J Arthropod-Borne Dis, June 2015, 9(1): 104–115 F Golfakhrabadi et al.: Biological Activities … 104 Original Article Biological Activities and Composition of Ferulago carduchorum Essential Oil Fereshteh Golfakhrabadi 1, Mahnaz Khanavi 1,2, Seyed Nasser Ostad 3, Soodabeh Saeidnia 4, Hassan Vatandoost 5, Mohammad Reza Abai 5, Mitra Hafizi 1, Fatemeh Yousefbeyk 6, Yaghoob Razzaghi Rad 1, Ameneh Baghenegadian 1, *Mohammad Reza Shams Ardekani 1,2 1Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 2Department of Traditional Pharmacy, Faculty of Traditional Medicine and Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran 3Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran 4Medicinal Plants Research Center, Tehran University of Medical Sciences, Tehran, Iran 5Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 6Department of Pharmacognosy, Faculty of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran (Received 18 Aug 2013; accepted 28 June 2014) Abstract Background: Ferulago carduchorum Boiss and Hausskn belongs to the Apiaceae family. This plant grows in west part of Iran that local people added it to dairy and oil ghee to delay expiration date and give them a pleasant taste. The aim of this study was to investigate the antioxidant, antimicrobial, acetyl cholinesterase inhibition, cytotoxic, larvicidal activities and composition of essential oil of F. carduchorum. Methods: Acetyl cholinesterase (AChE) inhibitory, larvicidal activities and chemical composition of essential oil of F. carduchorum were investigated. Besides, antioxidant, antimicrobial and cytotoxic activities of essential oil were tested using DPPH, microdilution method and MTT assay, respectively. Results: The major components of essential oil were (z)-β-ocimene (43.3%), α-pinene (18.23%) and bornyl acetate (3.98%). Among 43 identified components, monoterpenes were the most compounds (84.63%). The essential oil had noticeable efficiency against Candida albicans (MIC= 2340 µ g ml-1) and it was effective against Anopheles stephensi with LC50 and LC90 values of 12.78 and 47.43 ppm, respectively. The essential oil could inhibit AChE (IC50= 23.6 µ l ml -1). The essential oil showed high cytotoxicity on T47D, HEP-G2 and HT-29 cell lines (IC50< 2 μg ml-1). Conclusion: The essential oil of F. carduchorum collected from west of Iran had anti-Candida, larvicidal and cyto- toxicity effects and should be further investigated in others in vitro and in vivo experimental models. Keywords: Ferulago carduchorum, Essential oil, Antimicrobial, Antiacetyl cholinesterase, Larvicidal activity Introduction Ferulago carduchorum Boiss and Hausskn (Apiaceae) known as an endemic plant of Iran, grows in west part of Iran (Mozaffarian 2007). In west of Iran, F. carduchorum has been traditionally added to dairy and oil ghee to increase hold time and give them a pleasant taste. In the past, this plant was used as natural preservative to delay expiration date of meat, too. Some species of Ferulago are benefit for remedy of digestive pains, hem- orrhoid (Sodeifian et al. 2011), disease of spleen, headache, ulcers and snake bites (Demetzos et al. 2000). Phytochemical studies on Ferulago species have led to identification of different coumarins (Ognyanov et al. 1969, Andrianova et al. 1975, Serkerov et al. 1976, Sklyar et al. 1982, De Pascual et al. 1979, Doganca et al. 1991, *Corresponding author: Dr Mohammad Reza Shams Ardekani, Email: shams@tums.ac.ir http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 104–115 F Golfakhrabadi et al.: Biological Activities … 105 Doganca et al. 1992, Ruberto et al. 1994, Jimenez et al. 2000, Khalighi-Sigaroodi et al. 2006). There are also some reports about the acetyl cholinesterase inhibitory (Dall'Acqua et al. 2010), cytotoxic (Rosselli et al. 2009), antimicrobial and antioxidant (Basile et al. 2009) activities of coumarins of F. campestris Furthermore composition of essential oil of 6 different Ferulago species (Erdurak