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

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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 minutacis- -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.
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