J Arthropod-Borne Dis, September 2017, 11(3): 414–426 S Tavakoli et al.: Gas Chromatography … 414 http://jad.tums.ac.ir Published Online: September 08, 2017 Original Article Gas Chromatography, GC/Mass Analysis and Bioactivity of Essential Oil from Aerial Parts of Ferulago trifida: Antimicrobial, Antioxidant, AChE Inhibitory, General Toxicity, MTT Assay and Larvicidal Activities Saeed Tavakoli 1, 2, Hassan Vatandoost 3, 4, Reza Zeidabadinezhad 2, Reza Hajiaghaee 2, Abbas Hadjiakhoondi 1,2, Mohammad Reza Abai 3,4, *Narguess Yassa 1,2 1Department of Pharmacognosy, Faculty of Pharmacy and Medicinal Plant Research Center, Tehran University of Medical Sciences, Tehran, Iran 2Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran 3Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4Department of Environmental Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran (Received 21 Nov 2016; accepted 4 Sep 2017) Abstract Background: We aimed to investigate different biological properties of aerial parts essential oil of Ferulago trifida Boiss and larvicidal activity of its volatile oils from all parts of plant. Methods: Essential oil was prepared by steam distillation and analyzed by Gas chromatography and GC/Mass. Anti- oxidant, antimicrobial, cytotoxic effects and AChE inhibitory of the oil were investigated using DPPH, disk diffusion method, MTT assay and Ellman methods. Larvicidal activity of F. trifida essential oil against malaria vector Anoph- eles stephensi was carried out according to the method described by WHO. Results: In GC and GC/MS analysis, 58 compounds were identified in the aerial parts essential oil, of which E-ver- benol (9.66%), isobutyl acetate (25.73%) and E-β-caryophyllene (8.68%) were main compounds. The oil showed (IC50= 111.2µg/ml) in DPPH and IC50= 21.5 mg/ml in the investigation of AChE inhibitory. Furthermore, the oil demonstrated toxicity with (LD50= 1.1µg/ml) in brine shrimp lethality test and with (IC50= 22.0, 25.0 and 42.55 µg/ml) on three cancerous cell lines (MCF-7, A-549 and HT-29) respectively. LC50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of An. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. In antimicrobial activities, essential oil was effective on all specimens except Escherichia coli, Asper- gillus niger and Candida albicans. Conclusion: The essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and MTT assay on three cancerous cell lines. Keywords: Ferulago trifida, Larvicidal property, Antioxidant activity, Antimicrobial effect, MTT assay Introduction The use of medicinal plants to treatment of diseases and improve the health has al- ways been attractive for men. In addition to traditional medicinal purposes, the plants are of most interest to researchers because of isolation and identification of bioactive com- pounds as the lead compound in the devel- opment and production of new drugs with efficacy and safety. Ferulago trifida Boiss, from Apiaceae family is an Iranian indigenous plant and no study has been done for identification of its compounds in Iran, therefore phytochemical study on its extract, essential oil and some investigation e.g. antimicrobial and antioxi- dant effects, inhibition of the acetyl-cholin- esterase and cytotoxic properties are indis- pensable on this valuable plant. *Corresponding author: Dr Narguess Yassa, E-mail: yasa@tums.ac.ir mailto:yasa@tums.ac.ir J Arthropod-Borne Dis, September 2017, 11(3): 414–426 S Tavakoli et al.: Gas Chromatography … 415 http://jad.tums.ac.ir Published Online: September 08, 2017 Ferulago trifida is an endemic plant in Iran that grows in Qazvin Province near the village of Qvanin Alamut region. This genus consists of seven species in