Anti–Oxidative and Anti–Inflammatory Effects of Tagetes Minuta Essential Oil in Activated Macrophages

Asian Pac J Trop Biomed 2014; 4(3): 219-227 219

Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Biomedicine

journal homepage: www.elsevier.com/locate/apjtb

Document heading doi:10.1016/S2221-1691(14)60235-5 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved. 襃 Anti-oxidative and anti-inflammatory effects of Tagetes minuta essential oil in activated macrophages

1 1 2 Parastoo Karimian , Gholamreza Kavoosi *, Zahra Amirghofran 1Biotechnology Institute, Shiraz University, Shiraz, 71441-65186, Iran 2Department of Immunology, Autoimmune Disease Research Center and Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

PEER REVIEW ABSTRACT

Peer reviewer Objective: Tagetes minuta T. minuta To investigate antioxidant and anti-inflammatory effects of ( ) Hasan Salehi, University of Shiraz, essentialMethods: oil. T. minuta T. minuta Shiraz, Iran. In the present study essential oil was obtained from leaves of via E-mail: [email protected] hydro-distillation andT. minutathen was analyzed by gas chromatography-mass spectrometry. The anti- Comments oxidant capacity of essential oil was examined by measuring reactive oxygen,T. reactive minuta nitrogen species and hydrogen peroxide scavenging. The anti-inflammatory activity of TMO displayed an anti-oxidant essential αoil was determined through measuring NADH oxidase, inducible nitric oxide synthase property by scavenging superoxide, and TNF- mRNA expression in lipopolysacharide-stimulated murine macrophages using real- H2O2 and NO radicals, and reduced Results:time PCR. G oxidative stress. The decreased T. minutaas chromatography-mass spectrometry analysis indicated that the main components in ROS NOS (33 86%) E (19 92%) (16 15%) formation of and radicals in the β essential oil were dihydrotagetone . , -ocimene . , tagetone . T., (7 94%) Z (5 27%) (3 1%) (2 03%) macrophages was possibly due to the cis-minuta-ocimene . , -ocimene . , limonene . and epoxyocimene . . The radical scavenging activity of phenolic essential oil hadμ the ability to scavenge all reactive oxygen/reactive nitrogen species 50 12 15 groups present in the oil and/or due T.radicals minuta with IC - g/mL, which indicated a potent radical scavenging activity. In addition, NOS NOX NADH to an inhibition of i and α essential oil significantly reduced oxidase, μinducible nitric oxide synthaseand gene expressions. Furthermore, TMO TNF- mRNA expression in the cells at concentrations of 50 g/mL, indicating a capacity of this decreased the expression of the genesα productConclusions: to potentiallyT. minuta modulate/diminish immune responses. for pro-inflammatory cytokine TNF- . essential oil has radical scavenging and anti-inflammatory activities Details on Page 226 and could potentially be used as a safe effective source of natural anti-oxidants in therapy against oxidative damage and stress associated with some inflammatory conditions. KEYWORDS Tagetes minuta , Essential oil, Macrophages, Anti-inflammatory, Antioxidant

1. Introduction goods, confectionery, dairy products, ice cream, yogurt, citrus [1-3] T. minuta Tagetes minuta T. minuta juice, mustard and as colorant in poultry feed . is ( ) is a Tagetestall upright marigold plant in also extensively used medicinally as a condiment and herbal the sunflower (Asteraceae) family. species originally has tea in a wide variety of fields in its native region and as a ( been used as a source of essential oil extracted from leaves, popular traditional folkT. remediesminuta and in the complementary ) stalks and flowers for theTagetes flavoring in the food industries. The and medical therapy. has several medical benefits powders and extracts of are rich in the orange-yellow such as remedy for colds, respiratory inflammations, stomach carotenoid and are used as a food color for foods such as pasta, problem, anti-spasmodic, anti-parasitic, anti-septic, vegetable oil, margarine, mayonnaises, salad dressing, baked insecticide and sedative. It is used for chest infections, coughs

