Essential Oil Constituents of Tanacetum cilicicum : Antimicrobial and Phytotoxic Activities

Aerial parts ofTanacetum cilicicumwere hydrodistillated for 3 h using Clevenger. Essential oil (EO) yield was 0.4% (v/w). According to the GC/MS analyses, EO of T. cilicicum consisted of monoterpenes [α-pinene (2.95 ± 0.19%), sabinene (2.32 ± 0.11%), and limonene (3.17 ± 0.25)], oxygenated monoterpenes [eucalyptol (5.08 ± 0.32%), camphor (3.53 ± 0.27%), linalool (7.01 ± 0.32%), α-terpineol (3.13 ± 0.23%), and borneol (4.21 ± 0.17%)], and sesquiterpenes [sesquisabinene hydrate (6.88 ± 0.41%), nerolidol (4.90 ± 0.33%), α-muurolol (4.57% ± 0.35), spathulanol (2.98 ± 0.12%), juniper camphor (2.68 ± 0.19%), (-)-caryophyllene oxide (2.64 ± 0.19%), 8-hydroxylinalool (2.62 ± 0.15%), and Δ-cadinene (2.48 ± 0.16%)]. In the antimicrobial assay, MIC/MBC values of the EO were themost significant on B. subtilis (0.39/0.78 μL/mL) and B. cereus (0.78/1.56 μL/mL).Themost prominent phytotoxic activities of the EO were observed on L. sativa, L. sativum, and P. oleracea. The results of the present study indicated that EO of T. cilicicum includes various medicinally and industrially crucial phytoconstituents that could be in use for industrial applications. The finding of this study is the first report on this species from the East Mediterranean region.

Essential oils of aromatic and medicinal plants have been receiving increasing attention in many industries such as medicine, agriculture, food, and cosmetics [25].In nature, many plants have been still awaiting for the exploration of their bio-benefits.To date, there has been no previous study indicating the chemical composition as well as antimicrobial and phytotoxic activities of T. cilicicum from the East Mediterranean region.Hence, the essential oil composition as well as bioactivities of the aerial parts of T. cilicicum could be the first report.[16] T. chiliophyllum EO yield: 0.22% Main components: camphor (17.9), 1,8-cineole (16.6), borneol (15.4), dihydro--cyclogeranyl pentanoate (3.0), dihydro--cyclogeranyl hexanoate (10.1) [16] T. alyssifolium Main components: borneol (35.2), -thujone (24.6), camphor (12.4), -eudesmol (6.E).Essential oil (EO) from air-dried aerial parts of T. cilicicum was obtained with Clevenger for 3 h distillation period, which was followed by calculating the oil yield (v/w).A total of 100 g plant material was used during each distillation cycle.The blue coloured EO was dried with anhydrous sodium sulphate to remove the water from the distillate and then preserved in amber vials at +4 ∘ C for further analyses.

Analyses of the EO of T. cilicicum Using Gas Chromatography and Mass Spectrometry (GC/MS).
The characterization of the compounds in the EO was obtained with Gas Chromatography and Mass Spectrometry.The conditions used during the analyses were described in Table 2 [26].n-Alkane mixture was used to determine and calculate the linear retention index (Kovats Indices) of each compound in the EO under the same temperature programme used for the analyses [27].Mass spectra of the EO constituents were compared with those of the references documented in NIST (National Institute of Standards and Technology, 2013, Gaithersburg, MD, USA) and Wiley database.Tentative identification as well as the retention indices of the compounds in this assay was compared with those of NIST.In addition, quantitative analyses of the EO constituents (% area) as average of 3 repeated analytical assays were carried out with the measurement of the peak space normalization.27) juniper camphor (the peak numbering given here is not in accordance with peak numbers in Table 3).were used in the direct-contact assay of the essential oil [30,31].Two round filter papers were placed into each glass petri dish (90 mm in diameter) and then sterilized at 121 ∘ C for 15 min.Afterwards, sterilized seeds with aqueous sodium hypochlorite solution (1.5%, v/v) for 10 min were aseptically placed onto double layered membranes [32].EO of T. cilicicum was dissolved in 0.5% Tween 80 at the concentrations from 0.062 to 2.0 mg/mL.A total of 10 mL from each concentration was aseptically placed to lower layer of the filter paper by using a sterile glass pipette.Throughout the assay, control did include only distilled water.All petri plates were sealed with parafilm.Treated petri dishes as well as the control groups were maintained for 7 days at 24 ∘ C, 12/12 h, 1.500 lux light/dark period, and about 80% relative humidity.At the final day of maintenance, germinated seeds were counted and then recorded.The length of seedling growths was measured as mm.Glyphosate N-(phosphonomethyl) glycine was tested as the commercial herbicide.

