research article hepatoprotective effect of pretreatment with ...mhz) spectra were recorded in...

Research ArticleHepatoprotective Effect of Pretreatment withThymus vulgaris Essential Oil in Experimental Model ofAcetaminophen-Induced Injury

Renata Grespan1 Rafael Pazinatto Aguiar1 Frederico Nunes Giubilei1

Rafael Rocco Fuso1 Marcio Joseacute Damiatildeo1 Expedito Leite Silva2 Jane Graton Mikcha3

Luzmarina Hernandes4 Ciomar Bersani Amado1 and Roberto Kenji Nakamura Cuman1

1 Department of Pharmacology andTherapeutics State University of Maringa 87020-900 Maringa PR Brazil2 Department of Chemistry State University of Maringa 87020-900 Maringa PR Brazil3 Department of Clinical Analysis State University of Maringa 87020-900 Maringa PR Brazil4Department of Morphophysiology Sciences State University of Maringa 87020-900 Maringa PR Brazil

Correspondence should be addressed to Renata Grespan rgrespan2uembrand Roberto Kenji Nakamura Cuman rkncumanuembr

Received 9 September 2013 Accepted 23 December 2013 Published 4 February 2014

Academic Editor Luigi Gori

Copyright copy 2014 Renata Grespan et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Acute liver damage caused by acetaminophen overdose is a significant clinical problem and could benefit from new therapeuticstrategies Objective This study investigated the hepatoprotective effect of Thymus vulgaris essential oil (TEO) which is usedpopularly for various beneficial effects such as its antiseptic carminative and antimicrobial effects The hepatoprotective activityof TEO was determined by assessing serum aspartate aminotransferase (AST) alanine aminotransferase (ALT) and alkalinephosphatase (ALP) in mice Their livers were then used to determine myeloperoxidase (MPO) enzyme activity and subjected tohistological analysis In vitro antioxidant activitywas evaluated by assessing the free radical 22-diphenyl-1-picrylhydrazyl (DPPH∙)-scavenging effects of TEO and TEO-induced lipid peroxidation TEO reduced the levels of the serum marker enzymes AST ALTand ALP and MPO activity The histopathological analysis indicated that TEO prevented acetaminophen-induced necrosis Theessential oil also exhibited antioxidant activity reflected by its DPPH radical-scavenging effects and in the lipid peroxidation assayThese results suggest that TEO has hepatoprotective effects on acetaminophen-induced hepatic damage in mice

1 Introduction

Acetaminophen (APAP) at large doses causes serious liverinjury that may develop into liver failure [1] Hepatotoxicityinduced by acetaminophen occurs through a biotransforma-tion reaction that forms the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) through the cytochromeP-450 mixed function of oxidase system The metaboliteis normally detoxified through a conjugation reaction withreduced glutathione (GSH) However at large doses ofacetaminophen NAPQI levels increase ultimately depletingGSH levels Subsequently sulfhydryl groups of hepatic pro-teins may react with the reactive metabolite resulting in hep-atic necrosis [2 3] Hepatocellular degeneration and necrosis

are also associated with elevated enzyme markers such asserum alanine aminotransferase (ALT) and aspartate amino-transferase (AST) that indicate hepatotoxicity [4] Liverinjury induced by acetaminophen in mice is a commonlyused experimental model for screening substances withpotential hepatoprotective activity [5] Growing interest hasbeen observed in the analysis of these natural entities for theirpotential benefits to human health Accelerating researchof plants used in folk medicine to treat liver diseases andboost liver function has been performed In plants essentialoils are natural mixtures of terpenes mainly monoterpenesand sesquiterpenes which have been increasingly used incomplementary therapies because essential oils are usuallyrich sources of phytochemical mixtures [6]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 954136 8 pageshttpdxdoiorg1011552014954136

2 Evidence-Based Complementary and Alternative Medicine

Essential oils extracted from fresh leaves and flowerscan be used as aroma additives in food pharmaceuticalsand cosmetics [7] The leaves of thyme (Thymus vulgaris)can be used fresh or dried as a spice Thyme also possessesvarious beneficial effects including antiseptic carminativeantimicrobial and antioxidative effects [8] Recently in ourlaboratory it was showed that constituents thymol andcarvacrol of Thymus vulgaris L essential oil present effectson the inflammatory response [9] Besides essential oils andphenolic compounds such as thymol in thyme have antiox-idative properties and may have hepatoprotective properties[8 10] To our knowledge scarce information is availableabout the effects of Thymus vulgaris essential oil (TEO) inexperimental hepatotoxicity models Therefore the presentstudy investigated the hepatoprotective effect of TEO onacetaminophen-induced hepatic damage in mice