*Corresponding author: Gholamreza Kavoosi, Biotechnology Institute, Shiraz Article history: University, P.O.71441-65186, Shiraz, 71441-65186, Iran. Received 1 Jan 2014 Tel: +98-711-2272805 Received in revised form 10 Jan, 2nd revised form 16 Jan, 3rd revised form 22 Jan 2014 E-mail: [email protected] Accepted 26 Feb 2014 Foundation Project: Supported by the funding from Shiraz University (Grant no. 88- Available online 28 Mar 2014 GR-AGRST-108) and Shiraz University of Medical Science (Grant No. 3937). Parastoo Karimian et al./Asian Pac J Trop Biomed 2014; 4(3): 219-227 220 Escherichia coli and catarrh, dilating the bronchi, facilitating the flow of mucus 0111; B4) were purchased from Sigma-Aldrich and dislodging congestion and can be used in cases of skin (St, Louis, MO, USA) and Fluka (Heidelberg, Germany). RNX- infections. It also has a healing effect on wounds, cuts, calluses Plus buffer was obtained from Cinagen (Tehran, Iran). All other and bunions[4-9]. However, such practices are largely based on used chemicals and reagents were of the purest commercially folklore and train of traditional medicine rather than evidence- available products. based research. T. minuta 2.2. Plant materials and T. minuta essential oil preparation The most abundant components in essential ( oil are dihydrotagetone unsaturated acyclic monoterpene T. minuta ketone), ocimene (unsaturated acyclic monoterpene Seed of was obtained from Institute of Medicinal hydrocarbon), tagetone (unsaturated acyclic monoterpene Plants, Isfahan, Iran and was grown in green house conditions ) ( ketone and limoneneT. minutaunsaturated monocyclic monoterpene in Sadra near Shiraz, Iran. Seed ofT. medicinalminuta plant was hydrocarbon[10-14]. essential oil has a significant grown in sterile soil. The Seeds of were sown in antibacterial activity against both Gram-positive and experimental greenhouse, in September 2011. One month later [15-17] Gram-negative bacteria . SeveralT. minuta studies have also obtained seedlings were transferred to experimental field and described antifungal activities of essential oil distributed homogenously. The aerial parts of plants were Candida Penicillium Aspergilus [18-20] T. againstminuta , and species . harvested at the flowering stage. The leaves of the plants were 72 essential oil has been shown to possess anti-oxidant’ separated from the stem and were dried in the shade for activity in 2, 2-diphenyl-1-picrylhydrazyl and 2, 2 -azino-di h. The air-dried leaves (100 g) were hydro-distilled for 3 h (3-ethylbenzthiazoline-6- sulphonate) (ABTS) assay[21,22]. using an all-glass Clevenger-type apparatus (Herbal Exir Co., Advances in chemical and pharmacological evaluations Mashhad, Iran) according to the method outlined by the British T. minuta [23] T. minuta of essential oil have occurred in the paste.g. recent Pharmacopeia . The yield of essential oil from ( 1% ( ) years; however, several useful features of this plant the leaf material was near w/w . The obtained essential oil was° 4 mechanisms underlying its antioxidant and anti-inflammatory dehydrated over anhydrous sodium sulphate- and stored at C effects) have remained unknown. Macrophages play a pivotal until analyzed by gas chromatography mass spectrometry (GC- role in inflammatory responses. Overproduction of reactive MS) and was then used. (ROS) (RNS) oxygen species and nitrogen by macrophagesin situ is 2.3. Identification of the T. minuta essential oil components a classic indicator during inflammatory events . The production of ROS and RNS radicals are under the control of nicotinamide adenine dinucleotide phosphate oxidase (NOX) GC analysis was carried out using Agilent technology ( NOS) T HP 5 (30 伊0 32 and inducible nitric oxide synthase i , respectively. he chromatographμ with - column m . mm, internal 0 25 ) O aim of the present studyT. wasminuta to investigate the level of potential diameter° . ° m . ° ven temperature° was° performed° as follows: 60 210 3 210 240 20 modulating effects of essential oil on macrophages C to C at C/min; C to ° C at C/min and hold NOX 8 5 280 and their related functions including expression of subunits for °. min, injector temperature C; detector temperature, 22 ( ) 40 47 67 290 C N2 (1 L ) 1 50 T [p phox phagocyteα oxidase , p phox, p phox and p phox], ; carrier gas, m /min ; split ratio of : . he NOS TNF RNA (LPS) OBO 7890 and - m s in lipopolysacharidein vitro -stimulated was analyzed using an Agilent model -A series gas 5975 macrophages.T. minuta In addition, anti-oxidant capacity of chromatography and Agilent model -C mass spectrometry. ROS T HP 5 MS ( 30 essential oil was examined by assessments of , he - capillary column phenylμ methyl siloxane, m RNS and hydrogen peroxide (H2O2) scavenging ability using 伊0.25 mm, internal diameter伊 25 m) was used with helium ABTS H2O2 I 1 L GC , sodium nitrite, and scavenging, respectively.T. minuta t at m /min as the °carrier gas.° oven temperature° was 60 210 3 was expected that these studies would reveal that programmed from C °to C° at a rate of °C/min and was ( 210 240 20 essential oil exhibits radical scavenging activity against then increased from ° C to C at rate of C/min and was superoxide anion, H2O2, and NO radicals) in macrophages, in kept constant at 240 C for 8.5 min. The split ratio was adjusted NOS NOX 1 50 1 L T part, due to an inhibition of i and gene expression.T. to : and the injection° volume was m . he injector F 280 C T minutaurthermore, it was hypothesized for theα first time that temperature was . he quadrupole mass spectrometer essential oil would decrease TNF- mRNA expression as was scanned over 40-550 amu with an ionizing voltage of 70 part of its known anti-inflammatory character and secondarily eV.n Retention indices were determined using retentionT. minuta times due to the ongoing quenching of radicals known to trigger of -alkanes (C8-C25) that were injected after the formation of these pro-inflammatory cytokines. essential oil under the same chromatographic conditions. The retention indices for all componentsn were determined according 2. Materials and methods to the method that uses -alkanes as standard. The compounds were identified by comparison of retention indices with those 2.1. Chemicals and reagents reported in the literature and by comparison of their mass spectra with the Wiley GC-MS Library, Adams Library, Mass Finder 2.1 Library data published mass spectra data[24]. S ABTS G odium nitrite, sodium sulphate, , riess reagent 2.4. ROS scavenging assay (naphthylethylenediamine, sulfanilamide, phosphoric acid), 3 (4 5 2 ) 2 5 - , -dimethylthiazol- -yl - , -diphenyltetrazolium’ T. minuta bromide (MTT), fetal calf serum, Dulbecco s modified eagle The ROS scavenging activity of the essential oil medium, L-glutamine, dimethysulfoxide (DMSO) and LPS (from was determined as previously described[25]. Briefly, Parastoo Karimian et al./Asian Pac J Trop Biomed 2014; 4(3): 219-227 221 μ T. minuta μ 10 (0 500 DMSO) DMSO (0 1%) 24 L of the essential oil - g/mL in was solvent . ° were used as negative controls. After h of added to 1.0 mL of diluted ABTS radical solution (7 mmol/L incubation at 37 C, the culture supernatants in each well were ABTS and 2.54 mmol/L potassium persulfate). After mixing, removed and the cells harvested were used for RNA extraction the absorbance (A) was read at 734 nm using an Ultrospec and real-time PCR analysis. 