Asian Pacific Journal of Tropical Biomedicine

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1 Asin P J Trop Biome 4; 4(3): Contents lists ville t SieneDiret Asin Pifi Journl of Tropil Biomeiine journl homepge: Doument heing oi:.6/s-69(4)63- 襃 4 y the Asin Pifi Journl of Tropil Biomeiine. All rights reserve. Anti-oxitive n nti-inflmmtory effets of Tgetes minut essentil oil in tivte mrophges Prstoo Krimin, Gholmrez Kvoosi *, Zhr Amirghofrn Biotehnology Institute, Shirz University, Shirz, , Irn Deprtment of Immunology, Autoimmune Disese Reserh Center n Meiinl n Nturl Prouts Chemistry Reserh Center, Shirz University of Meil Sienes, Shirz, Irn PEER REVIEW Peer reviewer Hsn Slehi, University of Shirz, Shirz, Irn. E-mil: hslehi@shirzu..ir Comments TMO isplye n nti-oxint property y svenging superoxie, H O n NO rils, n reue oxitive stress. The erese formtion of ROS n NOS rils in mrophges ws possily ue to the ril svenging tivity of phenoli groups present in the oil n/or ue to n inhiition of inos n NOX gene expressions. Furthermore, TMO erese the expression of the genes for pro-inflmmtory ytokine TNF-α. Detils on Pge 6 ABSTRACT Ojetive: To investigte ntioxint n nti-inflmmtory effets of Tgetes minut (T. minut) essentil oil. Methos: In the present stuy T. minut essentil oil ws otine from leves of T. minut vi hyro-istilltion n then ws nlyze y gs hromtogrphy-mss spetrometry. The ntioxint pity of T. minut essentil oil ws exmine y mesuring retive oxygen, retive nitrogen speies n hyrogen peroxie svenging. The nti-inflmmtory tivity of T. minut essentil oil ws etermine through mesuring NADH oxise, inuile nitri oxie synthse n TNF-α mrna expression in lipopolyshrie-stimulte murine mrophges using reltime PCR. Results: Gs hromtogrphy-mss spetrometry nlysis inite tht the min omponents in the T. minut essentil oil were ihyrotgetone (33.86%), E-oimene (9.9%), tgetone (6.%), is-β-oimene (7.94%), Z-oimene (.7%), limonene (3.%) n epoxyoimene (.3%). The T. minut essentil oil h the ility to svenge ll retive oxygen/retive nitrogen speies rils with IC - μg/ml, whih inite potent ril svenging tivity. In ition, T. minut essentil oil signifintly reue NADH oxise, inuile nitri oxie synthsen TNF-α mrna expression in the ells t onentrtions of μg/ml, initing pity of this prout to potentilly moulte/iminish immune responses. Conlusions: T. minut essentil oil hs ril svenging n nti-inflmmtory tivities n oul potentilly e use s sfe effetive soure of nturl nti-oxints in therpy ginst oxitive mge n stress ssoite with some inflmmtory onitions. KEYWORDS Tgetes minut, Essentil oil, Mrophges, Anti-inflmmtory, Antioxint. Introution Tgetes minut (T. minut) is tll upright mrigol plnt in the sunflower (Asteree) fmily. Tgetes speies originlly hs een use s soure of essentil oil (extrte from leves, stlks n flowers) for the flvoring in the foo inustries. The powers n extrts of Tgetes re rih in the ornge-yellow rotenoi n re use s foo olor for foos suh s pst, vegetle oil, mrgrine, myonnises, sl ressing, ke *Corresponing uthor: Gholmrez Kvoosi, Biotehnology Institute, Shirz University, P.O , Shirz, , Irn. Tel: E-mil: ghkvoosi@shirzu..ir Fountion Projet: Supporte y the funing from Shirz University (Grnt no. 88- GR-AGRST-8) n Shirz University of Meil Siene (Grnt No. 3937). goos, onfetionery, iry prouts, ie rem, yogurt, itrus juie, mustr n s olornt in poultry fee[-3]. T. minut is lso extensively use meiinlly s oniment n herl te in wie vriety of fiels in its ntive region n s populr tritionl folk remeies n in the omplementry n meil therpy. T. minut hs severl meil enefits suh s remey for ols, respirtory inflmmtions, stomh prolem, nti-spsmoi, nti-prsiti, nti-septi, insetiie n setive. It is use for hest infetions, oughs Artile history: Reeive Jn 4 Reeive in revise form Jn, n revise form 6 Jn, 3r revise form Jn 4 Aepte 6 Fe 4 Aville online 8 Mr 4

