Hindwi Pulishing Corportion Biochemistry Reserch Interntionl Volume 214, Article ID 493183, 7 pges http://dx.doi.org/1.1155/214/493183 Reserch Article Effect of Inhling Cymopogon mrtinii Essentil Oil nd Gerniol on Serum Biochemistry Prmeters nd Oxidtive Stress in Rts Brun Fernnd Murch Teles Andrde, 1 Cmil Pereir Brg, 2 Klinsmnn Crolo dos Sntos, 2 Lidine Nunes Bros, 1 Ver Lúci Mores Rll, 1 José Murício Sforcin, 1 An Angélic Henrique Fernndes, 2 nd Ary Fernndes Júnior 1 1 Deprtment of Microiology nd Immunology, Institute of Biosciences, UNESP, 18618-97 Botuctu, SP, Brzil 2 Deprtment of Chemistry nd Biochemistry, Institute of Biosciences, UNESP, 18618-97 Botuctu, SP, Brzil Correspondence should e ddressed to Ary Fernndes Júnior; ry@i.unesp.r Received 13 Octoer 214; Accepted 23 Novemer 214; Pulished 9 Decemer 214 Acdemic Editor: Tzi Bun Ng Copyright 214 Brun Fernnd Murch Teles Andrde et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. The effects of the inhltion of Cymopogon mrtinii essentil oil (EO) nd gerniol on Wistr rts were evluted for iochemicl prmeters nd heptic oxidtive stress. Wistr rts were divided into three groups (n = 8): ws control group, treted with sline solution; received gerniol; nd received C. mrtinii EOy inhltion during3 dys. No significnt differences were oserved in glycemi nd tricylglycerol levels; nd decresed (P <.5) totl cholesterol level. There were no differences in serum protein, ure, sprtte minotrnsferse ctivity, nd totl heptic protein. Cretinine levels incresed in ut decresed in. Alnine minotrnsferse ctivity nd lipid hydroperoxide were higher in thn in. Ctlse nd superoxide dismutse ctivities were higher in. C. mrtinii EO nd gerniol incresed glutthione peroxidse. Oxidtive stress cused y gerniol my hve triggered some degree of heptic toxicity, s verified y the increse in serum cretinine nd lnine minotrnsferse. Therefore, the eneficil effects of EO on oxidtive stress cn prevent the toxicity in the liver. This proves possile interctions etween gerniol nd numerous chemicl compounds present in C. mrtinii EO. 1. Introduction Plnts synthesize round 2, secondry metolites or specilized phytochemicls, of which essentil oils (EOs) constitute n importnt group [1]. These compounds cn e extrcted from plnt tissues (e.g., stem, leves, flowers, nd roots) y severl procedures (e.g., hydrodistilltion nd stem distilltion) [2]. These compounds re mostly terpenes, which re commonly used in phrmceuticl industries nd hve therpeutic enefits nd promote welfre, especilly when used in romtherpy procedures [3]. Cymopogon mrtinii (Rox.), Wtson, populrly known s plmros, exhiits eneficil effects on severl centrl nervous system pthologies, minly neurlgi, epileptic, nd norexi [4]. There re few reports on its effects; still C. mrtinii hs ttrcted mny reserchers ttention due to its ntimicroil, ntigenotoxic, nd ntioxidnt ctivities [5 8]. Countries such s Indi, Brzil nd Mdgscr hve the prctice to produce EOs from this plnt. Gerniol, the mjor constituent of C. mrtinii EO, is n cyclic monoterpenoid tht is undnt in mny plnts [9]. It my represent new clss of therpeutic gents ginst pncretic [1] nd colon cncers [11] nd hs severl iologicl properties, including ntimicroil, ntioxidnt nd ntiinflmmtory ctivities [12]. Gerniol is lso n importnt constituent of ginger, lemon, lime, lvender, nutmeg, ornge nd rose EOs [13]. Also, it is used s flvoring gent nd ws determined to e sfe t the current levels of intke y the Joint Expert Committee on Food Additives