2000 (P U S ) T spectrophotometer harmacia, ppsala, weden . he 2.8. Cell viability assay percentage of ROS scavenging was calculated as [(A734blank- A734sample) A734blank 伊100 T / ] . he concentrations that could provide T. minuta 50% (IC50) T inhibition were calculated from theT. graph minuta that plotted he effect of essential oil on the viability of the inhibition percentage against different essential J774A.1 cells was determined by MTT assay as described × 4 [27] C (2 10 ) 24 oil concentrations. previously° . ells cells per well were incubated forμ h (at 37 C in 5% CO2) with different concentrations (0-200 g/ 2.5. H O scavenging assay T. minuta μ 2 2 mL) of essential oil. Thereafter, 10 L of MTT (5 mg/ mL) was added to each well and incubated for an additional T. minuta ° μ H2O2 scavenging activity of the essential oil was 4 h at 37 C followed by treatment with 100 L of lysis buffer μ T. determined as previously described[26]. Briefly, 10 L of the (10% SDS in 10 mmol/L HCl). The absorbance of each well was minuta μ essential oil (0-500 g/mL in DMSO) was incubated with determined by spectrophotometer at dual wavelengths of 570 and 1 0 H2O2 (50 100 630 ELISA (B E 808 W . mL of° mmol/L in mmol/L phosphate buffer pH nm on a microplate reader ioTek lx , inooski, 7.4) at 37 C for 60 min. After incubation, the absorbance (A) was VT, 05403, USA). Viability percentage was calculated by the 230 (A A read at nm against a blank solution containing phosphate following formula bsorbance of treated cells/ bsorbanceT. minuta buffer without H2O2 using a spectrophotometer. The percentage of corresponding control)伊100. The control was of H2O2 scavenging was calculated as [(A230blank-A230test)/ essential oil-untreated cells containing DMSO at the highest A230blank 伊100 IC50 (0 1%) T ] . was calculated from theT. graph minuta that plotted the concentration used . . he concentration that provided a 50% (IC50) inhibition percentage against different essential oil inhibition was calculated fromT. aminuta graph, plotting the concentrations. inhibition percentage against different essential oil concentrations. 2.6. RNS scavenging assay 2.9. RNA extraction and cDNA synthesis T. minuta RNS scavenging activity of the essential oil was μ T. determined as previously described[26]. Briefly, 10 L of the Total RNA was extracted using RNX-plus buffer from Cinagen, minuta μ 6 (0 500 L DMSO) T I B 2伊10 essential oil - μg/m in was incubated with ehran, ran. riefly, about cells were transferred to 0 5 (10 100 1 RNX . mL of sodium° nitrite g/mL in mmol/L sodium citrate mL of -plus buffer in an RNase-free microtube, mixed H 5) 37 C 2 A 0 5 L G 5 A p at for h. fter incubation, . m of riess reagent thoroughlyμ and left at room temperature for min. volume was added and the absorbance (A) was read at 540 nm using of 200 L of chloroform was added to the slurry and was mixed g ° a spectrophotometer. The percentage of RNS scavenging was gently. The mixture was centrifuged at 13 200 at 4 C for 15 calculated as follows: [(A540blank-A540sample)/A540blank伊100. IC50 was min, the supernatant was transferred to a new tube and was 15 calculated from theT. minutagraph that plotted the inhibition percentage precipitated with an equal volume of isopropanol for min on T RNA 75% against different essential oil concentrations. ice. he pellet wasμ washed using ethanol, briefly dried 15 L RN T 2.7. Macrophages cell culture and resuspended in of ase free water. he purified total RNA was quantified by NanoDrop ND 1000 spectrophotometer (Wilmington, DE). A sample (0.005 mg) of RNA was used for first The J774.1A murine macrophage cell line was obtained strand cDNA synthesis, using 100 pmoL oligo-dT (18 mer), 15 NTP 20 U RN 200 U M-M from the cell bank of the Pasteur’ Institute of Iran, Tehran. pmoL d s, ase inhibitor, and ulv reverse C D ( F H MD) 0 02 L ells were cultured in ulbecco s modified eagle mediumμ transcriptase all from ermentas, anover, in a . m containing 2 mmol/L L-glutamine, 100 IU/mL penicillin, 100 g/ final volume. mL streptomycin and 10% heat-inactivated fetal calf serum at ° 2.10. Quantitative real-time PCR 37 C in a humidified CO2 incubator. Cultures were allowed to grow until confluence at which point adherent macrophages were scraped° from the flask and were washed with warm Primer design, in the form of exon junction was carried medium (25 C). Cells were counted and their viability was out using AlleleID 7 software (Premier Biosoft Intl., Palo Alto, T CA) 3 determined by trypan blue dye6 exclusion. he cells were for the internal controls glyceraldehydes-β -phosphate seeded at concentration of 2伊10 cells per millilitre in 24-well dehydrogenase (GAPDH) (NM-010927) and -actin (NM-007393.3) tissue culture plates in triplicate (Jet Biofil, Kyoto, Japan). After and tested genes NOX p22phox (NM-007806), NOX p40phox (NM- 18 008677) NOX 47 (NM 010876) NOX 67 (NM 010877) culturing for h to allow cells to adhere, non-adherent cells , p phox -α , p phox - , R NOS (NM 008084) TNF (NM 013693) (T 1) T GAPDH were removed by gentle rinsing with medium. emaining i β - and - - able . he adherent cells were then cultured in the presence or absence and -actin were used as internal control (whose expression μ T. minuta LPS (1 L) A 2 LPS) [28] of medium bearing g/m . fter h, μ essential proved not to be influenced by for data normalizationμ . oil was added at a final concentration of 0-200 g/mL. Two sets Relative real-time PCR was performed in a 20 L volume T. minuta μ of wells without essential oil but containing LPS and containing 1 L cDNA, 1伊Syber Green buffer (Qiagen, Hilden, Parastoo Karimian et al./Asian Pac J Trop Biomed 2014; 4(3): 219-227 222 et Germany) and 4 pmol of each primer. The amplification using the Line-gene K