2 Prstoo Krimin et l./asin P J Trop Biome 4; 4(3): 9-7 n trrh, ilting the ronhi, filitting the flow of muus n isloging ongestion n n e use in ses of skin infetions. It lso hs heling effet on wouns, uts, lluses n unions[4-9]. However, suh prties re lrgely se on folklore n trin of tritionl meiine rther thn evienese reserh. The most unnt omponents in T. minut essentil oil re ihyrotgetone (unsturte yli monoterpene ketone), oimene (unsturte yli monoterpene hyroron), tgetone (unsturte yli monoterpene ketone) n limonene (unsturte monoyli monoterpene hyroron[-4]. T. minut essentil oil hs signifint ntiteril tivity ginst oth Grm-positive n Grm-negtive teri[-7]. Severl stuies hve lso esrie ntifungl tivities of T. minut essentil oil ginst Cni, Peniillium n Aspergilus speies[8-]. T. minut essentil oil hs een shown to possess nti-oxint tivity in, -iphenyl--pirylhyrzyl n, -zino-i (3-ethylenzthizoline-6- sulphonte) (ABTS) ssy[,]. Avnes in hemil n phrmologil evlutions of T. minut essentil oil hve ourre in the pst reent yers; however, severl useful fetures of this plnt (e.g. the mehnisms unerlying its ntioxint n nti-inflmmtory effets) hve remine unknown. Mrophges ply pivotl role in inflmmtory responses. Overproution of retive oxygen speies (ROS) n nitrogen (RNS) y mrophges is lssi initor uring inflmmtory events in situ. The proution of ROS n RNS rils re uner the ontrol of niotinmie enine inuleotie phosphte oxise (NOX) n inuile nitri oxie synthse (inos), respetively. The im of the present stuy ws to investigte the level of potentil moulting effets of T. minut essentil oil on mrophges n their relte funtions inluing expression of NOX suunits [pphox (phgoyte oxise), p4phox, p47phox n p67phox], NOS n TNF-α mrnas in lipopolyshrie (LPS)-stimulte mrophges. In ition, in vitro nti-oxint pity of T. minut essentil oil ws exmine y ssessments of ROS, RNS n hyrogen peroxie (H O ) svenging ility using ABTS, soium nitrite, n H O svenging, respetively. It ws expete tht these stuies woul revel tht T. minut essentil oil exhiits ril svenging tivity (ginst superoxie nion, H O, n NO rils) in mrophges, in prt, ue to n inhiition of inos n NOX gene expression. Furthermore, it ws hypothesize for the first time tht T. minut essentil oil woul erese TNF-α mrna expression s prt of its known nti-inflmmtory hrter n seonrily ue to the ongoing quenhing of rils known to trigger formtion of these pro-inflmmtory ytokines.. Mterils n methos.. Chemils n regents Soium nitrite, soium sulphte, ABTS, Griess regent (nphthylethyleneimine, sulfnilmie, phosphori i), 3-(4, -imethylthizol--yl)-, -iphenyltetrzolium romie (MTT), fetl lf serum, Duleo s moifie egle meium, L-glutmine, imethysulfoxie (DMSO) n LPS (from Esherihi oli ; B4) were purhse from Sigm-Alrih (St, Louis, MO, USA) n Fluk (Heielerg, Germny). RNX- Plus uffer ws otine from Cingen (Tehrn, Irn). All other use hemils n regents were of the purest ommerilly ville prouts... Plnt mterils n T. minut essentil oil preprtion See of T. minut ws otine from Institute of Meiinl Plnts, Isfhn, Irn n ws grown in green house onitions in Sr ner Shirz, Irn. See of meiinl plnt ws grown in sterile soil. The Sees of T. minut were sown in experimentl greenhouse, in Septemer. One month lter otine seelings were trnsferre to experimentl fiel n istriute homogenously. The eril prts of plnts were hrveste t the flowering stge. The leves of the plnts were seprte from the stem n were rie in the she for 7 h. The ir-rie leves ( g) were hyro-istille for 3 h using n ll-glss Clevenger-type pprtus (Herl Exir Co., Mshh, Irn) oring to the metho outline y the British Phrmopei[3]. The yiel of T. minut essentil oil from lef mteril ws ner % (w/w). The otine essentil oil ws ehyrte over nhyrous soium sulphte n store t 4 C until nlyze y gs hromtogrphy-mss spetrometry (GC- MS) n ws then use..3. Ientifition of the T. minut essentil oil omponents GC nlysis ws rrie out using Agilent tehnology hromtogrph with HP- olumn (3 m 伊.3 mm, internl imeter. μm). Oven temperture ws performe s follows: 6 C to C t 3 C/min; C to 4 C t C/min n hol for 8. min, injetor temperture 8 C; etetor temperture, 9 C; rrier gs, N ( ml/min); split rtio of :. The OBO ws nlyze using n Agilent moel 789-A series gs hromtogrphy n Agilent moel 97-C mss spetrometry. The HP- MS pillry olumn (phenyl methyl siloxne, 3 m 伊. mm, internl imeter 伊 μm) ws use with helium t ml/min s the rrier gs. GC oven temperture ws progrmme from 6 C to C t rte of 3 C/min n ws then inrese from C to 4 C t rte of C/min n ws kept onstnt t 4 C for 8. min. The split rtio ws juste to : n the injetion volume ws ml. The injetor temperture ws 8 C. The qurupole mss spetrometer ws snne over 4- mu with n ionizing voltge of 7 ev. Retention inies were etermine using retention times of n-lknes (C 8 -C ) tht were injete fter the T. minut essentil oil uner the sme hromtogrphi onitions. The retention inies for ll omponents were etermine oring to the metho tht uses n-lknes s stnr. The ompouns were ientifie y omprison of retention inies with those reporte in the literture n y omprison of their mss spetr with the Wiley GC-MS Lirry, Ams Lirry, Mss Finer. Lirry t pulishe mss spetr t[4]..4. ROS svenging ssy The ROS svenging tivity of the T. minut essentil oil ws etermine s previously esrie[]. Briefly,