Iran, most of them are endemic to Iran or have spread in regions of Anatolia, Syria, Lebanon, and Iraq, they are valuable pasture plants (1, 2). In certain parts of Iran, some species of this genus are traditionally added to dairy prod- ucts, especially in the oil made of animal fats for a pleasant taste and presentation of cor- ruption (3). Different species of this plant in some parts of Turkey were used as a sedative, ton- ic, gastrointestinal pain, cure hemorrhoids etc. There is a report about the use of the es- sential oil of various species of this genus for preparation of cosmetic products (3). In re- cent years, researchers have studies on im- mune modulatory and cytotoxic effects of some medicinal plants that F. angulata (Schlecht) Boiss, is one of them (4, 5). During the studies on essential oil of F. carduchorum, antibacte- rial and antifungal effects on Staphylococcus aureus and Candida albicans as well as strong antioxidant effects have been observed (6). Chemical analysis and antimicrobial ac- tivity of essential oil of F. Bernardii shown volatile oil of the plant contains 2, 4, 5- trimethylbenzaldehyde, caryophyllene oxide, spathulenol, cis-chrysanthenyl acetate, and α-pinene. Anti-microbial activity of the plant compared to fluconazole and gentamicin rep- resents weak effects against C. albicans, As- pergillus niger, Bacillus subtilis, S. aurous and Escherichia coli. The volatile oil did not show any activity on Pseudomonas aeruginosa (7). In several studies on some species of genus Ferulago different coumarins have been iden- tified, from the two species F. capillaris and F. brachyloba, four new coumarins: (+)-3'-hy- droxyprantschimgin, (-)-3'-senecioyloxyma- rmesin, senecioyloxymarmesin and (+)-sene- cioylprangol, have been detected (8). Acetyl- cholinesterase inhibitory effects of three isolat- ed coumarins (umbelliprenin, coladonin, co- ladin), phenolic compounds, polyacetylene and daucane from F. campestris were studied; all of these compounds showed inhibitory ef- fects on AChE (9). In a study from Turkey on chloroform ex- tract of F. aucheri, two coumarins: osthenol, prantschimgin and two flavonoids: isorhamnet- in- 3- glucoside, 6-hydroxyapigenin-6-methyl ether and two new aromatic compounds: quin- olmono acetate, 1-acetylhydroquinone-4-ga- lactoside were identified (10). In this project, we studied larvicidal prop- erties against Anopheles stephensi of F. trifi- da essential oils from its different parts (flow- er, leaf, aerial parts, root, stem, and fruits), and identified the essential oil components of aerial parts. In addition, we investigated an- timicrobial, antioxidant, cytotoxic effects on Brine shrimp and three tumor cell lines, and acetylcholine esterase inhibitory effect of the volatile oil from aerial parts of F. trifida. Materials and Methods Plant material The aerial parts of F. trifida were col- lected in July 2014 from Qazvin Province near the village of Ovan in Alamut region, Iran. The plant was identified by Dr V Mozaf- farian (Research Institute of Forest and Range- lands, Tehran, Iran) and a voucher specimen was deposited in the herbarium of Faculty of Pharmacy, Tehran University of Medical Sci- ences, Tehran, Iran (No.THE-6562). Preparation of essential oils The collected plant was dried in shade and powdered, 100g of powdered plant was sub- jected to hydro-distillation with a Clevenger type apparatus for 4h. The yellow color essen- tial oil was dried over anhydrous sodium sul- fate and was kept in refrigerator until analyses. Gas chromatography and GC/Mass spec- troscopy Essential oil of the aerial parts of F. trifida J Arthropod-Borne Dis, September 2017, 11(3): 414–426 S Tavakoli et al.: Gas