software and the method of Larionov al[29] G (B A RNAΔΔ reactions were carried out in a line ene k thermal cycler ioer . ccordingly, fold-expression of target m - s CTover the T C H C ) 2 echnology° o., angzhou, hina with initial° denaturing of referenceΔ values were calculated by equation , where 94 C 2 40 94 C 10 CT for min, followed by cycles of for seconds, was determined by subtracting the corresponding ΔΔ internal 15 CT CT CT annealing temperature of each primer pair was done° for control value from the specificΔ of targets, and was seconds and 30 seconds for extension to occur at 72 C. After 40 obtained by subtracting the CT of each experimental sample cycles, the specificity of the amplifications was checked based from that of the control sample[30]. on the melting curves resulting from heating the amplicons ° ° 2.11. Statistical analysis from 50 C to 95 C. All amplification reactions were repeated twice under identical conditions beside a negative control and 5 standard samples. To ensure that the PCR was generated All data are expressed as means plus standard deviations of at from cDNA and not genomic DNA, proper control reactions were least three independent experiments. The significant differences F O carried out without the reverse transcriptase treatment. or between treatments wereP< analyzed by ne-way analyses of PCR NOX (ANOVA) 0 05 quantitative realα time data, relative expression of s, variance test at . using statistical package for the iNOS and TNF- gene were calculated based on the threshold social sciences (SPSS, Abaus Concepts, Berkeley, CA) and Prism cycle (CT) method. The CT for each sample was calculated 5 (Graph Phad, San Diego, USA) software. Table 1 Primer used for real-time analysis. 1 Genes Accession Sense sequence Anti sense sequence ’ ’ ’ GAPDH NM-010927 5 -CGGTGTGAACGGATTTGGC-3 5 -TGAGTGGAGTCATACTGGAAC-3 β ’ ’ ’ ’ -actin NM-007393.3 5 -CCACACCCGCCACCAGTTCG-3 5 -CTAGGGCGGCCCACGATGGA-3 ’ ’ ’ ’ NOX p22 NM-007806 5 - ATGGAGCGATGTGGACAG-3 5 - ACCGACAACAGGAAGTGG-3 ’ ’ ’ ’ NOX p40 NM-008677 5 -CAACAAAGACTGGCTGGAG-3 5 -CCGCAATGTCCTTGATGG-3 ’ ’ ’ ’ iNOX p47 NM-010876 5 - ATGGCACAAAGGACAATC-3 5 - ACCTGAGGCTATACACAAG-3 ’ ’ ’ ’ NOX p67 NM-010877 5 - CAGCCACAGTCAGCAGAG-3 5 -GCACAAAGCCAAACAATACG-3 ’ ’ ’ ’ iNOS NM-008084 5 - CTGGAGGTTCTGGATGAG-3 5 - CTGAGGGCTGACACAAGG -3 α ’ ’ ’ ’ TNF- NM-013693 5 -GTCTCAGCCTCTTCTCATTC-3 5 - GGAACTTCTCATCCCTTTGG-3 1 β P A ID 7 GAPDH rimer design, in form of exon junction was carried out using llele software for the internal controls α and -actin and test genes NADH oxidase p22 phagocytes oxidase (NOX p22phox), NOX p47phox, NOX p47phox, NOX p67phox, iNOS and TNF- genes from Mus musculus sequence. 3. Results Table 2 T. minuta 3.1. Plant materials Chemical composition of essential oil. Compounds Retention index % of compounds α T. minuta -Pinene 933.526 0.327 92 The essential oil was prepared by water-distillation, Sabinene 973.444 0.397 03 and its chemical composition was determined by GC-MS. As cis-3-Hexenyl acetate 1 005.680 0.148 38 p shown in Table 2, GC-MS analysis indicated that the main -Cymene 1 025.210 0.933 76 (33 86%) E (19 92%) Limonene 1 029.490 3.102 93 components were dihydrotagetoneβ . , -ocimene . , β tagetone (16.15%), cis- -ocimene (7.94%), Z-ocimene (5.27%), cis- -Ocimene 1 037.830 7.940 67 limonene (3.1%) and epoxyocimene (2.03%). GC-MS analysis of the Dihydrotagetone 1 061.290 33.862 97 T. minuta Chrysanthenone 1 103.260 0.146 46 essential oil indicated the main components β essential oil were dihydrotagetone, E-ocimene, tagetone, cis- -ocimene, Allo-cimene 1 135.730 0.358 52 Z-ocimene, limonene and epoxyocimene. E, Z-Epoxyocimene 1 149.090 2.035 34 Tagetone 1 160.520 16.155 09 3.2. Antioxidant activity of T. minuta essential oil cis-Tagetone 1 167.400 0.198 29 p -Mentha-1,8 dien-3-one 1 208.060 0.188 94 T. minuta Z O 1 238 290 5 275 42 essential oil displayed a concentration dependent - cimene . . E-Ocimene 1 250.790 19.928 7 ROS, RNS and H2O2 scavenging activity. IC50 for ROS, RNS and H2O2 μ T. Thymol 1 282.780 0.477 33 scavenging were (12.0依3.0), (15.0依2.5) and (13.0依4.0) g/mL of minuta μ Carvacrol 1 298.190 0.470 14 essential oil, respectively. At concentrations >30 g/ T. minuta cis-Isoeugenol 1 398.530 0.865 60 mL, the essential oil significantly scavenges ROS, T. minuta E-Caryophyllene 1 420.970 0.299 45 RNS H2O2 100% T α , and by .in hevitro essential oil analyzed -Humulene 1 455.070 0.149 81 here possessed potent ROS, RNS and H2O2 scavenging T. minuta μ Germocrene D 1 497.900 0.398 97 30 activity. The essential oil at > g/mL had the ability Spathulenol 1 582.190 0.373 19 to scavenge all ROS, RNS and H2O2 radicals, an indicator of its potency as a radical scavenger. Parastoo Karimian et al./Asian Pac J Trop Biomed 2014; 4(3): 219-227 223 3.3. T. minuta essential oil reduced cell viability at high significantly decreased the NOX p22phox mRNA expression in concentrations LPS-treated cells from (21.0依2.7) to (4.5依0.8) fold of the control P ( <0.001) dose-dependently, indicating the inhibitory effect T. minuta The MTT assay results indicated that low concentrations of essential oil on p22phox mRNA induction/ μ T. minuta (1-50 g/mL) of essential oil had no effect on formation (Figure 2). J774A.1 cell viability. However, at higher concentrations μ (100-200 g/mL), cell viability was significantly reduced in 3.5. T. minuta essential oil reduced NOX p40phox mRNA a concentration-related manner, with the maximum effect μ Expression in LPS-Stimulated Macrophages (100% cell death) at concentrations >200 g/mL (Figure 1). μ Non-cytotoxic concentrations (<50 g/mL) were thus used for the subsequent studies including expression of genes. The un-stimulated (control) cells showed low level of NOX p40phox mRNA expression while, the expression of NOX 3.4. T. minuta essential oil reduced NOX p22phox mRNA p40phox mRNA in LPS-treated cells was (6.3依0.4) fold of the expression in LPS-stimulated macrophages P T. minuta control ( <0.001). The addition of essential oil at 1 μ to 50 g/mL significantly decreased the NOX p40phox mRNA The un-stimulated (control) cells showed low level of NOX expression in LPS-treated cells from (5.1依0.7) to (2.4依0.2) fold P< p22phox mRNA expression. LPS stimulation of macrophages of the control, dose-dependently ( 0.05), indicating the T. minuta resulted in an increase in NOX p22phox mRNA expression inhibitory effect of essential oil on p40phox mRNA P (26.5依1.7) fold of LPS-untreated control cells ( <0.001). induction/formation (Figure 3). T. minuta μ The addition of essential oil at 1 to 50 g/mL