3 Prstoo Krimin et l./asin P J Trop Biome 4; 4(3): 9-7 μl of the T. minut essentil oil (- μg/ml in DMSO) ws e to. ml of ilute ABTS ril solution (7 mmol/l ABTS n.4 mmol/l potssium persulfte). After mixing, the sorne (A) ws re t 734 nm using n Ultrospe spetrophotometer (Phrmi, Uppsl, Sween). The perentge of ROS svenging ws lulte s [(A734 lnk - A734 smple )/A734 lnk ] 伊. The onentrtions tht oul provie % inhiition (IC ) were lulte from the grph tht plotte the inhiition perentge ginst ifferent T. minut essentil oil onentrtions... H O svenging ssy H O svenging tivity of the T. minut essentil oil ws etermine s previously esrie[6]. Briefly, μl of the T. minut essentil oil (- μg/ml in DMSO) ws inute with. ml of H O ( mmol/l in mmol/l phosphte uffer ph 7.4) t 37 C for 6 min. After inution, the sorne (A) ws re t 3 nm ginst lnk solution ontining phosphte uffer without H O using spetrophotometer. The perentge of H O svenging ws lulte s [(A3 lnk -A3 test )/ A3 lnk ] 伊. IC ws lulte from the grph tht plotte the inhiition perentge ginst ifferent T. minut essentil oil onentrtions..6. RNS svenging ssy RNS svenging tivity of the T. minut essentil oil ws etermine s previously esrie[6]. Briefly, μl of the T. minut essentil oil (- μg/ml in DMSO) ws inute with. ml of soium nitrite ( μg/ml in mmol/l soium itrte ph ) t 37 C for h. After inution,. ml of Griess regent ws e n the sorne (A) ws re t 4 nm using spetrophotometer. The perentge of RNS svenging ws lulte s follows: [(A4 lnk -A4 smple )/A4 lnk 伊. IC ws lulte from the grph tht plotte the inhiition perentge ginst ifferent T. minut essentil oil onentrtions..7. Mrophges ell ulture The J774.A murine mrophge ell line ws otine from the ell nk of the Psteur Institute of Irn, Tehrn. Cells were ulture in Duleo s moifie egle meium ontining mmol/l L-glutmine, IU/mL peniillin, μg/ ml streptomyin n % het-intivte fetl lf serum t 37 C in humiifie CO inutor. Cultures were llowe to grow until onfluene t whih point herent mrophges were srpe from the flsk n were wshe with wrm meium ( C). Cells were ounte n their viility ws etermine y trypn lue ye exlusion. The ells were seee t onentrtion of 伊 6 ells per millilitre in 4-well tissue ulture pltes in triplite (Jet Biofil, Kyoto, Jpn). After ulturing for 8 h to llow ells to here, non-herent ells were remove y gentle rinsing with meium. Remining herent ells were then ulture in the presene or sene of meium ering LPS ( μg/ml). After h, T. minut essentil oil ws e t finl onentrtion of - μg/ml. Two sets of wells without T. minut essentil oil ut ontining LPS n DMSO solvent (.%) were use s negtive ontrols. After 4 h of inution t 37 C, the ulture superntnts in eh well were remove n the ells hrveste were use for RNA extrtion n rel-time PCR nlysis..8. Cell viility ssy The effet of T. minut essentil oil on the viility of J774A. ells ws etermine y MTT ssy s esrie previously[7]. Cells ( 4 ells per well) were inute for 4 h (t 37 C in % CO ) with ifferent onentrtions (- μg/ ml) of T. minut essentil oil. Therefter, μl of MTT ( mg/ ml) ws e to eh well n inute for n itionl 4 h t 37 C followe y tretment with μl of lysis uffer (% SDS in mmol/l HCl). The sorne of eh well ws etermine y spetrophotometer t ul wvelengths of 7 n 63 nm on miroplte ELISA reer (BioTek Elx 88, Winooski, VT, 43, USA). Viility perentge ws lulte y the following formul (Asorne of trete ells/asorne of orresponing ontrol) 伊. The ontrol ws T. minut essentil oil-untrete ells ontining DMSO t the highest onentrtion use (.