Statistical Analysis.
Assessment of the test results was carried out using SPSS17 programme.Variance analyses (ANOVA) of the results of the test parameters were performed.Tukey Multiple Comparative test was employed to determine the differences in test parameters.The mean and standard deviations of the results were calculated.The statistical differences were indicated as a superscript in the mean values.All assays in this study were carried out in triplicate.1).It seemed that yield and composition differed from previous reports owing to the species differences belonging to this genus.

Antimicrobial Activity Results of the EO of T. cilicicum.
Antibiotics raise serious concerns in relation to antimicrobial resistance problems for all group of organisms.Therefore, natural sources of the environments have been intensively studied by many scientists to combat their undesirable effects to the health of living organisms and their environments as well [33][34][35].In this study, EO of T. cilicicum was tested on twelve microorganisms, with two methods: disc diffusion and macrobroth dilution.In addition, standard antibiotics were used for comparison during the assays.As shown in Table 4, the results of the disc diffusion assay indicated that the most sensitive microorganisms were B. subtilis and S. aureus 29213.This was followed by B. cereus = S. aureus BAA > E. faecalis > E. casseliflavus.The susceptibilities of the Gramnegative bacteria towards the essential oil were less than Gram-positive bacteria.The most susceptible Gram-negative bacterium was E. hormaechei > E. coli > K. pneumonia = P. aeruginosa.In addition, C. parapsilosis was also more susceptible than that of C. albicans.3.12 ± 0.00 ab 12.5 ± 0.00 c --300 ± 0.00 a >300 ± 0.00 a C. albicans ATCC 14053 1.56 ± 0.00 a 6.25 ± 0.00 b -->300 ± 0.00 a >300 ± 0.00 a In each column, small case letters as the superscript in the columns showed the statistical differences ( < 0.05).-indicates not tested.MIC: Minimum Inhibitory Concentration; MFC: Minimum Fungicidal Concentration; AMP: Ampicillin; CLO: Clotrimazole.

Phytotoxicity Assay of T. cilicicum. As shown in Table
None of the doses of the herbicide revealed any inhibitory effects on the seed germination of P. oleracea.As the EO, lower concentrations of the herbicide ranging from 0.062 to 0.25 mg/mL had no inhibitory effect on the seed germination of L. sativa and L. sativum.At 0.50 mg/mL and higher doses, herbicide inhibited the seed germination by 10, 10, and 20% in L. sativa and by 13, 13, and 13% in L. sativum, respectively.It appeared that the inhibitory effects of the essential oil on the germination of test seeds seem to be more noteworthy than those of the herbicide.