2 Biochemistry Reserch Interntionl of Food nd Agriculture Orgniztion FAO/World Helth Orgniztion WHO [14]. Aromtherpy is trditionl tretment tht uses EOs. Its effects egin when the romtic molecule psses through the nsl cvity nd dheres to the olfctory epithelium, cusing nerve stimultion directly to the hippocmpus nd limic mygdloidl ody. This consequently triggers stimuli tht control the utonomic nervous system nd internl secretory control y chnging numer of vitl rections [15]. The inhltion of romtic compounds present in EOs is the reson for the nme romtherpy nd this therpy my hve sedting or stimulting effects on the individul [16]. Reports in the literture descrie the enefits of using EOs in romtherpy on the welleing of individuls, including improvements in mood, stress, nxiety, depression, nd chronic pin, nd promote so therpeutic, psychologicl, nd physiologicl effects [17]. The inhltion of EOs elevted lood pressure nd renl sympthetic ctivity, which enforces the ide tht these components ct in the centrl nervous system nd pss through the lood-rin rrier [18]. Voltile orgnic compounds re highly lipophilic nd my esily cross the lood-rin rrier nd esily exert their neurophrmcologicl nd toxicologicl effects. While studiesonthetoxiceffectsofthesecompoundsrereltively esy to perform, the centrl effects induced y the perception of odor (e.g., in romtherpy) re inherently complex. This is why the toxicologicl studies performed using voltile compounds re much more dvnced [19]. Mny studies hve een conducted in vitro with the purpose of verifying the iologicl properties of EOs [2, 17, 2]; usully they re performed using in vitro ssys. On the other hnd, when the tests re performed in vivo, the products re usully dministered in their liquid forms (e.g., y gvge or intrperitonel), with few studies in the voltile stte (i.e., y inhltion) [21]. Furthermore, EOs re used s flvoring gents in food products [14] ndrelsousedin dermtology nd in the frgrnce nd cosmetics industries. Specificlly, gerniol is extensively used in the mnufcture of oth household nd cosmetics products [12]. Since people re often use EOs, it is importnt to evlute the possile heptotoxic effects of these oils. Liver is the min detoxifiction orgn; the ctolism of oth endogenous nd exogenous compounds tkes plce in the liver. As result it is exposure to toxic gents which cn cuse drug-induced heptic dysfunction. Therefore, studies on serum ctivity of lnine minotrnsferse (ALT) nd sprtte minotrnsferse (AST), which re iomrkers of liver dmge, re importnt. The serum ctivity of ALT nd AST is frequently used in clinicl settings for dignostic heptic toxicity [22]. Numerous chronic degenertive diseses re ssocited with oxidtive stress, which occurs when there is excess formtion of rective oxygen species (e.g., superoxide, hydroxyl, nd hydrogen peroxide) nd insufficient defense y the ntioxidnt system (enzymtic nd nonenzymtic). This imlnce etween pro- nd ntioxidnts my cuse cell injury nd deth, which consequently led to tissue dysfunction [23, 24]. It is well estlished tht oxidtive stress plys fundmentl role in the pthogenesis of heptic disese, especilly nonlcoholic stetoheptitis [25]. In ddition, during heptic ctolism of xenoiotics, excessive production of rective oxygen species (ROS) occurs [26]. Our im ws to investigte the effect of inhltion of the C. mrtinii EO nd gerniol on serum iochemicl prmeters, iomrkers of heptotoxicity, nd oxidtive stress in heptic tissue. 2. Mterils nd Methods 2.1. Cymopogon mrtinii EO nd Gerniol. C. mrtinii EO ws supplied y the compny By Smi Aromterpi (São Pulo, SP) nd showed the following chemicl composition: gerniol (57.5%), gernyl cette (13.6%), linlool (1.7%), β-cryophyllene (1.1%), nd ocimene (.3%) found y gs chromtogrphy-mss spectrometer (GC-MS). The gerniol with 98% of purity ws purchsed from Sigm Aldrich (St. Louis, MO, USA). 2.2. Animls nd Experimentl Procedure. The experimentl procedure ws pproved y the Ethicl Committee from Institute of Biologicl Science, São Pulo Stte University, Botuctu, Brzil, nd the nimls experiments were crried out in ccordnce with the principles nd guidelines of the Cndin Council on Animl Cre s outlined in the Guide to the Cre nd Use of Experimentl Animls. Mle Wistr rts (29 31 g) were rered in polypropylene cges mintined in controlled environment (temperture 22 ± 3 C; 5 55% humidity; nd 12-hour light : drk cycle), with free ccess to wter nd food (Purin Ltd., Cmpins, SP, Brzil). The rts were rndomly