et al. 2006, Kilic et al. 2010) and antimicrobial activity of essential oil of F. bernardii (Khalighi-Sigaroodi et al. 2005) and F. campestris (Cecchini et al. 2010) have been reported later. Alzheimer’s disease (AD) is the most com- mon and important degenerative disease of brain among the elderly and is the fourth leading cause of death in western countries (Shen et al. 2005, Zhou et al. 2008). Reports suggest that AD is caused by reduced syn- thesis of acetylcholine (Weinstock et al. 1997), so the use of acetyl cholinesterase enzyme inhibitors (AChEIs) could help improve AD (Grutzendler et al. 2001). Traditional AD drugs exhibit side effects and many efforts have been made to achieve a variety of nat- ural AChEIs with less adverse effects (Karimi et al. 2010). Furthermore, a lot of medica- tions for cancer treatment are not effective enough so, researchers are trying to find the most effective medicinal plants to treat can- cers (Rahimifard et al. 2009). Mosquitoes are important vectors in trans- mission of some human diseases such as ma- laria, dengue fever, yellow fever and filariasis, which they are substantially among the great- est health problems all over the world (James et al. 1992). Different species of Anopheles transmit malaria, filariasis and certain arbo- viruses (Sedaghat et al. 2005). Malaria is one of the most important diseases in southern Iran (Vatandoost et al. 2011). In previous reports different plants showed toxic effects against public health pests (Hadjiakhoondi et al. 2005, Hadjiakhoondi et al. 2006, Hadji- akhoondi et al. 2008a, Hadjiakhoondi et al. 2008b, Vatandoost et al. 2008) The in vitro investigation of essential oils is important to estimate their potential to use as an antibiotic or as a drug for AD, cancers, malaria and as a supplement to edible or pharmaceutical products. The aim of this study was to investigate the antioxidant, antimicrobial, acetyl cholin- esterase inhibition, cytotoxic, larvicidal ac- tivities and composition of essential oil of F. carduchorum. Materials and Methods Plant material The aerial parts of Ferulago carduchorum (Apiaceae family) were collected from Manesht mountain of Illam Province in June 2011. The longitude and latitude of Manesht Moun- tain are 33°40'60" N and 46°28'0" E. This plant is an endemic plant of Iran, which grows in west part of Iran. The plant was identified and authenticated by Mr Yousef Ajani using flora iranica (Rechinger 1978). The voucher specimen is deposited in herbar- ium Institute of Medicinal Plants (ACECR), Karaj, Iran (Herbarium number: 1450). Isolation of the volatile oil The essential oil was obtained by hyd- rodistillation using a Clevenger type appa- ratus (Advanced Technocracy Inc., India) for 4 h according to the European Pharmaco- poeia (1975) (Maisonneune1975). The aerial parts of Ferulago carduchorum (Apiaceae family) were dried under shade and pow- dered. The air-dried parts of F. carduchorum (150 g) and 1,000 ml distilled water placed in a round bottom flask (using a fire source from below) connected to a Clevenger-type apparatus. Hydrodistillation is often used to isolate non-water soluble compounds. Hyd- rodistillation is a method that the plant parts being boiled in water, using a heating source http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 104–115 F Golfakhrabadi et al.: Biological Activities … 106 from below the vessel. In the presence of boiling water volatile compounds are volat- ilized at a temperature close to 100 °C, at atmospheric pressure. The volatile materials escape in vapor form through some tubes and then are cool. The essential oil is re- moved from the top of the hydrosol. 1 ml essential oil obtained duration 1 h hydrodistillation. The oil was dried over an- hydrous sodium sulphate and kept at 4 °C in the sealed brown vial until required. 