Chromatography … 416 http://jad.tums.ac.ir Published Online: September 08, 2017 was analyzed on an HP-6890 gas chromato- graph with an HP-5MS column (30×0.25mm id, 0.25 μmfilm thickness), equipped with HP-5973 mass detector (Ionization energy: 70 eV) under the following conditions, tem- perature program: 60 °C (0-3min), 60 °C to 250 °C at the rate of 3 °C/min (3-65 min), injector temperature: 220 °C, detector tem- perature: 290 °C, injection volume: 1.0μl, split ratio: 1:90, carrier gas: helium (Flow rate: 1ml min-1). The Kovats retention indices (KI) were calculated for all identified compounds using a homologous series of n-alkanes (C8-C24) injected under the same chromatographic con- ditions described for samples. The compo- nents of the oils were identified by compari- son of their mass spectra and retention indi- ces with Wiley library and those published in the literature (11). For quantitative analysis, essential oil was also injected to HP-6890 gas chromatograph with an HP-5MS column fitted with FID detector in conditions equal to GC/MS analysis (Table 1). Antibacterial activity Bacterial strains Antimicrobial activity of the essential oils was individually assessed against a set of sev- en bacterial strains, Gram-positive bacteria S. aureus (ATCC 29737), S. epidermidis (ATCC 12228) and Bacillus subtilis (ATCC 6633), Gram-negative bacterial Pseudomonas aeru- ginosa (ATCC 27853), E. coli (ATCC 10536), Klebsiella pneumonia (ATCC 10031), Shigella dysenteriae (PTCC 1188), Salmonella par- atyphi-A (ATCC 5702) and Proteus vulgaris (PTCC 1182), as well as tree fungi including two mold, Aspergillus brasiliensis (ATCC 1015) and A. niger (ATCC 16404) and one yeast, C. albicans (ATCC 10231), provided from Iranian Research Organization for Sci- ence and Technology (IROST). Disk diffusion assay Disc diffusion method was applied for the evaluation of antimicrobial activity of es- sential oil (12). The essential oil was filtered through 0.45µm Millipore filters for sterili- zation. 100µl of suspension containing 108 CFU/ml of bacteria, 104 spore/ml of mold and 106 CFU/ml of yeast were spread on the nutrient agar (NA), potato dextrose agar (PD) and sabouraud dextrose (SD) agar mediums, respectively. The impregnated discs (6mm in diameter) with 10µl of the essential oil were placed on the inoculated agar. The diameters of inhibition zones (IZ) (mm) were measured following incubation of all plates at 37 °C (bacteria) and at 30 °C (fungi) for 24h, gen- tamicin (10µg/disc) and rifampin (5 µg/disc) were used as positive controls for bacteria and nystatin (100I.U./disc) for fungi. Each assay was repeated twice and diameters of inhibition zones were represented as mean (Table 2). Micro-well dilution assay Essential oil was subjected to micro-well dilution assay in order to determination of minimal inhibition concentration (MIC) val- ues, for microbial strains found susceptible in disc diffusion assay (13). The suspensions of microbial strains were prepared from their 12h broth cultures at 0.5Mc Farland standard turbidity. The serial two-fold dilutions of es- sential oil sample were made in a concen- tration range from 7.8 to 500µg/ml in sterile test tubes containing brain heart infusion broth (BHI) for bacteria and sabouraud dex- trose broth (SD) for fungi strains. Ninety five µl of the cultures media and 5µl of the in- oculum were dispensed into each well of the 96-well plates. Then 100µl from essential oil dilutions was added to the wells. A well-con- taining 195µl of the cultures media and 5µl of the inoculum without the test sample were used as negative control. Gentamicin and ri- fampin for bacteria and nystatin for fungi were also used as positive control in same