120 30 a

110 ) 100 25 b )

% 90 ( 80 20 70

fold of control of fold cd 60 * ( 15 50 ell viability viability ell C 40 RNA d * 10 30 20

phox m phox 5 10 22 0 p e 0 0 1 T. minuta 10 50 μ 100 200 essential oil ( g/mL) Control LPS LPS+TMO1 LPS+TMO10 LPS+TMO50 Figure 1. T. minuta Treatment Effect of the essential oil on the viability of J774 cell Figure 2. T. minuta Effects of essential oil on NOX p22phox mRNA lines. T. minuta expression in LPS-stimulated macrophages. TMO: essential oil. The cells were treated with various concentrations T. minuta μ μ TMO: essential oil. The cells were cultured in 24-well plates and of essential oil (0-200 g/mL) and incubated for 24 h. Control (0 g/mL) was cells treated with and without LPS. Various concentrations of essential oil (1-50 treated only with the solvent (DMSO) at concentration of 0.1%. Data represent μ g/mL) were added. After 24 h, the expression of NOX p22phox mRNA was mean依SD from three sets of independent experiments. analyzed by real-time PCR. Cells treated with DMSO as the solvent (control) 8 and cells treated with the solvent and LPS was considered as positive controls. ) 7 a 6 a

) a b 6 5 a b 5 fold of control of fold

( 4

4 c b fold of control of fold (

RNA 3 3

RNA 2 2

phox m phox c

40 d p 1 1 phox m phox 47 0 p 0 Control LPS LPS+TMO1 LPS+TMO10 LPS+TMO50 Control LPS LPS+TMO1 LPS+TMO10 LPS+TMO50 Treatment Treatment Figure 3. T. minuta Figure 4. T. minuta Effects of essential oil on NOX p40phox mRNA Effects of essential oil on NOX p47phox mRNA expression in LPS-stimulated macrophages. expression in LPS-stimulated macrophages. T. minuta T. minuta TMO: essential oil. The cells were cultured in 24-well plates and TMO: essential oil. The cells were cultured in 24-well plates and μ treated with and without LPS. Various concentrations of essential oil (1-50 treated with and without LPS. Various concentrations of TMO (1-50 g/mL) μ g/mL) were added. After 24 h, the expression of NOX p47phox mRNA was were added. After 24 h, the expression of NOX p47phox mRNA was analyzed analyzed by real-time PCR. Cells treated with DMSO as the solvent (control) by real-time PCR. Cells treated with DMSO as the solvent (control) and cells and cells treated with the solvent and LPS was considered as positive controls. treated with the solvent and LPS was considered as positive controls. Parastoo Karimian et al./Asian Pac J Trop Biomed 2014; 4(3): 219-227 224 3.6. T. minuta essential oil reduced NOX p47phox mRNA 35 expression in LPS-stimulated macrophages ) 30 a

25 The un-stimulated (control) cells showed low level of NOX bc 20 c fold of control of fold 47 RNA NOX 47 ( p phox m expression while, the expression of p phox RNA LPS (5 00 0 25) 15 m in -treated cells was . 依 . fold of the control RNA P T. minuta induction/formation ( <0.001). The addition of m 10 μ d NOS

1 50 i essential oil at to g/mL significantly decreased this gene 5 expression in LPS-treated cells from (5.00依0.50) and (1.60 e P 0 依0.28) fold of control, dose-dependently ( <0.01) indicating the Control LPS LPS+TMO1 LPS+TMO10 LPS+TMO50 T. minuta Treatment inhibitory effect of essential oil on p47phox mRNA Figure 6. T. minuta Effects of essential oil on iNOS mRNA expression in ( 4) induction/formation Figure . LPS-stimulated macrophages. T. minuta TMO LPS T 3.7. T. minuta essential oil reduced NOX p67phox mRNA : essential oil. -stimulated macrophages. he cells were cultured in 24-well plates and treated with and without LPS. Various LPS μ expression in -stimulated macrophages concentrations of TMO (1-50 g/mL) were added. After 24 h, the expression of NOS mRNA was analyzed by real-time PCR. Cells treated with DMSO as the solvent (control) and cells treated with the solvent and LPS was considered as W NOX 67 ith respect to p phox, a decrease in the gene positive controls. expression was detected in LPS-stimulated macrophages which T. minuta were treated with essential oil. The relative NOX 3.9. T. minuta essential oil reduced TNF-α mRNA expression in p67phox mRNA expression in cells treated with LPS alone was P LPS-stimulated macrophages (5.00依0.25) fold of LPS-untreated the control cells ( <0.001). The T. minuta μ addition of essential oil at 1 to 50 g/mL significantly decreased the NOX p67phox mRNA expression in LPS-treated LPS stimulation of macrophages resulted in an increase in α cells from (4.0依0.5) and (1.0依0.4) fold of the control, dose- TNF- mRNA expression compared to the conditions in LPS- P< T. P T. dependently ( 0.01), indicating the inhibitory effect of untreated cells (9.8依0.5) fold ( <0.001). The addition of minuta minuta μ essential oil on p67phox mRNA induction/formation essential oil at 1 to 50 g/mL significantly decreased α (Figure 5). the TNF- mRNA expression from (9.7依0.4) and (3.3依0.6) fold of

) 6 the control, dose-dependently (Figure 7). These data indicated T. minuta α a the inhibitory effect of essential oil on TNF- mRNA 5 b induction/formation. 4 fold of control of fold (