1.3 ml essential oil has been obtained from 100 gram dried plant. The oil yield of the plant was determined as 1.3% v/w. The isolation of the volatile oil has been carried out in la- boratory of Pharmacognosy, Faculty of Phar- macy, Tehran University of Medical Sci- ences, Tehran, Iran. Gas chromatography mass spectroscopy Analytical gas chromatography (GC) was carried out using a Shimadzu 15A gas chro- matograph with capillary column HP-5ms (60m×0.25mm, ft 0.25mm), carrier gas, He, split ratio 1:25 and using a flame ionization detector (FID). The column temperature was programmed at 60 °C for 3 min and then it was heated to 260 °C at a rate of 5 °C min-1 and the temperature was then kept constant at 260 °C for 15 min. Gas chromatography mass spectroscopy (GC/MS) was carried out on a HP 68900 with a HP 5973 quadruple detector, on capillary column HP-5ms (5% phenyl methyl siloxane) (60m×0.25mm, ft 0.25mm), carrier gas, He, flow rate, 1 ml min-1. The column was held at 60 °C for 3 min and programmed up to 260 °C at the rate of 5 °C min-1, then kept constant at 260 °C for 15 min. The MS was operated at 70 eV ionization energy. Retention indices were calculated using the retention time of n-al- kanes that were injected after the oil at the same chromatographic conditions. Quantita- tive data were obtained from the electronic integration of the FID peak areas. The com- ponents of the oils were identified by com- parison of their mass spectra and retention indices with Wiley library and those pub- lished in the literature (Adams 1995). DPPH radical scavenging activity The 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH) (Merck, Germany) has a maximum absorption at 517 nm which was used for the investigation of the free radical-scavenging activity of the essential oil (Yokozawa et al. 1998, Khanavi et al. 2009). Antimicrobial activity Antimicrobial activities of essential oil of aerial parts of F. carduchorum was deter- mined against both Gram-positive (Staphy- lococcus aureus ATCC 6538), Gram-nega- tive (Escherichia coli ATCC 8739, Pseudo- monas aeruginosa ATCC 9027) bacteria and a fungal strain (Candida albicans ATCC 1023) by microdilution method. Negative con- trol was prepared using dimethylsulphoxide (DMSO), which was solvent used to dissolve the essential oil. Gentamycin was used as positive control against S. aureus, E. coli, P. aeruginosa while, Nystatin was used as positive control against C. albicans. Their dilutions ranged from 10 to 0.009 μg/ml concentrations in microtitre plates. The an- timicrobial activity of essential oil was as- sessed by the agar well diffusion method. The plates were incubated at 37 oC for 24 hours for bacteria and 20–25 oC for C. albicans. Inhibition was detected by meas- uring clear zones around the wells in milli- meters. Minimum Inhibitory Concentration (MIC) of the essential oil was assessed by broth microdilution method with visible growth observed by using 96 U-shaped wells plates (NCCLS 2006). After 24 h of incu- bation at 35 oC (bacteria) and 20–25 oC (C. albicans), the microdilution plates were test- ed for the absence or presence of visible growth in comparison with that of the growth in drug-free control well. The end- point of MIC is the lowest concentration of http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 104–115 F Golfakhrabadi et al.: Biological Activities … 107 the compound at which the test strain does not demonstrate visible growth. Acetyl cholinesterase (AChE) inhibition The enzymatic activity was measured using by the method described by Aazza et al. (2011) with minor modifications. 