distriuted into three groups (n = 8). The rts in the control group () received sline solution y inhltion (sline =.9% g/v). The group received gerniol y inhltion nd the group received C. mrtinii EO y inhltion. The rts from ll groups were plced individully into chmers (18 mm 3 mm 29 mm) dpted from de Almeid et l. [27] nd sumitted to inhltion of gerniol (8.36 mg gerniol/l of ir, which corresponds to 136.2 μl of gerniol/perspex ox 14.5 L of ir) nd C. mrtinii EO (13.73 mg of C. mrtinii EO/L of ir, which corresponds to 227 μl of C. mrtinii EO/perspex ox 14.5 L of ir) for 1 minutes every 48 hours for 3 dys. The gerniol concentrtion ws clculted from the mount of gerniol found in the C. mrtinii EO. Food nd wter consumption were mesured dily t the sme time nd ody weights were determined once week. 2.3. Biochemicl Mesurements nd Oxidtive Stress. After 3dys,thenimlswerefstedovernight(12 14h)nd euthnized y cervicl decpittion under nesthesi (solution contining 1% ketmine chloride nd 2% xylzine chloride with dose of.1 ml/1 g ody weight). Blood ws collected nd the serum ws otined y centrifugtion t 6 rpm for 15 minutes. Serum glucose ws determined using n enzymtic colorimetric method fter incution with glucose oxidse/peroxidse. The totl mount of protein
Biochemistry Reserch Interntionl 3 Tle 1: Generl chrcteristics nd serum protein levels fter 3 dys for ll experimentl groups. Prmeters Groups Finl ody weight g 348.57 ± 43.65 328.4 ± 29.82 32.98 ± 39.9 Body weight gin g 37.47 ± 9.25 36.86 ± 8.22 38.53 ± 17.6 Finl food consumption g/dy 2.3 ± 4.4 19.25 ± 4.14 18.52 ± 3.78 Finl wter consumption ml/dy 248.75 ± 21.84 c 248.75 ± 16.2 c 211.88 ± 1.67, Serum protein g/dl 5,13 ±,83 5,39 ± 1,39 5,95 ± 1,13 Vlues re given s the men ± SD for ech group of eight nimls. Significntly different from ; P.5; significntly different from ; P.5;nd c significntly different from ; P.5. : untreted control; : treted with gerniol; nd : treted with essentil oil. ws estimted using the iuret regent nd the totl cholesterol concentrtion ws determined using the cholesterol esterse/oxidse enzymtic procedure. Tricylglycerols levels were mesured y enzymtic hydrolysis nd the finl formtion of quinoneimine, which is proportionl to the concentrtion of tricylglycerols present in the smple. Serum ure ws determined y ddition of urese nd phenol-hypochloride, which leds to the formtion of n indophenol-lue complex. The serum cretinine levels were estimted using rection with picric cid in lkline uffer to form yellow-ornge complex, whose color intensity is proportionl to the cretinine concentrtion in the smple. ALT nd AST ctivities were determined y using pyruvte nd oxlocette s sustrtes, wherein NADH is converted into NAD+ proportionl to the ctivities of these enzymes. Heptic smples (2 mg) were removed nd homogenized in.1 M phosphte uffer, ph 7.4, using Teflon-glss Potter-Elvehjem homogenizer. The homogente ws centrifuged (1, g for 15 minutes) nd the superntnt ws used to determine the concentrtion of heptic lipid hydroperoxide (LH) nd ctivities of ntioxidnt enzymes. Lipid hydroperoxide ctivity ws determined y the oxidtion of Fe +2 in the presence of rective mixture contining methnol, xylenol ornge, sulfuric cid, nd utylted hydroxytoluene. Ctlse ctivity ws ssyed using phosphte uffer contining hydrogen peroxide. The ctivity of glutthione peroxidse (GSH-Px) ws determined in the presence of phosphte uffer, NADPH 2, reduced glutthione, nd glutthione reductse. Superoxide dismutse (SOD) ctivity ws ssyed ccording to the method y mesuring the rte of reduction of nitrolue-tetrzole (NBT) in the presence of free rdicls generted y hydroxylmine. 2.4. Sttisticl Anlysis. Results re expressed s the men ± SD.Thesttisticlsignificnceetweenthegroupsws ssessed using one-wy nlysis of vrince (ANOVA) with Tukey s test to compre the mens of the experimentl group. The proility with P.5 ws considered significnt. 