2 Methods

21 Extraction of Essential Oil The fresh leaves of Thymusvulgaris L were collected from the Proffessor Irenice SilvaMedicinal Plant Garden on the campus of the StateUniversityof Maringa Parana Brazil The leaves were identified andauthenticated by botanist Maria Aparecida Sert A voucherspecimenwas deposited in theHerbarium of the Departmentof Botany State University of Maringa (number 11329)The essential oil was obtained by hydrodistillation using aClevenger-type apparatus Approximately 556 g of the leaveswas subjected to steam distillation for 2 h The oil was driedover sodium sulfate and stored in an amber flask at 4∘C TheTEO yield was 176 vw

22 Essential Oil Analysis

221 Gas Chromatography-Mass Spectrometry (GC-MS)Gas chromatographic (GC) analysis was performed witha Thermo Electron Corporation Focus GC model underthe following conditions DB-5 capillary column (30m times032mm 050mm) column temperature 60∘C (1min) to180∘C at 3∘Cmin injector temperature 220∘C detector tem-perature 220∘C split ratio 1 10 carrier gas He flow rate10mLmin The volume injected 1 120583L diluted in chloroform(1 10) The GCMS analysis was performed in a Quadrupolemass spectrometer (Thermo Electron Corporation DSQ IImodel) operating at 70V Identification of the individualcomponents was based on comparison of their GC retentionindices (RI) on apolar columns and comparison with massspectra of authentic standard purchased from Sigma-Aldrichliterature data

222 NuclearMagnetic Resonance (NMR) 1H (30006MHz)and 13C NMR (7545MHz) spectra were recorded in deuter-ated chloroform (CDCl

3) solution in aMercury-300BB spec-

trometer with 120575 (ppm) and spectra referred to CDCl3(120575 727

for 1H and 7700 for 13C) as internal standard

23 Animals Male Balbc mice weighing 24 plusmn 2 g wereprovided by the Central Animal House of the State University

of Maringa The animals were housed at 22 plusmn 2∘C under a1212 h lightdark cycle Prior to the experiments the animalsfasted overnight with water provided ad libitum The experi-mental protocols were approved by the Ethical Committee inAnimal Experimentation of the State University of Maringa(CEAEUEM 1262010)

24 Treatment of Animals The experimental animals weredivided into six groups of five animals each Firstly eachgroup received orally during seven days the following treat-ment Group I the mice did not receive any treatmentIn Group II the mice received TEO vehicle (saline thatcontained 01 Tween 80) In Groups IIIndashV the mice werepretreated with TEO at doses of 125 250 and 500mgkgrespectively In Group VI the mice were pretreated with thestandard drug silymarin (200mgkg) After this time theanimals fasted for 8 h and then received oral acetaminophenon the seventh day at a dose of 250mgkg in Groups IIndashVIGroup I orally received saline that contained 01 Tween 80(APAP vehicle) After 12 h the mice were anesthetized withhalothane and blood was collected for the determinationof serum AST ALT and alkaline phosphatase (ALP) Thelivers were then used to determine myeloperoxidase (MPO)enzyme activity and for histological analysis

25 Determination of Serum ALT AST and ALP LevelsBlood samples were collected and centrifuged at 3000timesg for15min at 4∘C Serum ALT AST and ALP levels were thenmeasured using the Analyze Gold enzymatic test kit

26 Determination of MPO Activity The livers were used todetermine MPO enzyme activity in the homogenate super-natant of the liver sections which were placed in potassiumphosphate buffer that contained hexadecyltrimethylammo-nium bromide in a Potter homogenizer The homogenatewas stirred in a vortex and centrifuged Ten microliters ofthe supernatant was added to each well in triplicate in a96-well microplate Two hundred microliters of the buffersolution that contained 167mg O-dianisidine dihydrochlo-ride (Sigma) 90mL double-distilled water 10mL potassiumphosphate buffer and 50120583L of 1 H

2O2

was added Theenzymatic reaction was stopped by the addition of sodiumacetate Enzyme activity was determined by absorbancemeasured at 460 nm using a Spectra Max Plus microplatespectrophotometer