50 µ l of buffer 0.1 M (pH 8), 25 µl of essential oil dis- solved in dimethyl sulphoxide (DMSO) with different concentrations and 25 µ l of 0.22 U/ml of AChE enzyme were mixed. After 15 min incubation at 37 °C, 25 µl of 15 mM acetyl thiocholine iodide (AChI) and 125 µ l of 3 mM 5,5’- dithiobis [2-nitrobenzoic acid] (DTNB) were added and the resulting mix- ture incubated for 30 min at room temper- ature. Absorbance of the mixture was meas- ured at 405 nm by using a microplate reader (ELX808, BioTek, USA). The inhibitory ef- fect of test compound was calculated by com- paring to the negative control: %= [(A0–A1)/ A0]* 100 where A0 was the absorbance of the blank sample and A1 was the absorbance of the sample. The test was repeated three times. The in- hibition of enzyme activity was expressed as IC50 (the concentration of the sample (µ l ml - 1), required to inhibit 50% of enzyme), cal- culated by a linear regression analysis. Bioassays and larval mortality Fourth instar larvae of Anopheles stephensi Bandar-Abbas strain was exposed to test concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 ppm of essential oil (solvent: Eth- anol) for 24 hours according to standard method described by WHO (1981). In brief- ly 1 ml of appropriate dilution of essential oil with 224 ml of water and 25 larvae in 25 ml water mixed and total volume was 250 ml (Dharmagadda et al. 2005). For control, only 1 ml of ethanol with 224 ml of water and 25 larvae in 25 ml water mixed and total volume was 250 ml. The experiment was repeated four times on different days. The percentage of mortality was reported from the average for the four replicates after 24 hour exposure period. From the regression line between logarithmic dose and probit mor- tality, the LC50 was determined. The inves- tigation of larvicidal activity has been car- ried out in the insectarium of Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Cytotoxicity assay The colon carcinoma (HT-29), breast car- cinoma (T47D), hepatocellular carcinoma (HepG2) was obtained from Pasture Institute of Iran, Tehran, Iran. The colon carcinoma HT-29 and T47D (breast carcinoma) cell lines were mentioned as exponentially growing cultures in RPMI 1640 cell culture medium (PAA, Germany), supplemented with 10% fetal bovine serum (FBS: Gibco, USA), for HT-29 cells and 15% FBS for T47D cells. The hepatocellular carcinoma (HepG2) were cultured in Dulbecco’s modified Eagle’s me- dium (DMEM, PAA, Germany) supplement- ed with 10% FBS. The Swiss mouse embryo fibroblast (NIH 3T3) cell line was kept in Dulbecco’s modified Eagle’s medium (DMEM, PAA, Germany) supplemented with 10% FBS. 100 IU/ml penicillin and 100 μg/ml strep- tomycin (Roche, Germany) were added to the media. All the cell lines were cultured at 37 °C in air /carbon dioxide (95:5) atmosphere. Cytotoxic activity was measured using modified MTT assay (Newman DJ 2007). 1×104 cells/well were plated in 96-well plates (Nunc, Denmark) and incubated for 24 h be- fore the addition of drugs. After 48 h of incubation in HT-29, NIH/3T3 and MCF-7 cells, 20 μl of MTT (Merck, Germany) re- agent (5 mg/ml) in phosphate buffered saline (PBS) was added to each well. The plates were incubated at 37 °C for 4 h. The me- dium was discharged and the formazan blue, which had been formed in the cells, were dissolved with 100 μl dimethyl sulphoxide (DMSO). After the incubation at 37 °C for http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 104–115 F Golfakhrabadi et al.: Biological Activities … 108 10 min, absorbance at 570 nm at the dis- solved solutions was detected using a micro plate reader (Anthos, Austria). The cell via- bility in MTT assay was calculated as the percentage of control value (Khanavi et al. 2012b). Methotrexate was used as the posi- tive control. Cytotoxicity was expressed as the concentration of extract inhibiting cell growth with 50% (IC50±SD). All tests and analysis were run in triplicate. Statistical analysis In antioxidant and antiacetyl cholinester- ase assays, analyses were carried out in trip- licate and the data were expressed as mean ±SD. One-way ANOVA and Tukey post-hoc multicomparison tests were used for the anal- yses after data normality test. In cytotoxicity assay, IC50 (the median growth inhibitory concentration) values were calculated from the IC50 of dose-response curve in the sigma plot 11 software. Data representative of three independent experiments with similar results were