12 3 c ) a b RNA 2 d 10 d

phox m phox 1 8 c 67 p fold of control of fold

( d

0 6 Control LPS LPS+TMO1 LPS+TMO10 LPS+TMO50 T RNA reatment 4 Figure 5. T. minuta m Effects of essential oil on NOX p67phox mRNA expression in LPS-stimulated macrophages. TNF T. minuta 2 TMO: essential oil. The cells were cultured in 24-well plates and e μ treated with and without LPS. Various concentrations of TMO (1-50 g/mL) 0 were added. After 24 h, the expression of NOX p67phox mRNA was analyzed Control LPS LPS+TMO1 LPS+TMO10 LPS+TMO50 Treatment by real-time PCR. Cells treated with DMSO as the solvent (control) and cells Figure 7. T. minuta Effects of essential oil on TNF mRNA expression in LPS- treated with the solvent and LPS was considered as positive controls. stimulated macrophages. T. minuta TMO: essential oil. The cells were cultured in 24-well plates and 3.8. T. minuta essential oil reduced iNOS mRNA expression in μ LPS-stimulated macrophages treated with and without LPS. Various concentrations of TMO (1-50 g/mL) were added. After 24 h, the expression of TNF mRNA was analyzed by real- time PCR. Cells treated with DMSO as the solvent (control) and cells treated LPS stimulation of macrophages resulted in an increase in iNOS with the solvent and LPS was considered as positive controls. P mRNA expression (27.5 依1.4) fold of LPS-untreated cells ( <0.001). T. minuta The addition of essential oil at concentrations 1 to μ 4. Discussion 50 g/mL significantly decreased the iNOS mRNA expression in LPS-treated cells from (20.4依1.4) to (3.8依1.0) fold of untreated P T. minuta cells, dose-dependently ( >0.05) indicating the inhibitory effect The antioxidant and anti-inflammatory effects of T. minuta of essential oil on iNOS mRNA induction/formation essential oil were investigated in the present study. GC-MS (Figure 6). analysis of the essential oil indicated the main components Parastoo Karimian et al./Asian Pac J Trop Biomed 2014; 4(3): 219-227 225 T. minuta E R G (R 1 R 2 R R in essentialβ oil were dihydrotagetone, -ocimene, and the small ho protein ac or ac , ac: ho-related tagetone, cis- -ocimene, Z-ocimene, limonene and C3 botulinum toxin substrate), which assemble on the cellular epoxyocimene. Previous study reported the main components membrane to activate the enzyme[33]. Studies have shown that T. minuta β of essential oil were -ocimene, dihydrotagetone, phosphorylation of p47phox leads to conformational changes, Z E [11] A 22 tagetone, -ocimene and -ocimene . nother study reportedTagetes allowing its translocation and interaction with p phox. T 47 thiophenes T.and minuta polyacetylenic compounds in the ranslocation of p phox brings with it the other subunits, [12] 67 40 [34] A species and had the highest totalT. minuta thiophene yield . p phox and p phox to the membrane . ctivation of this Accordingly, the main components of essential oil enzyme complex leads to fusion of the vesicles containing NOX could be tagetone (cis/trans, ketone/alcohol, aldehyde/alcohol), with the plasma membrane or the phagosomal membrane. The ( ) ocimene cis/trans, ketone/alcohol, aldehyde/alcohol and active enzyme converts molecular oxygen to superoxide anionT. [11-14] F [35] A thiophene derivatives . or the reasons that, essential oils minutathrough a one-electron transfer . s our study showed, composition depend on the species, climate, altitude, time of essential oil was able to decrease the expression of key collection and growth stage, thus the plants analyzed in this components of NOX. It has been shown that the assembly of 47 67 22 research hadT. minuta roughly same components with other previously p phox, p phox and p phox at the membrane is necessary analyzed essential oil however, showed important for oxidase function[36]. Thus, it can be assumed that reduced ROS differencesT. minuta in their quality and quantity of components. T. minuta generation by stimulated macrophages in the presence of T in vitrohe essential oil analyzed here possessedT. minuta potent essential oil might be, in part, due to the modulation ROS RNS H2O2 T NOX , andμ scavenging activity. he of the expression of subunits. essential oil at >30 g/mL had the ability to scavenge all ROS, In addition to ROS, the overproduction of RNS by activated RNS and H2O2 radicals, an indicator of its potency as a radical macrophages seems to play an important role in different scavenger. ROS are oxygen-derived small molecules, including steps of many inflammatory processes[37]. RNS are nitrogen- oxygen radicals such as superoxide, hydroxyl and peroxyl and containing oxidants, mainly NO which is a free radical playing some non-radicals that are easily converted into radicals, such a key role in the pathogenesis of pain and inflammation. as hydrogen peroxide. ROS, once produced, can interact with NO in macrophages is generated by activation of iNOS. This various molecules including other small inorganic molecules enzyme has the ability to produce high concentrations of NO D as well as macromolecules such as proteins and lipids. uring after stimulation with bacterial endotoxinsα or a variety of pro- ROS TNF IL 1 IL 6[38] NO these interactions, may destroy or change the functionT. minuta of inflammatory cytokines such as - , - and - . [31] ROS the target molecule . The reducing activity of generation involves several stepsκ including the activation of (NF) B NOS essential oil observed in our study imply the beneficial role of nuclear transcriptionκ factor - and subsequent i gene this product for reducing damages in biological tissues. The expression[39]. NF- B regulates the expression of various genes I radical scavenging activity of compounds is mainly due to their involved in inflammatory responses. ts activation canα also oxidation-reduction potential, which can play an important role be regulated by various cytokines, among which TNF- is the T I in neutralizing free radicals.T. minuta his activity is related to phenolic most important. n response to inflammatory stimuli such as [32] LPS hydroxyl groups . essential oil mainly contains , macrophagesα secreteβ a variety of inflammatoryα mediators dihydrotagetone, ocimene, tagetone and limonene which all such as TNF- and IL-1 . The production of TNF- cytokine is are monoterpenes. This antioxidant activity was confirmed by important for the induction of NO synthesis in LPS-stimulated μ T. T. minuta previous research with IC50 between 35-344 g/mL[21,22]. Thus, macrophages[40]. As results of this study showed that minuta α TNF essential oil analyzed in this research showed strongerIn vitro essential oil was able to reduce inducible expression of - antioxidant activity rather than previously analyzed one. gene, which indicated that the reduced NO production seen inhibition of the NO radical is a measure of antioxidant activity in the macrophage cultures might be partly related to the T. α α A TNF TNF minutaof plant extracts. s results of the present study show, the suppression of - expression. - is known to play a essential oil usedμ here have the ability to scavenge total crucial role in inflammatory responses and is involved in the RNS 30 L [41] A at concentration > g/m . pathogenesis ofT. inflammatory minuta diseases . s results of our The MTT assay results indicated that low concentrations study showed, essential oil significantly reduced μ T. minuta (1 50 L) NOS RNA S - g/m of essentialμ oil had no effect on i m α expression in stimulated macrophages. uppression J774A 1 (IC50 95 L) I TNF NOS . cell viability = g/m . n order to determineT. minuta of - expression in macrophagesT. minuta as well as reduced i the antioxidant and anti-inflammatory effects of μ gene expression, due to the essential oil indicates 50 L essentialT. minuta oil on macrophages, the concentrations of > g/m the ability of this product to diminish immune reactions and essential oil which were overtly cytotoxic to the cells provides further evidence that this plant may have potent A were not used. lthough the constituents of essential oils can immuno-modulatory properties. T. minuta act as antioxidants, they may also act as pro-oxidants and affect Considering all these finding, essential oil inner cell membranes and organelles such as mitochondria in displayed an anti-oxidant property by scavenging superoxide, eukaryotic cells. Depending on the type and concentration, this H2O2 and NO radicals, and reduced oxidative stress. This effect may result in cellular cytotoxicity. suggested that there was a potential for use of this product In macrophages ROS production is under the control of NOX. in the therapy of oxidative damage, a process that usually This multi-component enzyme consists of several cytosolic accompanies inflammatory conditions. The decreased formation components including p91phox, p67phox, p40phox, p47phox of ROS and NOS radicals in macrophages was possibly due to the Parastoo Karimian et al./Asian Pac J Trop Biomed 2014; 4(3): 219-227 226 radical scavenging activity of phenolic groups present in the oil an inhibition of iNOS and NOX gene expression. NOS NOX and/or due to T.an minuta inhibition of i and gene expressions. Applications F urthermore, essential oil decreased theα expression TNF T T of the genes for pro-inflammatory cytokine - . herefore, a T. minutahese data suggest a potential therapeutic usefulness for reduced expression of the above-noted inflammatory enzymes in the modulation ofT. macrophages minuta and provides and cytokines could be attributed to a suppression of the NF- evidence to support the use of as a tea/additive/ B T k pathway in the treated cells.T. minutahese data suggest a potential traditional remedy for treatment of inflammatory diseases. therapeutic usefulness for in the modulation T.of Peer review macrophagesminuta and provides evidence to support the use of T. minuta as a tea/additive/traditionalin vivo remedy for treatment of essential oil displayed an anti-oxidant property F H2O2 NO inflammatory diseases. urther studies are recommendedT. minuta by scavenging superoxide, and radicals, and to more fully understand the therapeutic potential of reduced oxidative stress. The decreased formation of ROS essential oil in a multitude of inflammatory disorders. and NOS radicals in macrophages was possibly due to the radical scavenging activity of phenolic groups present in NOS NOX Conflict of interest statement the oil and/or due to an inhibitionT. minuta of i and gene expressions. Furthermore, essential oil decreased