%). The onentrtion tht provie % inhiition (IC ) ws lulte from grph, plotting the inhiition perentge ginst ifferent T. minut essentil oil onentrtions..9. RNA extrtion n DNA synthesis Totl RNA ws extrte using RNX-plus uffer from Cingen, Tehrn, Irn. Briefly, out 伊 6 ells were trnsferre to ml of RNX-plus uffer in n RNse-free mirotue, mixe thoroughly n left t room temperture for min. A volume of μl of hloroform ws e to the slurry n ws mixe gently. The mixture ws entrifuge t 3 g t 4 C for min, the superntnt ws trnsferre to new tue n ws preipitte with n equl volume of isopropnol for min on ie. The RNA pellet ws wshe using 7% ethnol, riefly rie n resuspene in μl of RNse free wter. The purifie totl RNA ws quntifie y NnoDrop ND spetrophotometer (Wilmington, DE). A smple (. mg) of RNA ws use for first strn DNA synthesis, using pmol oligo-t (8 mer), pmol NTPs, U RNse inhiitor, n U M-Mulv reverse trnsriptse (ll from Ferments, Hnover, MD) in. ml finl volume... Quntittive rel-time PCR Primer esign, in the form of exon juntion ws rrie out using AlleleID 7 softwre (Premier Biosoft Intl., Plo Alto, CA) for the internl ontrols glyerlehyes-3-phosphte ehyrogense (GAPDH) (NM-97) n β-tin (NM ) n teste genes NOX pphox (NM-786), NOX p4phox (NM- 8677), NOX p47phox (NM-876), NOX p67phox (NM-877), inos (NM-884) n TNF-α (NM-3693) (Tle ). The GAPDH n β-tin were use s internl ontrol (whose expression prove not to e influene y LPS) for t normliztion[8]. Reltive rel-time PCR ws performe in μl volume ontining μl DNA, 伊 Syer Green uffer (Qigen, Hilen,

4 Prstoo Krimin et l./asin P J Trop Biome 4; 4(3): 9-7 Germny) n 4 pmol of eh primer. The mplifition retions were rrie out in line Gene k therml yler (Bioer Tehnology Co., Hngzhou, Chin) with initil enturing of 94 C for min, followe y 4 yles of 94 C for seons, nneling temperture of eh primer pir ws one for seons n 3 seons for extension to our t 7 C. After 4 yles, the speifiity of the mplifitions ws heke se on the melting urves resulting from heting the mplions from C to 9 C. All mplifition retions were repete twie uner ientil onitions esie negtive ontrol n stnr smples. To ensure tht the PCR ws generte from DNA n not genomi DNA, proper ontrol retions were rrie out without the reverse trnsriptse tretment. For quntittive rel time PCR t, reltive expression of NOXs, inos n TNF-α gene were lulte se on the threshol yle (CT) metho. The CT for eh smple ws lulte using the Line-gene K softwre n the metho of Lrionov et l[9]. Aoringly, fol-expression of trget mrnas over the referene vlues were lulte y eqution -ΔΔCT, where ΔCT ws etermine y sutrting the orresponing internl ontrol CT vlue from the speifi CT of trgets, n ΔΔCT ws otine y sutrting the ΔCT of eh experimentl smple from tht of the ontrol smple[3]... Sttistil nlysis All t re expresse s mens plus stnr evitions of t lest three inepenent experiments. The signifint ifferenes etween tretments were nlyze y One-wy nlyses of vrine (ANOVA) test t P<. using sttistil pkge for the soil sienes (SPSS, Aus Conepts, Berkeley, CA) n Prism (Grph Ph, Sn Diego, USA) softwre. Tle Primer use for rel-time nlysis. Genes Aession Sense sequene Anti sense sequene GAPDH NM-97 -CGGTGTGAACGGATTTGGC-3 -TGAGTGGAGTCATACTGGAAC-3 β-tin NM CCACACCCGCCACCAGTTCG-3 -CTAGGGCGGCCCACGATGGA-3 NOX p NM ATGGAGCGATGTGGACAG-3 - ACCGACAACAGGAAGTGG-3 NOX p4 NM CAACAAAGACTGGCTGGAG-3 -CCGCAATGTCCTTGATGG-3 inox p47 NM ATGGCACAAAGGACAATC-3 - ACCTGAGGCTATACACAAG-3 NOX p67 NM CAGCCACAGTCAGCAGAG-3 -GCACAAAGCCAAACAATACG-3 inos NM CTGGAGGTTCTGGATGAG-3 - CTGAGGGCTGACACAAGG -3 TNF-α NM GTCTCAGCCTCTTCTCATTC-3 - GGAACTTCTCATCCCTTTGG-3 Primer esign, in form of exon juntion ws rrie out using Allele ID 7 softwre for the internl ontrols GAPDH n β-tin n test genes NADH oxise p phgoytes oxise (NOX pphox), NOX p47phox, NOX p47phox, NOX p67phox, inos n TNF-α genes from Mus musulus sequene. 3. Results 3.. Plnt mterils The T. minut essentil oil ws prepre y wter-istilltion, n its hemil omposition ws etermine y GC-MS. As shown in Tle, GC-MS nlysis inite tht the min omponents were ihyrotgetone (33.86%), E-oimene (9.9%), tgetone (6.%), is-β-oimene (7.94%), Z-oimene (.7%), limonene (3.%) n epoxyoimene (.3%). GC-MS nlysis of the essentil oil inite the min omponents T. minut essentil oil were ihyrotgetone, E-oimene, tgetone, is-β-oimene, Z-oimene, limonene n epoxyoimene. 3.. Antioxint tivity of T. minut essentil oil T. minut essentil oil isplye onentrtion epenent ROS, RNS n H O svenging tivity. IC for ROS, RNS n H O svenging were (. 