condi- tions as described to test samples. The con- tent of plates was mixed on a plate shaker at 300rpm for 20sec and then incubated at ap- J Arthropod-Borne Dis, September 2017, 11(3): 414–426 S Tavakoli et al.: Gas Chromatography … 417 http://jad.tums.ac.ir Published Online: September 08, 2017 propriate temperatures for 24h. Microbial growth was determined by the presence of a white pellet on the well bottom and confirmed by plating 5µl samples from clear wells on NA medium. The MIC value was defined as the lowest concentration of the plant essen- tial oil required for inhibiting the growth of microorganisms. All tests were repeated two times (Table 2). Acetylcholinesterase inhibitory assay Acetylcholinesterase inhibitory activities of the sample were determined (14), with slight modification in a 96-well microplate. Brief- ly, 125µl of 3 mMDTNB [5, 5’- dithiobis (2- nitrobenzoic acid)], 25µl of 15m MATCI (acetylthiocholine iodide), 50µl of phosphate buffer (pH 8) and 25µl of the essential oil sample solution (3mg ml-1, in methanol) were added to 96-well plates. The absorbance was recorded at 405nm in 13sec intervals for 65 sec using a TECAN microplate reader. After that, 25μL of AChE enzyme (0.22 U ml-1) was added and the absorbance was measured again in 13sec intervals for 104sec. the enzyme ac- tivity was calculated from the slope of the line obtained from plotting of the absorbance against time. Any increase in the absorbance caused by non-enzymatic hydrolysis of ATCI was corrected by the recorded absorbance be- fore addition of enzyme. Percentage of en- zyme inhibition was calculated by comparing the rates for the sample to the blank (using methanol without tested sample). Physostig- mine was used as the positive control (Table 3). Free radical scavenging assay Antioxidant capacities of the essential oil of F. trifida were determined (15). Briefly, 2.5ml of DPPH (2, 2-diphenyl-1-picrylhydra- zyl radical, Merck, Germany) solution (80µg ml-1 in methanol) was added to 2.5ml of sam- ple solutions prepared in concentrations rang- ing from 5.0 to 9.5× 10-3mg/ml in methanol, and test tubes were kept in dark for 30min at 25 °C, then UV absorptions were recorded on an Optizen 2120 UV PLUS spectropho- tometer at 517nm. BHT (Butylated hydroxy- toluene) was used as a positive control. All tests were done in triplicate and IC50 values were reported as Mean± SD (Table 3). Brine shrimp lethality test General toxicity of the essential oil was eval- uated by brine shrimp lethality test (BSLT) (16). For preparation of artificial seawater, 38g of sea salt was dissolved in 1.0L water and adjusted to pH 9 using sodium carbonate. The cysts of Artemia salina L. were hatched in sterile artificial seawater under constant aeration for 48 h at 30 °C. 50mg of essential oil were mixed with 250μl DMSO and one drop tween 80 and diluted with artificial sea- water to get 1000, 700, 500, 300, 100, 10, 2, 1, 0.5 and 0.25μg/ml concentrations in a se- ries of tubes containing about 20 active nau- plii in each. The tubes were placed in a water bath at 30 °C for 24h under light, and the sur- viving nauplii were then counted to obtain the concentration causing 50% lethality (LD50 val- ue). Podophyllotoxin, a known cytotoxic nat- ural compound, was applied as positive con- trol. The test was carried out three times and LD50 value was reported as Mean ± SD (Ta- ble 3). Cytotoxic activity Cytotoxic activities of the essential oil of F. trifida was evaluated by MTT [3-(4, 5- dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazo- lium bromide] colorimetric assay (17). Three tumor cell lines, MCF-7 (human breast ade- nocarcinoma), A-549 (non-small cell line car- cinoma) and