presented as mean ± SD. For larvicidal activity was used from MicroProbit software (version 3.0). The percentages of mortality were corrected for the mortality in controls by using Abbott's correction. From the re- gression line between the logarithmic dose and probit mortality, all the parameters in- cluding LC50, LC90, confidence interval (CI) and slope values were determined (Abbott 1925). Results Chemical composition of the essential oil The results of essential oil analysis of F. carduchorum were led to identification of 43 compounds (Table 1), represented 92.3% of the total oil. Gas chromatography-Mass dia- gram of F. carduchorum essential oil showed in figure 1. The oil yield of the plant was determined as 1.3% v/w. Major component of essential oil were identified as (Z)-β- ocimene (43.3%), α-pinene (18.23%), bornyl acetate (3.98%) and myrcene (3.15%). Antioxidant and antimicrobial activities In this research radical scavenging activ- ity of essential oil of F. carduchorum was determined and IC50 value was calculated as 29.61 µ l. Vitamin E also used as a reference and positive control compound. In our antimicrobial investigation, the re- sults of Minimum Inhibitory Concentration (MIC) are shown in Table 2. The results of gentamycin and nystatin were used as posi- tive control against bacterial and fungal strain, respectively. The results of positive (gentamycin and nystatin) and negative (DMSO) controls are shown in Table 2. Acetyl cholinesterase Inhibition Activity Acetyl cholinesterase inhibition activity of F. carduchorum essential oil was studied for the first time. The results showed that the IC50 of essential oil was 23.6 µ l ml -1. Larval mortality The larvicidal activity of our essential oil and methanol against Anopheles stephensi larvae under laboratory conditions are pre- sented in Table 3. Essential oil of F. carduchorum was effective against An. stephensi with LC50 and LC90 values of 12.78 and 47.43 ppm, respectively. Cytotoxicity effect The effects of F. carduchorum essential oil on the proliferative response of the HT- 29, HepG2 and T47D cell lines have been analyzed by treating the cells with different concentrations of the essential oil and sig- nificant decrease in cell lines proliferation were observed. The results of cytotoxic tests have been shown in Table 4. http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 104–115 F Golfakhrabadi et al.: Biological Activities … 109 Table 1. Chemical composition of essential oil of Ferulago carduchorum No. Component RTa RIb Composition (%) 1 α-Pinene 10.133 937 18.23 2 Camphene 10.403 946 1.63 3 Verbenene 10.485 958 Trace 4 β-Pinene 11.443 971 1.81 5 Myrcene 12.306 989 3.15 6 α-Phellandrene 12.697 997 0.4 7 p-Cymene 13.654 1017 0.21 8 o-Cymene 13.500 1020 0.61 9 -3-Carene 13 1029 0.25 10 β-Phelandrene 13.819 1031 2.07 11 (Z)-β-Ocimene 14.969 1045 43.3 12 (E)-β-Ocimene 15.47 1055 2.71 13 -Terpinene 16.807 1062 1.82 14 Terpinolene 16.917 1088 0.16 15 Linalool 17.236 1100 0.16 16 1,3,8-para-Menthatriene 18.155 1115 Trace 17 allo-Ocimene 18.744 1129 2.29 18 cis-Verbenol 18.904 1135 0.26 19 neo- allo-Ocimene 18.799 1150 Trace 20 trans-Verbenol 19.162 1156 1.01 21 p-Mentha -1,5-dien-8-ol 20.043 1165 0.39 22 4-Terpineol 20.516 1170 0.13 23 α –Terpineol 21.159 1180 0.06 24 Dodecane 22.234 1200 1.02 25 Bornyl acetate 25.446 1277 3.98 26 α - Cubebene 33.892 1366 2.65 27 α -Ylangene 31.025 1370 0.16 28 α -Copaene 29.649 1378 0.36 29 β–Bourbonene 29.952 1382 0.43 30 β -Cubebene 30.156 1389 0.14 31 β -Elemene 30.271 1399 0.14 32 α -Gurjunene 36.654 1421 0.32 33 -Elemene 31.944 1428 0.1 34 Aromadendrene 31.306 1433 0.31 35 α -Humulene 32.659 1446 Trace 36 trans- β-Farnesene 33.017 1452 Trace 37 Germacrene-D 33.198 1475 Trace 38 Bicyclogermacrene 34.431 1498 0.68 39 β - Bisabolene 35.047 1500 0.15 40 -Cadinene 35.482 1513 0.5 41 Spathulenol 37.424 1568 0.71 42 Salvial-4(14)-en-1-one 37.754 1589 Trace 43 α-Cadinol 39.862 1650 Trace Total 92.3 Monoterpenes