依 3.), (. 依.) n (3. 依 4.) μg/ml of T. minut essentil oil, respetively. At onentrtions >3 μg/ ml, the T. minut essentil oil signifintly svenges ROS, RNS, n H O y %. The T. minut essentil oil nlyze here possesse potent in vitro ROS, RNS n H O svenging tivity. The T. minut essentil oil t >3 μg/ml h the ility to svenge ll ROS, RNS n H O rils, n initor of its poteny s ril svenger. Tle Chemil omposition of T. minut essentil oil. Compouns Retention inex % of ompouns α-pinene Sinene is-3-hexenyl ette p-cymene Limonene is-β-oimene Dihyrotgetone Chrysnthenone Allo-imene E, Z-Epoxyoimene Tgetone is-tgetone p-menth-,8 ien-3-one Z-Oimene E-Oimene Thymol Crvrol is-isoeugenol E-Cryophyllene α-humulene Germorene D Spthulenol

5 Prstoo Krimin et l./asin P J Trop Biome 4; 4(3): T. minut essentil oil reue ell viility t high onentrtions The MTT ssy results inite tht low onentrtions (- μg/ml) of T. minut essentil oil h no effet on J774A. ell viility. However, t higher onentrtions (- μg/ml), ell viility ws signifintly reue in onentrtion-relte mnner, with the mximum effet (% ell eth) t onentrtions > μg/ml (Figure ). Non-ytotoxi onentrtions (< μg/ml) were thus use for the susequent stuies inluing expression of genes T. minut essentil oil reue NOX pphox mrna expression in LPS-stimulte mrophges The un-stimulte (ontrol) ells showe low level of NOX pphox mrna expression. LPS stimultion of mrophges resulte in n inrese in NOX pphox mrna expression (6. 依.7) fol of LPS-untrete ontrol ells (P<.). The ition of T. minut essentil oil t to μg/ml signifintly erese the NOX pphox mrna expression in LPS-trete ells from (. 依.7) to (4. 依.8) fol of the ontrol (P<.) ose-epenently, initing the inhiitory effet of T. minut essentil oil on pphox mrna inution/ formtion (Figure ). 3.. T. minut essentil oil reue NOX p4phox mrna Expression in LPS-Stimulte Mrophges The un-stimulte (ontrol) ells showe low level of NOX p4phox mrna expression while, the expression of NOX p4phox mrna in LPS-trete ells ws (6.3 依.4) fol of the ontrol (P<.). The ition of T. minut essentil oil t to μg/ml signifintly erese the NOX p4phox mrna expression in LPS-trete ells from (. 依.7) to (.4 依.) fol of the ontrol, ose-epenently (P<.), initing the inhiitory effet of T. minut essentil oil on p4phox mrna inution/formtion (Figure 3) * 4 * 3 T. minut essentil oil (μg/ml) Figure. Effet of the T. minut essentil oil on the viility of J774 ell lines. TMO: T. minut essentil oil. The ells were trete with vrious onentrtions of essentil oil (- μg/ml) n inute for 4 h. Control ( μg/ml) ws ells trete only with the solvent (DMSO) t onentrtion of.%. Dt represent men 依 SD from three sets of inepenent experiments. Cell viility (%) p4 phox mrna (fol of ontrol) Control LPS LPS+TMO LPS+TMO LPS+TMO Tretment Figure 3. Effets of T. minut essentil oil on NOX p4phox mrna expression in LPS-stimulte mrophges. TMO: T. minut essentil oil. The ells were ulture in 4-well pltes n trete with n without LPS. Vrious onentrtions of essentil oil (- μg/ml) were e. After 4 h, the expression of NOX p47phox mrna ws nlyze y rel-time PCR. Cells trete with DMSO s the solvent (ontrol) n ells trete with the solvent n LPS ws onsiere s positive ontrols. p phox mrna (fol of ontrol) 3 e Control LPS LPS+TMO LPS+TMO LPS+TMO Tretment Figure. Effets of T. minut essentil oil on NOX pphox mrna expression in LPS-stimulte mrophges. TMO: T. minut essentil oil. The ells were ulture in 4-well pltes n trete with n without LPS. Vrious onentrtions of essentil oil (- μg/ml) were e. After 4 h, the expression of NOX pphox mrna ws nlyze y rel-time PCR. Cells trete with DMSO s the solvent (ontrol) n ells trete with the solvent n LPS ws onsiere s positive ontrols. 6 p47 phox mrna (fol of ontrol) 4 3 Control LPS LPS+TMO LPS+TMO LPS+TMO Tretment Figure 4. Effets of T. minut essentil oil on NOX p47phox mrna expression in LPS-stimulte mrophges. TMO: T. minut essentil oil. The ells were ulture in 4-well pltes n trete with n without LPS. Vrious onentrtions of TMO (- μg/ml) were e. After 4 h, the expression of NOX p47phox mrna ws nlyze y rel-time PCR. Cells trete with DMSO s the solvent (ontrol) n ells trete with the solvent n LPS ws onsiere s positive ontrols.