3. Results Inhltion of gerniol () nd of C. mrtinii EO () hd no effects on finl ody weight, ody weight gin, nd food intke of the rts (Tle 1). No ltertion in totl heptic protein ws oserved. While no significnt differences were oserved in the glycemi nd tricylglycerol levels, gerniol (mg/dl) 18 16 14 12 1 8 6 4 2, c Glucose Totl cholesterol Triglycerides Figure 1: Serum glucose, totl cholesterol, nd triglycerides levels fter 3 dys for ll experimentl groups. Vlues re given s the men ± SD for ech group of eight nimls. Significntly different from ; P.5; significntly different from ; P.5; nd c significntly different from ; P.5. : untreted control; : tretedwithgerniol;nd:tretedwithessentiloil. () nd C. mrtinii EO () decresed (P.5) totl cholesterol levels when compred with the control group (Figure 1). There were no significnt differences in serum ure levels etween the groups. Cretinine levels incresed in the presence of gerniol ut decresed in the presence of C. mrtinii EO (; Figure2). ALT ctivity ws higher in the group exposed to gerniol when compred to the other groups, which did not differ from ech other. No chnge ws found in the AST ctivity etween the groups (Figure 3). LH ws higher in the group thn in the group (Figure 4). Ctlse nd SOD ctivities were higher in the group when compred to the other groups. Both gerniol nd C. mrtinii EO incresed GSH-Px when compred to the control rts (Figure 5). 4. Discussion EOs re widely used in romtherpy procedures nd there is interest in reports on the heptic toxicity of these nturl products. However, few studies hve explored the effects of
4 Biochemistry Reserch Interntionl 12 3 c (mg/dl) 1 8 6 4 2 c (nmol/g tissue) 25 2 15 1 Ure Cretinine 5 LH Figure 2: Serum ure nd cretinine levels fter 3 dys for ll experimentl groups. Vlues re given s the men ± SD for groups of eight nimls ech. Significntly different from ; P.5; significntly different from ; P.5; c significntly different from ; P.5. : untreted control; : treted with gerniol; : treted with essentil oil. 14 Figure 4: Heptic lipid hydroperoxide levels fter 3 dys for ll experimentl groups. Vlues re given s the men ± SD for ech group of eight nimls. Significntly different from ; P.5; significntly different from ; P.5; nd c significntly different from ; P.5. : untreted control; : treted with gerniol; nd : treted with essentil oil. (U/L) 12 1 8 6 4 2, c 7 6 5 4 3, c, c, c ALT AST Figure 3: Serum ctivity of ALT nd AST fter 3 dys for ll experimentl groups. Vlues re given s the men ± SD for ech group of eight nimls. Significntly different from ; P.5; significntly different from ; P.5; nd c significntly different from ; P.5. : untreted control; : treted with gerniol; nd : treted with essentil oil. EOsonnorgnismwhendministeredyinhltion.Since EOs hve een extensively used in romtherpy due to their therpeutic properties nd lso used in food products, in dermtology, nd in the frgrnce nd cosmetic industries [14], there is interest in investigting their heptic toxicity, s well s dyslipidemi. C. mrtinii EO reduced the finl wter intke of the rts, utwithoutlteringotherprmetersthtwestudied.no significnt chnges were oserved in finl ody weight, ody weight gin, finl food intke, or totl serum protein level, indicting no dehydrtion nd no deficiencies of nutritionlly 2 1 Ctlse (μg/tissue) SOD (nmol/mg tissue) GSH-Px (nmol/mg tissue) Figure 5: Heptic ctivities of ctlse, SOD, nd GSH-Px fter 3 dys for ll experimentl groups. Vlues re given s the men ± SD for ech group of eight nimls. Significntly different from ; P.5; significntly different from ; P.5; nd c significntly different from ; P.5. : untreted control; : treted with gerniol; nd : treted with essentil oil. importnt compounds in nimls in the experimentl groups. These results disgree with others studies, which showed tht the use of different EOs, for exmple, Citrus urntifoli EO, decreses food intke nd, consequently, weight gin [28, 29]. There were no chnges in serum glucose levels etween the groups, indicting mintennce of glycemic homeostsis in these nimls. However, other experimentl studies hve