27 Histopathological Analysis The livers were washed in09 (wv) sodium chloride solution and placed in 10neutral buffered formalin for fixation Subsequently the liverswere processed to paraffin embedded and sectioned in semis-erial at a 6 120583m thickness on a Leica rotary microtome (LeicaMicrosystems Gladesville New SouthWales Australia) Thesections were stained with hematoxylin and eosin to evaluatetissue morphology using light microscopy (Olympus BX-41Tokyo Japan) The graded lesions were subjectively classifiedas absent mild moderate or severe according to lesion area

Evidence-Based Complementary and Alternative Medicine 3

28 Lipid Peroxidation Assay A lipid peroxidation assay wasperformed as previously reported with a minor modification[11] Egg yolk homogenateswere prepared as lipid-richmediaBriefly 01mL of TEO (5 50 500 2500 and 5000 120583gmL)in methanol was thoroughly mixed with 05mL of egg yolkhomogenate (10 vv diluted with pure water) and madeup to 1mL with pure water Ferrous sulfate (50 120583L 70mM)was added to induce lipid peroxidation and the mixturewas incubated for 30min at 375∘C Afterward 15mL of20 acetic acid (vv pH 35 diluted with pure water) and15mL of 08 (wv) thiobarbituric acid in 11 sodiumdodecyl sulfate (wv diluted with pure water) were addedand the resulting mixture was vortexed and heated at 95∘Cfor 60min After cooling 5mL of 1-butanol was added toeach tube and centrifuged at 5000 rotations per minute for15min The organic upper layer was collected and measuredspectrophotometrically at 532 nm using a Beckman DU-65spectrophotometer The essential oil was diluted in methanol(the solvent expressed no antioxidant activity) Ascorbicacid was used as a positive control The inhibition of lipidperoxidation was calculated as follows Inhibition () = (1 minus119860 sample119860control)times100 119860control was considered the absorbanceof the control (ie methanol instead of the sample) TheIC50

value representing the concentration of the essentialoil that caused 50 inhibition of lipid peroxidation in theFe2+ascorbate system was determined by linear regressionanalysis from the obtained inhibition () values

29 DPPH Assay Free radical-scavenging capacity (RSC)was evaluated bymeasuring the 22-diphenyl-1-picrylhydrazil(DPPH)-scavenging activity of TEO The DPPH assay wasperformed as previously described [12] withminormodifica-tionsThe samples (60ndash2500 120583gmL) inmethanol weremixedwith 1mL of a 25mM DPPH∙ solution (Sigma St LouisMO USA) with the addition of 95 methanol to a finalvolume of 4mL The absorbance of the resulting solutionsand blank (ie with the same chemicals with the exceptionof the sample) were recorded against ascorbic acid (ChemCruz used as a positive control) after 30min at room tem-perature For each sample four replicates were recordedThedisappearance of DPPH∙ was measured spectrophotometri-cally at 515 nm using a Beckman DU-65 spectrophotometerThe percentage of RSC was calculated using the followingequation RSC () = 100 times (119860blank minus 119860 sample119860blank) TheIC50value representing the concentration of the essential oil

that caused 50 RSC inhibition was determined by linearregression analysis from the obtained RSC values

3 Results

The thyme essential oil showed a predominance of carvacrol(4554) 120572-terpineol (2296) and endo-borneol (1429)as the major components (data not shown) In the acutetoxicological study TEO tested orally showed an LD50 valueof 4000mgkg All doses used in the present study werelowest of LD50 values observed Consequently no apparentbehavioural side effects were observed in the animals duringour studies The high LD50 values also suggest that the TEOwas relatively safe and nontoxic to the animals

We evaluated the effects of TEO on serum enzyme mark-ers As shown in Table 1 acetaminophen-induced hepaticdamagemarkedly elevated serumALTAST andALP enzymelevels compared with the normal animals Pretreatment with250 and 500mgkg TEO but not 125mgkg TEO for 7 daysprior to acetaminophen administration markedly reducedserum ALT AST and ALP levels compared with vehicle-treated controls The effect of TEO was also comparable tosilymarin a standard hepatoprotective agent

The activity of MPO in TEO-pretreated mice thatreceived doses of 250 and 500mgkg was significantlydecreased (0073 plusmn 0008 and 0069 plusmn 0008 IUL resp)compared with the group that received acetaminophen only(0251 plusmn 0149 IUL Table 1)