6 4 Prstoo Krimin et l./asin P J Trop Biome 4; 4(3): T. minut essentil oil reue NOX p47phox mrna expression in LPS-stimulte mrophges The un-stimulte (ontrol) ells showe low level of NOX p47phox mrna expression while, the expression of NOX p47phox mrna in LPS-trete ells ws (. 依.) fol of the ontrol inution/formtion (P<.). The ition of T. minut essentil oil t to μg/ml signifintly erese this gene expression in LPS-trete ells from (. 依.) n (.6 依.8) fol of ontrol, ose-epenently (P<.) initing the inhiitory effet of T. minut essentil oil on p47phox mrna inution/formtion (Figure 4) T. minut essentil oil reue NOX p67phox mrna expression in LPS-stimulte mrophges With respet to NOX p67phox, erese in the gene expression ws etete in LPS-stimulte mrophges whih were trete with T. minut essentil oil. The reltive NOX p67phox mrna expression in ells trete with LPS lone ws (. 依.) fol of LPS-untrete the ontrol ells (P<.). The ition of T. minut essentil oil t to μg/ml signifintly erese the NOX p67phox mrna expression in LPS-trete ells from (4. 依.) n (. 依.4) fol of the ontrol, oseepenently (P<.), initing the inhiitory effet of T. minut essentil oil on p67phox mrna inution/formtion (Figure ). p67 phox mrna (fol of ontrol) Control LPS LPS+TMO LPS+TMO LPS+TMO Tretment Figure. Effets of T. minut essentil oil on NOX p67phox mrna expression in LPS-stimulte mrophges. TMO: T. minut essentil oil. The ells were ulture in 4-well pltes n trete with n without LPS. Vrious onentrtions of TMO (- μg/ml) were e. After 4 h, the expression of NOX p67phox mrna ws nlyze y rel-time PCR. Cells trete with DMSO s the solvent (ontrol) n ells trete with the solvent n LPS ws onsiere s positive ontrols T. minut essentil oil reue inos mrna expression in LPS-stimulte mrophges LPS stimultion of mrophges resulte in n inrese in inos mrna expression (7. 依.4) fol of LPS-untrete ells (P<.). The ition of T. minut essentil oil t onentrtions to μg/ml signifintly erese the inos mrna expression in LPS-trete ells from (.4 依.4) to (3.8 依.) fol of untrete ells, ose-epenently (P>.) initing the inhiitory effet of T. minut essentil oil on inos mrna inution/formtion (Figure 6). inos mrna (fol of ontrol) 3 3 e Control LPS LPS+TMO LPS+TMO LPS+TMO Tretment Figure 6. Effets of T. minut essentil oil on inos mrna expression in LPS-stimulte mrophges. TMO: T. minut essentil oil. LPS-stimulte mrophges. The ells were ulture in 4-well pltes n trete with n without LPS. Vrious onentrtions of TMO (- μg/ml) were e. After 4 h, the expression of NOS mrna ws nlyze y rel-time PCR. Cells trete with DMSO s the solvent (ontrol) n ells trete with the solvent n LPS ws onsiere s positive ontrols T. minut essentil oil reue TNF-α mrna expression in LPS-stimulte mrophges LPS stimultion of mrophges resulte in n inrese in TNF-α mrna expression ompre to the onitions in LPSuntrete ells (9.8 依.) fol (P<.). The ition of T. minut essentil oil t to μg/ml signifintly erese the TNF-α mrna expression from (9.7 依.4) n (3.3 依.6) fol of the ontrol, ose-epenently (Figure 7). These t inite the inhiitory effet of T. minut essentil oil on TNF-α mrna inution/formtion. TNF mrna (fol of ontrol) e Control LPS LPS+TMO LPS+TMO LPS+TMO Tretment Figure 7. Effets of T. minut essentil oil on TNF mrna expression in LPSstimulte mrophges. TMO: T. minut essentil oil. The ells were ulture in 4-well pltes n trete with n without LPS. Vrious onentrtions of TMO (- μg/ml) were e. After 4 h, the expression of TNF mrna ws nlyze y reltime PCR. Cells trete with DMSO s the solvent (ontrol) n ells trete with the solvent n LPS ws onsiere s positive ontrols. 4. Disussion The ntioxint n nti-inflmmtory effets of T. minut essentil oil were investigte in the present stuy. GC-MS nlysis of the essentil oil inite the min omponents

7 Prstoo Krimin et l./asin P J Trop Biome 4; 4(3): 9-7 in T. minut essentil oil were ihyrotgetone, E-oimene, tgetone, is-β-oimene, Z-oimene, limonene n epoxyoimene. Previous stuy reporte the min omponents of T. minut essentil oil were β-oimene, ihyrotgetone, tgetone, Z-oimene n E-oimene[]. Another stuy reporte thiophenes n polyetyleni ompouns in the Tgetes speies n T. minut h the highest totl thiophene yiel[]. Aoringly, the min omponents of T. minut essentil oil oul e tgetone (is/trns, ketone/lohol, lehye/lohol), oimene (is/trns, ketone/lohol, lehye/lohol) n thiophene erivtives[-4]. For the resons tht, essentil oils omposition epen on the speies, limte, ltitue, time of olletion n growth stge, thus the plnts nlyze in this reserh h roughly sme omponents with other previously nlyze T. minut essentil oil however, showe importnt ifferenes in their qulity n quntity of omponents. The T. minut essentil oil nlyze