the expressionα of the genes for pro-inflammatory cytokine We declare that we have no conflict of interest. TNF- . Acknowledgements References

This work was supported by the funding from Shiraz Iranian Herbal Pharmacopoeia University (Grant no. 88-GR-AGRST-108) and Shiraz [1] . Tehran: Iranian Ministry of University of Medical Science (Grant No. 3937). Health & Medical Education Publications; 2002. [2] N erio LS, Olivero-Verbel J, Stashenko E. Repellent activity of Bioresour Technol 101 essential oils: a review. 2010; : 372-378. Comments [3] R ahimi R, Shams-Ardekani MR, Abdollahi M. A review of the efficacy of traditional Iranian medicine for inflammatory bowel Background World J Gastroenterol 16 disease. 2010; : 4504-4514. T. minuta Tagetes erecta [4] N ikkon F, Habib MR, Saud ZA, Karim MR. and its is an aromatic plant that has several medical Culex quinquefasciatus Asian Pac mosquitocidal potency against . benefits such as a remedy for the colds, respiratory J Trop Biomed 1 inflammations, stomach problems, anti-spasmodic, anti- 2011; : 186-188. T [5] K amaraj C, Rahuman AA, Bagavan A, Elango G, Zahir AA, parasitic, antiseptic and sedative. his work aimed T.to Santhoshkumar T. Larvicidal and repellent activity of medicinal minutainvestigate antioxidant and anti-inflammatory effects of plant extracts from Eastern Ghats of South India against malaria essential oil. Asian Pac J Trop Med 4 and filariasis vectors. 2011; : 698-705. Research frontiers [6] G ovindarajan M. Larvicidal and repellent properties of some Culex tritaeniorhynchus Anopheles T essential oils against Giles and he aim of the present study wasT. to minutainvestigate the level subpictus Asian Pac J Trop Med Grassi (Diptera: Culicidae). 2011; of potential modulating effects of essential oil 4 : 106-111. on macrophages and their related functionsα including NOX NOS TNF RNA [7] A ristatile B, Al-Assaf AH, Pugalendi KV. Carvacrol expression of subunits, and -in vitrom s in LPS-stimulated macrophages. In addition, anti- suppresses the expression of inflammatory marker genes in T. minuta Asian Pac J Trop Med 6 oxidant capacity of essential oil was examined by D-galactosamine-hepatotoxic rats. 2013; : assessments of ROS, RNS and H2O2 scavenging ability. 205-211. [8] M aity N, Nema NK, Abedy MK, Sarkar BK, Mukherjee Related reports Tagetes erecta PK. Exploring Linn flower for the elastase, J Ethnopharmacol Advances in chemical and pharmacological evaluations of hyaluronidase and MMP-1 inhibitory activity. T. minuta 137 essential oil such as antioxidant and antimicrobial 2011; : 1300-1305. H [9] W ang M, Tsao R, Zhang S, Dong Z, Yang R, Gong J, et al. have occurred in the past recente.g. years. owever, several useful features of this plant ( the mechanisms underlying Antioxidant activity, mutagenicity/anti-mutagenicity, and ) clastogenicity/anti-clastogenicity of lutein from marigold flowers. its antioxidant and anti-inflammatory effects have remain Food Chem Toxicol 44 unknown. 2006; : 1522-1529. [10] B reme K, Tournayre P, Fernandez X, Meierhenrich UJ, Brevard Innovations and breakthroughs H, Joulain D, et al. Identification of odor impact compounds of T. minuta Tagetes minuta T essential oil: comparison of two GC-olfactometry hese studies would reveal that essential oil J Agric Food Chem 57 exhibits radical scavenging activity (against superoxide methods. 2009; : 8572-8580. anion, H2O2, and NO radicals) in macrophages, in part, due to [11] L opez SB, Lopez ML, Aragon LM, Tereschuk ML, Slanis AC, Parastoo Karimian et al./Asian Pac J Trop Biomed 2014; 4(3): 219-227 227 Zataria multiflora Feresin GE, et al. Composition and anti-insect activity of essential essential oil and its main components on nitric Tagetes Ceratitis capitata oils from species on Wiedemann and oxide and hydrogen peroxide production in lipopolysaccharide- Triatoma infestans J Agric Food Chem 59 J Pharm Pharmacol 64 Klug. 2011; : 5286-5292. stimulated macrophages. 2012; : 1492-1500. [12] M arotti I, Marotti M, Piccaglia R, Nastri A, Grandi S, Dinelli G. [27] N ouri AM, Thompson C, Cannell H, Symes M, Purkiss S, Tagetes Thiophene occurrence in different species: agricultural Amirghofran Z. Profile of epidermal growth factor receptor (EGFr) J Sci Food Agric biomasses as sources of biocidal substances. expression in human malignancies: effects of exposure to EGF 90 2010; : 1210-1217. and its biological influence on established human tumor cell Inter J Mol Med 6 [13] M ohamed MA, Harris PJ, Henderson J, Senatore F. Effect of lines. 2000; : 495-500. drought stress on the yield and composition of volatile oils of [28] B arber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a Tagetes drought-tolerant and non-drought-tolerant clones of housekeeping gene: analysis of GAPDH mRNA expression in a minuta Planta Med 68 Physiol Genomics 21 . 2002; : 472-484. panel of 72 human tissues. 