 6746-6751. DOI: 10.1128/JB.183.23.6746-6751.2001 TiriLLini, B., pAgioTTi, r., A ngeLini, p., p inTore, g., chessA, M., Menghini, L., 2009: Chemical composition and fungicidal activity of the essential oil of Laserpitium garganicum from Italy. Chem. Nat. Compd. 45, 103-10. DOI: 10.1007/s10600-009-9237-x TsukATAni, T., suenAgA, h., shigA, M., noguchi, k., ishiYAMA, M., ezoe, T., MATsuMoTo, k., 2012: Comparison of the WST-8 colorimetric me- 338 K.S. Mileski, A.D. Ćirić, J.D. Petrović, M.S. Ristić, V.S. Matevski, P.D. Marin, A.M. Džamić thod and the CLSI broth microdilution method for susceptibility testing against drug-resistant bacteria. J. Microbiol. Meth. 90, 160-166. DOI: 10.1016/j.mimet.2012.05.001 TuTin, T.g., 1968: Laserpitium L. In: Heywood, V.H., Burges, N.A., Moore, D.M., Valentine, D.H., Walters, S.M., Webb, D.A. (eds.), Flora Europaea, 2nd ed, 368-370. Cambridge, Cambridge University Press. Address of corresponding author: Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia E-mail: ksenija.mileski@bio.bg.ac.rs © The Author(s) 2017. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creativecommons.org/licenses/by-sa/4.0/).