here possesse potent in vitro ROS, RNS n H O svenging tivity. The T. minut essentil oil t >3 μg/ml h the ility to svenge ll ROS, RNS n H O rils, n initor of its poteny s ril svenger. ROS re oxygen-erive smll moleules, inluing oxygen rils suh s superoxie, hyroxyl n peroxyl n some non-rils tht re esily onverte into rils, suh s hyrogen peroxie. ROS, one proue, n intert with vrious moleules inluing other smll inorgni moleules s well s mromoleules suh s proteins n lipis. During these intertions, ROS my estroy or hnge the funtion of the trget moleule[3]. The ROS reuing tivity of T. minut essentil oil oserve in our stuy imply the enefiil role of this prout for reuing mges in iologil tissues. The ril svenging tivity of ompouns is minly ue to their oxition-reution potentil, whih n ply n importnt role in neutrlizing free rils. This tivity is relte to phenoli hyroxyl groups[3]. T. minut essentil oil minly ontins ihyrotgetone, oimene, tgetone n limonene whih ll re monoterpenes. This ntioxint tivity ws onfirme y previous reserh with IC etween μg/ml[,]. Thus, T. minut essentil oil nlyze in this reserh showe stronger ntioxint tivity rther thn previously nlyze one. In vitro inhiition of the NO ril is mesure of ntioxint tivity of plnt extrts. As results of the present stuy show, the T. minut essentil oil use here hve the ility to svenge totl RNS t onentrtion >3 μg/ml. The MTT ssy results inite tht low onentrtions (- μg/ml) of T. minut essentil oil h no effet on J774A. ell viility (IC =9 μg/ml). In orer to etermine the ntioxint n nti-inflmmtory effets of T. minut essentil oil on mrophges, the onentrtions of > μg/ml T. minut essentil oil whih were overtly ytotoxi to the ells were not use. Although the onstituents of essentil oils n t s ntioxints, they my lso t s pro-oxints n ffet inner ell memrnes n orgnelles suh s mitohonri in eukryoti ells. Depening on the type n onentrtion, this effet my result in ellulr ytotoxiity. In mrophges ROS proution is uner the ontrol of NOX. This multi-omponent enzyme onsists of severl ytosoli omponents inluing p9phox, p67phox, p4phox, p47phox n the smll Rho G protein (R or R, R: Rho-relte C3 otulinum toxin sustrte), whih ssemle on the ellulr memrne to tivte the enzyme[33]. Stuies hve shown tht phosphoryltion of p47phox les to onformtionl hnges, llowing its trnslotion n intertion with pphox. Trnslotion of p47phox rings with it the other suunits, p67phox n p4phox to the memrne[34]. Ativtion of this enzyme omplex les to fusion of the vesiles ontining NOX with the plsm memrne or the phgosoml memrne. The tive enzyme onverts moleulr oxygen to superoxie nion through one-eletron trnsfer[3]. As our stuy showe, T. minut essentil oil ws le to erese the expression of key omponents of NOX. It hs een shown tht the ssemly of p47phox, p67phox n pphox t the memrne is neessry for oxise funtion[36]. Thus, it n e ssume tht reue ROS genertion y stimulte mrophges in the presene of T. minut essentil oil might e, in prt, ue to the moultion of the expression of NOX suunits. In ition to ROS, the overproution of RNS y tivte mrophges seems to ply n importnt role in ifferent steps of mny inflmmtory proesses[37]. RNS re nitrogenontining oxints, minly NO whih is free ril plying key role in the pthogenesis of pin n inflmmtion. NO in mrophges is generte y tivtion of inos. This enzyme hs the ility to proue high onentrtions of NO fter stimultion with teril enotoxins or vriety of proinflmmtory ytokines suh s TNF-α, IL- n IL-6[38]. NO genertion involves severl steps inluing the tivtion of nuler trnsription ftor (NF)-κB n susequent inos gene expression[39]. NF-κB regultes the expression of vrious genes involve in inflmmtory responses. Its tivtion n lso e regulte y vrious ytokines, mong whih TNF-α is the most importnt. In response to inflmmtory stimuli suh s LPS, mrophges serete vriety of inflmmtory meitors suh s TNF-α n IL-β. The proution of TNF-α ytokine is importnt for the inution of NO synthesis in LPS-stimulte mrophges[4]. As results of this stuy showe tht T. minut essentil oil ws le to reue inuile expression of TNF-α gene, whih inite tht the reue NO proution seen in the mrophge ultures might e prtly relte to the suppression of TNF-α expression. TNF-α is known to ply ruil role in inflmmtory responses n is involve in the pthogenesis of inflmmtory iseses[4]. As results of our stuy showe, T. minut essentil oil signifintly reue inos mrna expression in stimulte mrophges. Suppression of TNF-α expression in mrophges s well s reue inos gene expression, ue to the T. minut essentil oil inites the ility of this prout to iminish immune retions n provies further eviene tht this plnt my hve potent immuno-moultory properties. Consiering ll these fining, T. minut essentil oil isplye n nti-oxint property y svenging superoxie, H O n NO rils, n reue oxitive stress. This suggeste tht there ws potentil for use of this prout in the therpy of oxitive mge, proess tht usully ompnies inflmmtory onitions. The erese formtion of ROS n NOS rils in mrophges ws possily ue to the