HT-29 (human colon adenocar- cinoma) were prepared from Pasture Institute of Iran. The cell lines were cultured in Dul- becco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a 5% CO2 incubator at 37 °C. Cells were seeded into 96-well plates at a density of 0.5-1.5×104 cells/well and incu- J Arthropod-Borne Dis, September 2017, 11(3): 414–426 S Tavakoli et al.: Gas Chromatography … 418 http://jad.tums.ac.ir Published Online: September 08, 2017 bated for 24h at 37 °C. The medium was then replaced with fresh medium containing dif- ferent concentrations of essential oil and in- cubated for 72h at 37 °C. Then, the medium was changed by fresh medium containing MTT and incubated for 4h. During this period, MTT is reduced to formazan by living cells. Final- ly, the precipitated formazan crystals (purple dye) were dissolved in 200µl DMSO and de- termined at 570nm, in a TECAN microplate reader. Cytotoxic activity of the essential oil was defined as a 50% reduction in viability of cells (IC50 value). Tamoxifen was used as positive control (Table 4). Bioassays and larval mortality According to the standard method described by WHO (18), fourth instar larvae of An. ste- phensi was used for this experiment. 1ml of different concentrations (0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 ppm) of essential oil (sol- vent: ethanol) was mixed with 224ml of wa- ter. About 25 larvae in 25ml water were add- ed to the diluted essential oil. For control, only 1ml of ethanol with 224ml of water and 25 larvae in 25ml water were mixed and vol- ume of the all tests and control were 250ml (19). All of the tests and control were ex- posed for 24h with larva. The experiment was repeated four times on different days. The percentage of mortality was reported from the average for the four replicates after 24h ex- posure period. From the regression line be- tween logarithmic dose and probit mortality, the LC50 was determined. This investigation has been carried out in the insectarium of Department of Medical Entomology and Vec- tor Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Fig. 1). Statistical analysis Larvicidal activity was calculated from Mi- cro Probit software (ver. 3.0). The percent- ages of mortality were corrected for the mor- tality in controls by using Abbott's correction. From the regression line between the logarith- mic dose and probity mortality, all the pa- rameters including LC50 and slope values were determined (20). In antioxidant and inhibition of acetylcho- linesterase assays, One-way ANOVA and Tuk- ey Post-hoc multi-comparison tests were used for the analyses after data normality test. Anal- yses were performed in triplicate and the da- ta were expressed as mean ±SD. In cytotoxi- city assay, LC50 (the median growth inhibito- ry concentration) values were calculated from the LC50 of dose-response curve in the sigma plot software. Results Components of the essential oil GC and GC/MS analysis of the aerial parts essential oil were led to the identification of (86.41%) of the oil with 58 compounds. It is reach of oxygenated monoterpenes (49.30%) such as cis-verbenol, trans-verbenol (9.66%), p-mentha-1, 5-dien-8-ol (3.23%), isobornyl ac- etate (25.73%), thymol and carvacrol. E-β- caryophyllene (8.68%), Germacrene D and caryophyllene oxide are the abundant ses- quiterpenes in the essential oil (Table 1). Antimicrobial activities The results of essential oil effects on elev- en bacteria and fungi were reported as Min- imum Inhibitory Concentration (MIC) and In- hibition zone in diameter (mm) (Table 2). Ri- fampin, gentamycin, and nystatin were used as positive control against bacterial and fun- gal strains, respectively. The results of posi- tive