Biochemistry Reserch Interntionl 5 demonstrted tht C. mrtinii extrcts exhiit ntihyperglycemic ctivitiesthroughinhiitionof α-glucosidse under dietic conditions [3]. Assys with C. citrtus queous extrct (5 mg/kg/dy; vi orl) showed tht the mechnism y which the extrct induced hypoglycemi could e ttriuted to incresed insulin synthesis nd secretion or incresed peripherl glucose utiliztion [31]. The inhltion of oth gerniol () ndc. mrtinii EO ()reducedtotlcholesterol,utnochngesintheserum tricylglycerol concentrtion were oserved. Our results re in greement with the result of Adeneye nd Agje [31]nd Burke et l. [1], who oserved hypocholesterolemic effects using n queous extrct of Cymopogon citrtus y orl dministrtion. This reduction cused y EO cn possily e ttriuted to inhiition of 3-hydroxy-3-methylglutryl CoA reductse, key enzyme tht regultes heptic cholesterol synthesis [32, 33], or y reduction in the expression of these enzymes [34]. Yu et l. [35] demonstrted tht gerniol inhiited the formtion of mevlonte, metolic intermedite in the iosynthesis of cholesterol, in heptoms. On the other hnd, the dministrtion of the highest EO dose (1 mg/kg) from Cymopogon resulted in no chnge in totl serum cholesterol [36]. Our results re lso in greement with Cost et l. [36] out totl serum cholesterol; they reported tht the iochemicl prmeters did not chnge fter tretment with Cymopogon ut showed tht there ws significnt reduction in it (F(4,27) = 3.61; P.5)fterthedministrtion of the highest EO dose (1 mg/kg) y gvge over period of 21 dys. Since ure is formed in the liver nd excreted y kidneys, estimtion of this nitrogenous compound in the loodstrem is importnt to estimte oth heptic nd renl functions. Animls treted with gerniol nd EO did not show ltered serum ure levels, suggesting norml degree of protein ctolism, which ws confirmed y norml concentrtion of heptic protein. Although there ws no ltertion in the levels of serum ure in the group, we cnnot exclude the involvement of possile chnges in the glomerulr filtrtion rte in these nimls. In clinicl prctice, serum cretinine, iomrker for renl filure, is used s n indictor of renl function [37]. Curiously, serum cretinine levels were higher in the group nd lower in the group, suggesting decrese in renl excretion nd some degree of renl insufficiency or erly stges of kidney dysfunction when the mjor compound of C. mrtinii EO ws dministrted lone. The nimls treted with oth products hd tendency to hve decresed levels of serum ure in our study. Serum cretinine ws higher in, which my indicte lower renl excretion since the cretinine production ws reltively constnt [38]. Plsm memrne dmge from some cells types, such s heptic cells, is ccompnied y relese of cytosolic enzymes into loodstrem, phenomenon tht lwys occurs under severl pthophysiologicl conditions [39]. The minotrnsferses ALT nd AST re used for dignosis of heptic injury fter toxic gents exposure [4]. There ws significnt increse in serum ALT ctivity in nimls tht inhled gerniol (Figure 3). Since serum enzymtic ctivity of ALT is often used s iomrker of heptic toxicity, we cn ssume tht there ws some injury in heptic tissue induced y gerniol fter 3 dys. ROS, such s superoxide nion (O 2 ), hydroxyl rdicls (OH ), nd hydrogen peroxide (H 2 O 2 ), re formed through mitochondril respirtion during norml cellulr metolism. However, the cells hve n enzymtic ntioxidnt defense system ginst ROS, ut, under certin pthologicl conditions the excess formtion of ROS results in suppression of ntioxidnt enzymes, the increse of ROS cn occur in this wy leding to oxidtive stress [41]. Lipid peroxidtion is n importnt toxic event ecuse involves the removl of hydrogen from ftty cid chins medited y ROS [42, 43]thiswycnledtocelldethnd tissue dmge. The endogenous ntioxidnt enzyme includes superoxide dismutse tht ctlyzes the dismuttion of superoxide rdicls [23]. Glutthione peroxidse ctlyzes the reduction of hydrogen peroxidse to wter through the oxidtion