8 6 Prstoo Krimin et l./asin P J Trop Biome 4; 4(3): 9-7 ril svenging tivity of phenoli groups present in the oil n/or ue to n inhiition of inos n NOX gene expressions. Furthermore, T. minut essentil oil erese the expression of the genes for pro-inflmmtory ytokine TNF-α. Therefore, reue expression of the ove-note inflmmtory enzymes n ytokines oul e ttriute to suppression of the NFkB pthwy in the trete ells. These t suggest potentil therpeuti usefulness for T. minut in the moultion of mrophges n provies eviene to support the use of T. minut s te/itive/tritionl remey for tretment of inflmmtory iseses. Further in vivo stuies re reommene to more fully unerstn the therpeuti potentil of T. minut essentil oil in multitue of inflmmtory isorers. Conflit of interest sttement We elre tht we hve no onflit of interest. Aknowlegements This work ws supporte y the funing from Shirz University (Grnt no. 88-GR-AGRST-8) n Shirz University of Meil Siene (Grnt No. 3937). Comments Bkgroun T. minut is n romti plnt tht hs severl meil enefits suh s remey for the ols, respirtory inflmmtions, stomh prolems, nti-spsmoi, ntiprsiti, ntisepti n setive. This work ime to investigte ntioxint n nti-inflmmtory effets of T. minut essentil oil. Reserh frontiers The im of the present stuy ws to investigte the level of potentil moulting effets of T. minut essentil oil on mrophges n their relte funtions inluing expression of NOX suunits, NOS n TNF-α mrnas in LPS-stimulte mrophges. In ition, in vitro ntioxint pity of T. minut essentil oil ws exmine y ssessments of ROS, RNS n H O svenging ility. Relte reports Avnes in hemil n phrmologil evlutions of T. minut essentil oil suh s ntioxint n ntimiroil hve ourre in the pst reent yers. However, severl useful fetures of this plnt (e.g. the mehnisms unerlying its ntioxint n nti-inflmmtory effets) hve remin unknown. Innovtions n rekthroughs These stuies woul revel tht T. minut essentil oil exhiits ril svenging tivity (ginst superoxie nion, H O, n NO rils) in mrophges, in prt, ue to n inhiition of inos n NOX gene expression. Applitions These t suggest potentil therpeuti usefulness for T. minut in the moultion of mrophges n provies eviene to support the use of T. minut s te/itive/ tritionl remey for tretment of inflmmtory iseses. Peer review T. minut essentil oil isplye n nti-oxint property y svenging superoxie, H O n NO rils, n reue oxitive stress. The erese formtion of ROS n NOS rils in mrophges ws possily ue to the ril svenging tivity of phenoli groups present in the oil n/or ue to n inhiition of inos n NOX gene expressions. Furthermore, T. minut essentil oil erese the expression of the genes for pro-inflmmtory ytokine TNF-α. Referenes [] Irnin Herl Phrmopoei. Tehrn: Irnin Ministry of Helth & Meil Eution Pulitions;. [] Nerio LS, Olivero-Verel J, Stshenko E. Repellent tivity of essentil oils: review. Bioresour Tehnol ; : [3] Rhimi R, Shms-Arekni MR, Aollhi M. A review of the effiy of tritionl Irnin meiine for inflmmtory owel isese. Worl J Gstroenterol ; 6: [4] Nikkon F, Hi MR, Su ZA, Krim MR. Tgetes eret n its mosquitoil poteny ginst Culex quinquefsitus. Asin P J Trop Biome ; : [] Kmrj C, Rhumn AA, Bgvn A, Elngo G, Zhir AA, Snthoshkumr T. Lrviil n repellent tivity of meiinl plnt extrts from Estern Ghts of South Ini ginst mlri n filrisis vetors. Asin P J Trop Me ; 4: [6] Govinrjn M. Lrviil n repellent properties of some essentil oils ginst Culex triteniorhynhus Giles n Anopheles supitus Grssi (Dipter: Culiie). Asin P J Trop Me ; 4: 6-. [7] Aristtile B, Al-Assf AH, Pugleni KV. Crvrol suppresses the expression of inflmmtory mrker genes in D-gltosmine-heptotoxi rts. Asin P J Trop Me 3; 6: -. [8] Mity N, Nem NK, Aey MK, Srkr BK, Mukherjee PK. Exploring Tgetes eret Linn flower for the elstse, hyluronise n MMP- inhiitory tivity. J Ethnophrmol ; 37: 3-3. [9] Wng M, Tso R, Zhng S, Dong Z, Yng R, Gong J, et l. Antioxint tivity, mutgeniity/nti-mutgeniity, n lstogeniity/nti-lstogeniity of lutein from mrigol flowers. Foo Chem Toxiol 6; 44: -9. [] Breme K, Tournyre P, Fernnez X, Meierhenrih UJ, Brevr H, Joulin D, et l. Ientifition of oor impt ompouns of Tgetes minut essentil oil: omprison of two GC-olftometry methos. J Agri Foo Chem 9; 7: [] Lopez SB, Lopez ML, Argon LM, Tereshuk ML, Slnis AC,

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