controls (rifampin, gentamycin, and nys- tatin) and negative control (DMSO) also are shown in Table 2. Antioxidant, AChE inhibitory and Brine shrimp lethality In free radical scavenging assay with DPPH method, the volatile oil (IC50= 111.5.2µg/ml) J Arthropod-Borne Dis, September 2017, 11(3): 414–426 S Tavakoli et al.: Gas Chromatography … 419 http://jad.tums.ac.ir Published Online: September 08, 2017 showed no antioxidant activity in comparison to the BHA (IC50=21.2µg/ml). AChE inhibitory of the oil (22.5mg ml-1) versus physostigmine as standard (0.8µg ml-1) was very low. General toxicity of the oil was evaluated by brine shrimp lethality test, and the sample with LD50= 1.1µg/ml demonstrated strong toxic effect in comparison to podophyl- lotoxin with LD50= 2.8µg/ml (Table 3). Cytotoxic effect The effects of F. trifida essential oil on the proliferative response of the MCF7 (human breast adenocarcinoma), A-549 (non-small cell line carcinoma) and HT-29 (human colon ade- nocarcinoma) cell lines have been analyzed by treating the cells with different concentrations of the volatile oil and significant decrease in cell lines proliferation in comparison with tamoxifen as positive control were observed in Table 4. Larval mortality The larvicidal activity of the essential oils from aerial parts, flower, fruit, leaf, stem, and root of the F. trifida against An. stephensi larvae were examined under laboratory conditions and the results are presented in Fig. 1. Stem oil of the F. trifida was the most effective one against An. stephensi with LC50= 6.51ppm. Table 1. Essential oil components of Ferulago trifida aerial parts by Gas Chromatography Mass spectroscopy No Compounds % KI No Compounds % KI 1 Hexanal 0.47 807 30 Isobornyl acetate 25.73 1292 2 2E-hexenal 2.55 860 31 Thymol 2.35 1303 3 Nonane 0.20 900 32 Carvacrol 1.30 1311 4 Heptanal 0.34 909 33 Undecanal 0.37 1316 5 Α-pinene 0.42 935 34 Α-copaene 0.52 1381 6 Thuja-2,4(10)-diene 0.21 958 35 Geranyl acetate 0.31 1384 7 1-octen-3-ol 1.26 986 36 Β-bourbonene 0.93 1389 8 Myrcene 0.41 992 37 Z-jasmone 0.38 1406 9 Mesitylene 1.16 1000 38 (Z)-caryophyllene 0.12 1411 10 N-octanal 0.13 1011 39 (E)-caryophyllene 8.68 1427 11 P-cymene 0.22 1030 40 Β-copaene 0.23 1437 12 Limonene 0.44 1034 41 Neryl acetone 0.39 1456 13 (Z)-β-ocimene 3.33 1040 42 Α-humulene 0.81 1464 14 (E)-β-ocimene 0.56 1050 43 Γ-muurolene 0.43 1482 15 Γ-terpinene 0.39 1063 44 Germacrene D 2.18 1489 16 Terpinolene 0.14 1090 45 Β-himachalene 0.64 1505 17 Linalool 0.69 1106 46 Β-bisabolene 0.38 1514 18 N-nonanal 1.43 1113 47 Δ-cadinene 0.35 1526 19 Allo-ocimene 0.33 1133 48 E-nerolidol 0.21 1567 20 Cis-verbenol 2.55 1151 49 Geranylbutanoate 0.33 1575 21 Trans-verbenol 9.66 1156 50 Spathulenol 0.51 1590 22 P-mentha-1,5-dien-8-ol 3.22 1183 51 Caryophyllene oxide 2.47 1595 23 Terpinen-4-ol 0.36 1190 52 Tetradecanal 0.14 1621 24 P-cymen-8-ol 0.40 1199 53 1-epi-cubenol 0.14 1640 25 Α-terpineol 0.30 1206 54 Caryophylla-4(14),8(15)-diene-5-α-ol 0.29 1649 26 N-decanal 1.43 1214 55 Caryophylla-4(14),8(15)-diene-5-β-ol 0.60 1652 27 Verbenone 0.22 1221 56 Α-muurolol 0.27 1656 28 Geraniol 1.59 1258 57 Α-cadinol 0.32 1670 29 Cis-verbenyl acetate 0.29 1288 58 14-hydroxy-9-epi-(E)-caryophyllene 0.33 1686 Total Identified 84.41 J Arthropod-Borne Dis, September 2017, 11(3): 414–426 S Tavakoli et al.: Gas Chromatography … 420 http://jad.tums.ac.ir Published Online: September 08, 2017 Table 2. Antibacterial and antifungal activities of the essential oil of Ferulago trifida aerial parts Microorganisms Essential oil of aerial parts Antibiotics Rifa Genb Nysc IZd MICe IZ MIC IZ MIC IZ MIC S. paratyphi-A 20 500 - - 21 500 NAf NA S. aureus 23 500 10 250 21 500 NA NA S. epidermidis 