hydrocarbons 78.64 Monoterpenes oxygenated 5.99 Sesquiterpenes hydrocarbons 5.94 Sesquiterpenes oxygenated 0.71 Nonterpenoids 1.02 Notes a Retention time, bRI Retention Index on HP-6890 with reference to n- alkanes injected after the oil at the same chromatographic conditions. http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 104–115 F Golfakhrabadi et al.: Biological Activities … 110 Table 2. Minimum inhibitory concentration (MIC) of Ferulago carduchorum essential oil against selected bacteria and Candida albicans MICa Positive control Positive control Negative control Microorganism Essential oil Gentamycin Nystatin DMSO Staphylococcus aureus 9000 0.62 - - Escherichia coli 23000 0.009 - - Pseudomonas aeruginosa 18000 2.48 - - Candida albicans 2340 - 0.02 - Notea: MIC was determined by broth micro dilution method and expressed in µ g ml-1. - No effect. Table 3. Probit regression line of Anopheles stephensi exposed to different interval concentrations of essential oil of Ferulago carduchorum Intercept Slope±SE* LC50 (ppm) 95% CI LC90 (ppm) 95% CI χ2 χ2table (df) p-value -2.4906 1.9303± 0.323 12.7818 19.5101– 33.4434 47.4356 89.9890– 357.8734 35.78 * 22.458 (6) 0.001 *: No heterogeneity; SE: Standard Error, LC50: Lethal Concentration to cause 50% mortality in population; LC90: Lethal Concentration to cause 90% mortality in population, CI: Confidence Interval, χ2 (df) = heterogeneity about the regression line (Degrees Of Freedom). Table 4. Cytotoxic activity of essential oil of Ferulago carduchorum Cell Linesa (MTT assay) Essential oil Methotrexate Doxorubicin NIH-3T3 0.64±0.02 0.24 ± 0.013 0.21 ± 0.03 HT-29 1.74±0.14 0.23 ± 0.02 - T47D 0.17±0.01 0.16 ± 0.09 - HEP-G2 0.35±0.02 - 1.04 ± 0.07 a: Results are expressed as IC50 values (μg ml -1), Key to cell Lines employed: HT-29 (colon carcinoma), T47D (breast carcinoma), HEP-G2 (hepatocellular carcinoma), NIH 3T3 (Swiss embryo fibroblast). Fig. 1. Gas chromatography-Mass diagram of Ferulago carduchorum essential oil http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 104–115 F Golfakhrabadi et al.: Biological Activities … 111 Discussion Among 43 identified components, mono- terpenes were the most identified compounds (84.63%) that only 5.99% of them were ox- ygenated whereas, sesquiterpenes were to- tally detected about 6.65% with 0.71% ox- ygenated sesquiterpenes. (Z)-β-ocimene and myrcene as acyclic monoterpene hydrocar- bons and α-pinene as a cyclic monoterpene hydrocarbon were identified as main com- pounds. Moreover, the major oxygen contain- ing monoterpene and sesquiterpene are bornyl acetate (3.98 %) and spathulenol (0.71 %), respectively. Composition of the oil obtained from air-dried aerial parts of F. carduchorum from Kerman Province, was found to contain (z)-β-ocimene as the major component (Samiee et al. 2006), which agrees with our research. The amount of (z)-β-ocimene in our investi- gation (43.3 %) was more than previous study (21.2 %). More ever F. carduchorum from Illam and Kerman Provinces had shown the constituent of α-pinene 18.23 % and 4.8 % respectively. Moreover, other reports showed that in F. humillis, F. trachycarpa and F. angulata (z)-β-ocimene is the main compo- nent (Baser et al. 2002, Khanahmadi et al. 2006). The important compound in three species (F. aucheri and F. mughlae and F. sandrasica) was α-pinene. The major com- ponents in essential oil of F. macroseiadia, F. sylvatica and F. bernardii were methyl carvacrol, p-cymene and 2, 4, 5-trimethyl benzaldehyde, respectively (Baser et al. 2002, Khalighi-Sigaroodi et al. 2005). Evaluation of MIC of the essential oil showed that sample had antimicrobial effect against both Gram-positive (Staphylococcus aureus), Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria and a fun- gal strain (Candida albicans). The essential oil indicated noticeable efficiency against C. albicans (MIC= 2340 µ g ml-1). The antioxidant assessment of fruits and roots of F. campestris