2005; : 389-395. [14] R anilla LG, Kwon YI, Apostolidis E, Shetty K. Phenolic [29] L arionov A, Krause A, Miller W. A standard curve based method in vitro BMC Bioinformatics compounds, antioxidant activity and inhibitory potential for relative real time PCR data processing. 6 against key enzymes relevant for hyperglycemia and hypertension 2005; : 62. 30 of commonly used medicinal plants, herbs and spices in Latin [ ] L ivak KJ, Schmittgen TD. Analysis of relative geneΔΔ expression Bioresour Technol 101 - CT America. 2010; : 4676-4689. data using real-time quantitative PCR and the 2 method. é í ó Methods 25 [15] C spedes CL, Avila JG, Mart nez A, Serrato B, Calder n-Mugica 2001; : 402-408. JC, Salgado-Garciglia R. Antifungal and antibacterial activities [31] C ircu ML, Aw TY. Reactive oxygen species, cellular redox Tagetes lucida J Agric Food Chem 54 Free Radic Biol Med 48 of Mexican tarragon ( ). 2006; : systems, and apoptosis. 2010; ; 749-762. 3521-3527. [32] H uang CC, Wang HF, Chen CH, Chen YJ, Yih KH. A study of four [16] T ereschuk ML, Baigori MD, Abdala LR. Antibacterial activity of antioxidant activities and major chemical component analyses of Tagetes terniflora Fitoterapia 74 J Cosmet Sci 62 . 2003; : 404-416. twenty-five commonly used essential oils. 2011; : é é á é é [17] H th lyi E, D nos B, T t nyi P, Koczka I. GC-MS analysis of the 393-404. Tagetes esssential oils of four species and the anti-microbial [33] G roemping Y, Rittinger K. Activation and assembly of the NADPH Tagetes minuta Flavor Fragr J 1 Biochem J 386 activity of . 1986; : 169-173. oxidase: a structural perspective. 2005; : 401-416. [18] D unkel FV, Jaronski ST, Sedlak CW, Meiler SU, Veo KD. [34] M inakami R, Sumimotoa H. Phagocytosis-coupled activation of Tagetes minuta Effects of steam-distilled shoot extract of and the superoxide-producing phagocyte oxidase, a member of the Tetanops myopaeformis Int J Hematol 84 entomopathogenic fungi on larval . NADPH oxidase (NOX) family. 2006; : 193-198. Environ Entomol 39 2010; : 979-988. [35] B edard K, Krause KH. The NOX family of ROS-generating NADPH Physiol Rev 87 [19] M ares D, Tosi B, Poli F, Andreotti E, Romagnoli C. Antifungal oxidases: physiology and pathophysiology. 2007; : Tagetes patula activity of extracts on some phytopathogenic fungi: 245-313. Pythium ultimum Microbiol Res ą ultrastructural evidence on . 2004; [36] K leniewska P, Piechota A, Skibska B, Gor ca A. The NADPH 159 Arch Immunol Ther Exp (Warsz) : 295-304. oxidase family and its inhibitors. 60 [20] T hembo KM, Vismer HF, Nyazema NZ, Gelderblom WC, Katerere 2012; : 277-294. DR. Antifungal activity of four weedy plant extracts against [37] Z ielonka J, Zielonka M, Sikora A, Adamus J, Joseph J, Hardy M, J Appl Microbiol 109 selected mycotoxigenic fungi. 2010; : 1479- et al. Global profiling of reactive oxygen and nitrogen species in J Biol 1486. biological systems: high-throughput real-time analyses. Chem 287 [21] G ong Y, Liu X, He WH, Xu HG, Yuan F, Gao YX. Investigation 2012; : 2984-2995. α into the antioxidant activity and chemical composition of [38] S ellers SL, Iwasaki A, Payne GW. Nitric oxide and TNF are Fitoterapia alcoholic extracts from defatted marigold residue. critical regulators of reversible lymph node vascular remodeling 83 PLoS One 8 2012; : 481-489. and adaptive immune response. 2013; : 60741. í [22] P arejo I, Bastida J, Viladomat F, Codina C. Acylated quercetagetin [39] L orenzo O, Picatoste B, Ares-Carrasco S, Ram rez E, Egido Tagetes maxima ñó glycosides with antioxidant activity from . J, Tu n J. Potential role of nuclear factor kB in diabetic Phytochemistry 66 Mediators Inflamm 2005; : 2356-2362. cardiomyopathy. 2011; doi: 10.1155/2011/652097. British Pharmacopeia [23] . London: The British Pharmacopoeia [40] Z ha LY, Mao LM, Lu XC, Deng H, Ye JF, Chu XW, et al. Anti- Commission; 1998, p. 137-138. inflammatory effect of soyasaponins through suppressing Identification of essential oil components by gas [24] A dams RP. nitric oxide production in LPS-stimulated RAW 264.7 cells by chromatography/mass spectrometery κ . 4th ed. Illinois, USA: Allured attenuation of NF- B-mediated nitric oxide synthase expression. Bioorg Med Chem Lett 21 Publishing Corporation; 2007, p. 456-494. 2011; : 2415-2418. [25] K avoosi G, Rowshan V. Chemical composition, antioxidant and [41] L eonard B, Maes M. Mechanistic explanations how cell-mediated Ferula antimicrobial activities of essential oil obtained from immune activation, inflammation and oxidative and nitrosative assa-foetida Food oleo-gum-resin: effect of collection time. stress pathways and their sequels and concomitants play a role Chem 138 Neurosci Biobehav 2013; : 2180-2187. in the pathophysiology of unipolar depression. Rev 36 [26] K avoosi G, Teixeira da Silva JA, Saharkhiz MJ. Inhibitory effects of 2012; : 764-785.