20 125 8 250 18 500 NA NA E. coli - - 11 500 20 500 NA NA K. pneumoniae 11 250 7 250 22 250 NA NA B. subtilis 16 125 13 15 21 500 NA NA P. vulgaris 11 250 10 125 23 500 NA NA S. dysenteriae 23 125 40 250 35 500 NA NA C. albicans - - NA NA NA NA 33 125 A. brasiliensis 12 500 NA NA NA NA 23 500 A. niger - - NA NA NA NA 27 31 adash (-) indicates no antimicrobial activity. aRifampin (5μg/disc), b Gentamicin (10μg/disc), cNystatin (100 I.U. /disc), dInhibition zone in diameter (mm) around the impregnated discs including diameter of the disc (6mm) [weak activity (<10 mm), moderate activity (10–15mm), strong activity (15-20mm), very strong activity (20˂ mm)], e Minimal inhibition concentrations as μg/ml, f Not applicable. Table 3. The results of free radical scavenging, Acetylcholine esterase inhibitory and brine shrimp lethality assays of the essential oil of Ferulago trifida Samples DPPH free radical scavenging assay IC50 (µg/ml) AChE inhibitory assay IC50 (µg/ml) Brine shrimp lethality test LD50 (µg/ml) Essential oil 111.2 ± 5.2 21.5 ± 2.2 (mg ml-1) 1.1 ± 0.3 BHT 21.2 ± 2.6 NA a NA Podophyllotoxin NA NA 2.80 ± 0.3 Physostigmine 0.80 ± 0.04 NA 0.8 ±0.04 NA aNot applicable. Data are presented as the mean ± SEM of three independent experiments (P< 0.05) Table 4. The results of MTT assay of the essential oil of Ferula gotrifida on different cell lines Cell lines, IC50(µg/ml) Samples MCF7a A-549b HT-29c oil 22.0 25.0 42.55 Tamoxifen 3.6 10.7 2.50 ahuman breast adenocarcinoma, b non-small cell line carcinoma, chuman colon adenocarcinoma J Arthropod-Borne Dis, September 2017, 11(3): 414–426 S Tavakoli et al.: Gas Chromatography … 421 http://jad.tums.ac.ir Published Online: September 08, 2017 Fig. 1. Comparison of equation, regression line and lethal concentration (LC50) of essential oil of different parts of Ferulago trifida (Apiaceae) against larvae of Anopheles stephensi Discussion The aerial part of F. trifida was collected around Ovan Lake located in Alamut region, Qazvin province, Iran in July 2014. Hydro- distillation of the air-dried of aerial parts of F. trifida, yielded 1.5% (v/w) of the oil. Analysis of the volatile oil by GC and GC/ MS resulted in fifty-eight compounds, repre- senting 86.41% of the total oil. The result showed the essential oil of the aerial part was rich in monoterpenes (55.75%) that ox- ygenated monoterpenes (49.30%) were dom- inant. The main components were isobornyl acetate (25.73%) and E-verbenol (9.66%) but amount of the Z-beta ocimene was 3.33% whereas it is the abundant monoterpene in the oil of some Ferulago species (21-24, 6). In F. aucheri, F. mughlae and F. sandrasica essential oils α-pinene were the major com- pound and in essential oil of F. macroseia- dia, F. sylvatica and F. bernardii, methyl car- vacrol, p-cymene and 2, 4, 5-trimethyl ben- zaldehyde were reported as important com- ponents (22, 7). In our study, amounts of the α-pinene were only (0.42%), and there were not methyl carvacrol, p-cymene and 2, 4, 5- trimethyl benzaldehyde in the essential oil. In F. trifida there was 20.41% sesquiterpenes, which beta-caryophyllene (8.68%), Germacrene D (2.18%) and caryophyllene oxide (2.47%) were abundant. Ferulago trifida aerial part volatile oil showed strong antimicrobial activity with MIC near gentamycin against all bacterial strains except E.coli, and antifungal activity on A. brasiliensis with MIC equal nystatin. A. niger and C. albicans had shown resistance to the oil. The essential oil of Ferulago genus showed different results depending on the compounds contained in oil (7, 21, 25, 26). In our study, on the AchE inhibitory prop- erties, F. trifida oil did not show significant effect versus physostigmine