ofreducedglutthione.ctlselsoprticiptesinthis conversion [44]. Significntly high LH ws oserved in rts exposed to gerniol (), while the eneficil effect of C. mrtinii EO ws evidenced y the reduced LH in these nimls. The reductions oserved in cn e ttriuted to synergistic mechnism: concomitnt ntioxidnt ction etween other compounds, for exmple, linlool nd β-cryophyllene, present in the C. mrtinii EO tht showed ntioxidnt ctivity in other reserches [45, 46]. Since free rdicl scvenger ility depends on the numer of hydroxyl rdicls in the molecule [43], the inhltion of the totl C. mrtinii EO contriuted to the reduction in the formtion of ROS. Rts exposed to gerniol () hd higher ctlse, SOD, nd GSH-Px ctivities, indicting tht ntioxidnt enzyme ctivities were not sufficient to inhiit the ROS ction nd, consequently, the lipoperoxide genertion in liver of these nimls. According to Koek et l. [47] the ctivity of ntioxidnts enzymes is incresed erly in nonlcoholic stetoheptitis ut tends to decrese with progression of pthogenesis. The ctivity of the SOD nd ctlse did not chnge in the group,whilegsh-pxincresedinthesenimls,which showed lower vlues for LH.Buch et l. [4] oserved increses in oth SOD nd ctlse in the rins of rts treted with C. mrtini EO. Terpenoids, which re importnt components of EOs, lowered mlondildehyde levels nd improved SOD ctivity in gstric mucos [48]. Experimentl dt hve shown tht terpenoids, which re min components of EOs, re responsile for their ntioxidnt ction [49, 5]. Since lipid hydroperoxide hs een widely studied s mrker of lipoperoxidtion [51], process tht involves removl of hydrogen from ftty cids side chins y ROS, the result is referring to the mixture of compounds present in C. mrtinii EO()thtwseffectiveincontrolling oxidtive stress nd, therefore, lipoperoxidtion y reducing the concentrtion of LH through mechnism independent of the endogenous ntioxidnt enzymtic system. In nother study, gerniol reduced lipid peroxidtion nd inhiited the relese of NO, indicting its possile
6 Biochemistry Reserch Interntionl ntioxidnt potentil in inflmmtory lung diseses, in which oxidtive stress plys key role in these pthogenesis [14]. Moreover, the possile synergism etween the compounds present in EOs cn influence iologicl responses [52]. This cn explin the results otined for the group. Thus, theeffectsweoservedcouldettriutedtoconstituent in smller proportion or synergism etween compounds tht re present in the oil [53]. For iologicl purposes, it ismoreinformtivetostudythewholeoilthnsomeofits components ecuse the concept of synergism ppers to e more significnt in the reserch on nturl products [2]. 5. Conclusion In conclusion, C. mrtinii EO nd gerniol mintined the glycemi, tricylglycerol protein, nd ure levels ut decresed cholesterol levels in Wistr rts. The oxidtive stress cused y gerniol lone ppers to trigger, to some degree, heptic toxicity, s cn e verified y the increse of serum cretinine nd ALT. These results suggest tht eneficil ctions of C. mrtinii EO on oxidtive stress cn prevent the toxicity in liver. This proves the possile interctions etween gerniol nd numerous chemicl compounds present in C. mrtinii EO. Arevitions C. mrtinii: Cymopogon mrtinii EO: Essentil oil EOs: Essentil oils ALT: Alnine minotrnsferse AST: Asprtte minotrnsferse ROS: Rective oxygen species LH: Lipid hydroperoxide GSH-Px: Glutthione peroxidse SOD: Superoxide dismutse NBT: Nitrolue-tetrzole. Conflict of Interests The uthors declre tht there is no conflict of interests regrding the puliction of this pper. References [1] P. Shrm, N. Sngwn, S. Bose, nd R. Sngwn, Biochemicl chrcteristics of novel vegettive tissue gerniol cetyltrnsferse from monoterpene oil grss (Plmros, Cymopogon mrtinii vr. Moti) lef, Plnt Science, vol. 23, pp. 63 73, 213. [2]F.Bkkli,S.Avereck,D.Avereck,ndM.Idomr, Biologicl effects of essentil oils review, Food nd Chemicl Toxicology,vol.46,no.2,pp.446 475,28. [3] M. Arrud, H. Vin, N. 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