were demonstrated that their IC50 were less than IC50 of essen- tial oil of F. carduchorum (Cecchini et al. 2010). Moreover, antimicrobial effect of F. campestris was better than F. carduchorum, that MIC of roots and fruits essential oils were less than MIC of F. carduchorum es- sential oil (Cecchini et al. 2010). The results of antimicrobial activity of F. bernardii es- sential oil were shown MIC of essential oil againt Gram-positive, Gram-negative bacte- ria and fungal strain were <1000 µ g ml-1 (Khalighi-Sigaroodi et al. 2005). So, antimi- crobial activity of F. bernardii is better than F. carduchorum. Anti-candida effect of Ferulago capillaris essential oil was investi- gated and the results were demonstrated that MIC is less than F. carduchorum (Pinto et al. 2013). There have been reports on the AchE inhibitory activity of some bicyclic monoterpenoides including α-pinene and 3- carene (Miyazawa et al. 2005). Hence in our study, AchE inhibitory activity of essential oil could be attributed to its α-pinene. In a previous study, larvicidal activity of Eucalyptus camaldulensis essential oil against An. stephensi was investigated and the LC50 and LC90 values were 89.85 and 215.26 ppm, respectively (Sedaghat et al. 2010). In one study, the larvicidal activities of methanolic extracts of some Iranian plants (Lawsonia inermis, Thymus kotschyanus, Cedrus deodara and eight species from Stachys) against ma- laria vector, An. stephensi were investigated (Khanavi et al. 2013). These results indi- cated that essential oil of F. carduchorum was more potent than these plants against An. stephensi. Compared to another studies on larvicidal activity of essential oils of Heracleum persicum, Foeniculum vulgare, Coriandrum sativum (Sedaghat et al. 2011), Cymbopogon olivieri (Hadjiakhoondi et al. 2003) and Nepeta menthoides (Khanavi et al. 2012a) it was found that F. carduchorum was most effective against An. stephensi http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 104–115 F Golfakhrabadi et al.: Biological Activities … 112 (Khanavi et al. 2011). Therefore, the essen- tial oil of F. carduchorum can be used in insect control as an alternative for chemical compounds on the environment. The essential oil of F. carduchorum showed a high cytotoxicity on T47D, HEP- G2 and HT-29 cell lines (IC50< 2 μg ml-1). The effect of essential oil on T47D cell line was much stronger than HT-29 and HEP-G2. It indicated that the essential oil had poten- tial cytotoxic selectivity on T47D cell line similar to Methotrexate (positive control), whereas, the essential oil had lower cyto- toxic effect than Methotrexate on NIH 3T3 (Swiss embryo fibroblast). So, the oil could be safer than this drug. Another study on Vinca minor revealed that its cytotoxicity activity on T47D (IC50= 1.34±0.29) and HT- 29 (IC50= 3.63±1.24) was less than F. carduchorum (Khanavi et al. 2010). In re- sults, this plant has active cytotoxic compo- nents. Conclusion The essential oil of F. carduchorum col- lected from west of Iran give good biological activity including anti-Candida, AchE in- hibitory, larvicidal and high cytotoxic effects and should be further investigated in others in vitro and in vivo experimental models. The determined biological activities of F. carduchorum essential oil are important in- formation for its future application in treat- ment. Furthermore, the determination of an- tioxidant and antibacterial activity of the es- sential oil have great importance in preser- vation of food products and pharmaceutical industries. Acknowledgements This research has been supported by Teh- ran University of Medical Sciences (Grant No: 90-02-33-13855). The authors declare that there is no conflict of interests. References Aazza S, Lyoussi B, Miguel MG (2011) An- tioxidant and antiacetyl cholinesterase activities of some commercial essential oils and their major compounds. Mole- cules. 6: 7672–7690. 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