as standard. We did not observe any report about AchE in- hibitory activity from essential oil of Fer- ulago genus. The antioxidant assessment of oil of the F. trifida has demonstrated it has low level free radical scavenging activity because it's IC50 (111.2µg/ml) was less than BHT (IC50= 21.2µg/ml) as standard. Such investigation has been done on some species of this genus (27). The brine shrimp lethality test is consid- ered as an inexpensive, simple, rapid and ef- fective method for preliminary assessment of toxicity and as a guide for the detection of cy- totoxic, anti-tumor and pesticidal compounds (28). Table 3 demonstrates the result of gen- era50 of l toxicity of the sample. Aerial part essential oil of the F. trifida with LD50= 1.1 µg/ml showed more toxicity against podo- phyllotoxin (IC50= 2.8µg/ml) as standard. The main sesquiterpenes of the essential oils such as β-caryophyllene, germacrene D, caryo- phyllene oxide etc. may be involved in the toxic effects of the tested essential oil (29). In this investigation, the MCF7 human breast adenocarcinoma, A-549 non-small cell line carcinoma and HT-29 human colon ade- nocarcinoma cell lines were treated with dif- ferent concentrations of F. trifida aerial parts essential oil and the cell viability were meas- ured for 24 and 72h as described in the ex- perimental part. The results of these meas- urements are shown in Table 4. Essential oil of F. trifida showed interesting toxicity with J Arthropod-Borne Dis, September 2017, 11(3): 414–426 S Tavakoli et al.: Gas Chromatography … 422 http://jad.tums.ac.ir Published Online: September 08, 2017 IC50≥40µg/ mlon cancerous cell lines, alt- hough it was not similar to tamoxifen activity. Other review on the literature for the cy- totoxic and anticancer effects of various spe- cies of Ferulago essential oil, (30) has demon- strated that the essential oil of F. carduchorum showed potential cytotoxic selectivity on T47D cell line similar to methotrexate (positive con- trol). The present study showed that the es- sential oils obtained from F. tifida different parts e.g. stem, root, aerial part, fruit and flow- er could induce 50% mortality in the larvae of An. stephensi at a very low concentrations (10.46, 22.27, 20.50, 31.93 and 79.87ppm) respectively. The essential oils of some plants such as Cymbopogon nardus, C. flexuosus, C. martini, Lavandula officinalis, Menthaarven- sis, Racinus communis, Eucalytus globules, Eugenia caryophyllus, Ocimum basilicum, Melia azardirachta, Cannabis sativa demon- strated LC50 values of 105.4, 91.4, 100.0, 83.6, 83.8, 113.0, 98.5, 96.5, 80.0, 88.5, and 27.0ppm respectively, against the larvae of the An. stephensi (31-34). There are several studies on larvicidal activities of different plants against malaria vectors in Iran (35-59, 30). Therefore, the F. tifida aerial parts essential oil has strong activity against An. stephensi. Conclusion The essential oil of F. trifida aerial parts, collected from Qazvin Province of Iran, has demonstrated some biological activities in- cluding antibacterial effects on Gram posi- tive, Gram-negative and 1 fungi, high gen- eral toxicity on the brine shrimp lethality test, cytotoxic effects on three cancerous cell lines, and has remarkable larvicidal properties on fourth instar larvae of An. stephensi. There- fore, it is worthwhile to study on the larvicidal properties of its essential oil by isolating and identifying the active components that cause larval mortality, and their field trials. Acknowledgements This research has been supported by Tehran University of Medical Sciences and Health Services grant (Grant No: 94-02-33-29402). 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