Effects on the Seedling Growth.
In the agriculture, herbicides have been utilised in the fields to overcome undesirable attack of various organisms to crops.Detrimental effects of these chemicals are not questionable anymore because of long term negative effects on the soil structure and community [36,37].Natural compounds are very attractive constituents to overcome the negative effects of the synthetic chemicals.In this study, inhibitory effects at the lowest dose of the EO (0.0625 mg/mL) were not observed on the radicle growth of L. sativa seeds.Two-fold increase onwards showed decreasing effects on the radicle.The most significant decreasing effects were 95% at 1.0 mg/mL and 100% at 2.0 mg/mL, respectively.Plumules of L. sativa were also inhibited significantly by 85% at 0.50 mg/mL; however, a complete inhibition was observed at 1.0 and 2.0 mg/mL.Herbicide in this assay was not as effective as the EO at the same doses (Table 6).A complete inhibition was not observed on neither the length of radicle nor the plumule of L. sativa.Herbicide seemed to have more inhibitory effects on the radicle growth of L. sativa rather than the plumule growth at 0.0625-2.0mg/mL.It appeared that herbicide was not found to be as effective as the essential oil.
EO revealed significant inhibitory effects on radicle growth of L. sativum by 93% at 0.50 mg/mL, 98% at 1.0 mg/mL, and 99% at 2.0 mg/mL.It was also found to be very active on the plumule growth of L. sativum by 91% at 0.50 mg/mL onwards.A complete inhibition of the plumule growth of L. sativum was observed at 1.0 and 2.0 mg/mL.It seemed that EO seemed to be more effective on the plumule growth of L. sativum.Compared to the EO, herbicide revealed similar inhibitory effects on the radicle growth, whereas inhibition on both the radicle and the plumule was not effective as the EO.
EO of T. cilicicum inhibited the growth of P. oleracea at all doses; however, the most significant inhibitory effects on the radicle growth of P. oleracea were observed at 0.5 mg/mL onwards.At 0.50, 1.0, and 2.0 mg/mL, inhibitions of the radicle were 95%, 97%, and 99%, respectively.The plumule growth of P. oleracea was also inhibited by 94% at 0.50 mg/mL; however, a total inhibition was observed at 1.0 and 2.0 mg/mL of the EO doses.The present findings indicated that effects of the EO were more prominent on the plumule growth.Unlike EO, different doses of the herbicide had no inhibitory effect on neither the radicle nor the plumule growth of P. oleracea; however, inhibitory effects of the herbicide were more prominent on radicle growth rather than plumule growth of P. oleracea.
In a previous study, the bioherbicidal potential of two species of the genus Tanacetum has been reported by Salamci et al. (2007), who found that EO of T. aucheranum and T. chiliophyllum at the dose of 30 L per petri receiving 50 test seeds completely inhibited the seed germination, radicle, and plumule growth of A. retroflexus, C. album, and R. crispus.The findings of the previous and the present results indicated that Tanacetum species has active bioherbical constituents and deserves to be studied in more detail in advance.

Conclusion
In the present study, EO of T. cilicicum includes significant amount of antimicrobial compounds against a wide range of microorganisms and therefore, it could be used in food, agricultural, and pharmaceutical applications and so forth.Furthermore, significant phytoconstituents of the EO from T. In the same column, different letters showed significant statistical differences for the applied doses of the essential oil ( < 0.05).
cilicicum could be suggested for use in the various formulation of biopesticides.However, further studies are required for isolating the bioactive constituents in the EO and testing alone and/or their synergistic, antagonistic relationships as well as determining their toxic effects on test organisms before potential industrial benefits.

Table 3 :
Essential oil composition of T. cilicicum.
parts of T. cilicicum was 0.4% (v/w).In previous reports, essential oil yield and chemical constituents of Tanacetum sp. were reported in different species belonging to genus Tanacetum (Table

Table 4 :
Antimicrobial activities of the EO from T. cilicicum using disc diffusion assay.
EO: essential oil; SXT5: Trimethoprim/Sulfamethoxazole; VA30: Vancomycin; TEC30: Teicoplanin; NS 100 IU: Nystatin.The small case letters as the superscript in the columns and capital letters as the superscript in the columns showed the statistical differences in each column and row, respectively ( < 0.05).indicatesnot tested.

Table 5 :
Minimum Inhibitor Concentration and Minimum Bactericidal/Fungicidal Concentration values of T. cilicicum essential oil.

Table 6 :
Effects of the essential oil from T. cilicicum on the seed germination (%), radicle, and plumule growth of L. sativa, L. sativum, and P. oleracea.