The histopathological analysis of control group (APAPvehicle) did not show hepatocellular damage (Figure 1(a))However the acetaminophen-treated group showed severeinjury characterized by hemorrhagic and necrotic areaspresence of inflammatory infiltrate and piknotic nucleus(Figure 1(b)) Considering the sylimarin group (standarddrug) although the hepatic parenchymal did not presenthomogenous necrotic areas were not observed Also cellularnucleus was more basophilic than of that control miceand many cells showed cytoplasm vacuolization showingminor injuries (Figure 1(c)) The group of animals treatedwith TEO 125mgKg showed interspersed necrotic areaswith non-necrotic areas with cytoplasm vacuolizationand hemorrhagic points characteristics of moderateinjuries (Figure 1(d)) differently to that observed after TEO250mgkg treatment where mild injuries were observedcharacterized by basophilic nucleus almost piknotic withshrinkage level (Figure 1(e)) Besides the group treatedwith 500mgkg of TEO showed the hepatic parenchymawith similar morphology to the control group (Figure 1(f))Therefore TEO appeared to provide significant protectionagainst hepatocyte injury

In the DPPH test the ability of TEO to act as a donorfor hydrogen atoms or electrons in the transformation ofDPPH∙ in its reduced form (DPPH-H) was measured spec-trophotometricallyThe RSC of TEO at concentrations of 60ndash2500 120583gmL showed significant antioxidant activity in vitro(IC50= 1377 plusmn 16970 120583gmL Figure 2(a) Table 2) The IC

50

value of ascorbic acid (ie the positive control) was 440 plusmn007928 120583gmL in the DPPH assay (Figure 2(b) and Table 2)

Egg yolk lipids undergo rapid lipid peroxidation whenincubated in the presence of ferrous sulfate The effect ofTEO on nonenzymatic peroxidation is shown in Figure 2(c)and Table 2 At concentrations of 5ndash5000120583gmL TEOsignificantly inhibited lipid peroxidation (IC

50= 8461 plusmn

7778 120583gmL) The IC50

value of ascorbic acid (ie thepositive control) was 6300 plusmn 33870 120583gmL in the lipidperoxidation assay (Figure 2(d) and Table 2)Therefore TEOwas significantly correlated with total antioxidant activity(119877 gt 099) demonstrating that TEO had antioxidant activity

4 Discussion

In the present study it was evaluated the hepatoprotectiveeffect of TEO using the hepatotoxicity model induced by


Table 1 The effect of Thymus essential oil on biomarkers of hepatic damage

Groups and designe of treatment ALT AST ALP MPO(IULminus1)

Group I control Tween 80 4888 plusmn 205 1133 plusmn 1539 13840 plusmn 1067 0058 plusmn 0009Group II APAP control (250mg kgminus1) 11130 plusmn 97340a 6860 plusmn 14000a 18190 plusmn 2475a 0251 plusmn 0149ordfGroup III 125mg kgminus1 of TEO + APAP 3847 plusmn 3673 3201 plusmn 2731 11040 plusmn 3574 0101 plusmn 0008Group IV 250mg kgminus1 of TEO + APAP 26110 plusmn 8441b 1760 plusmn 7650b 7137 plusmn 1149b 0073 plusmn 0009b

Group V 500mg kgminus1 of TEO + APAP 11070 plusmn 3579b 1108 plusmn 2253b 7920 plusmn 1591b 0069 plusmn 0009b

Group VI 200mg kgminus1 of SLM + APAP 38880 plusmn 14810b 2867 plusmn 15040b 10140 plusmn 4001b 0091 plusmn 0003b

Values are mean plusmn SEM 5 mice in each group (119899 = 5) 119875 lt 005 values are considered statistically significant a119875 lt 005 acetaminophen (APAP) treated groupcomparedwith animals in control groups b119875 lt 005mice treatedwith theThyme essential oil (TEO) or Silymarin (SLM) comparedwith acetaminophen group

(a)

(d)

(c)(b)

(f)(e)

Figure 1 Photomicrograph of the liver in mice that received orally (a) saline (b) acetaminophen on last day of treatment (250mgkg) (c)silymarin (200mgkg) and ((d)ndash(f)) acetaminophen after being treated for 7 days with the essential oil ofThymus vulgaris (TEO) 125 250and 500mgkg respectively In (a) the liver showed normal morphology (b) presence of necrosis and hemorrhagic points (lowast) in the definedarea ((c) and (e)) parenchyma stands out for having vacuolated hepatocytes (arrows) (d) observed necrotic areas (lowast) (f) hepatic parenchymamorphology similar to that observed in the control Original magnification 40x in (a) (c) (e) and (f) original magnification 20x in (b) and(d) The sections stained with hematoxylin and eosin

acetaminophen This drug in an overdose (ie at doses thatare different from analgesic doses that are safely and effec-tively used therapeutically) can induce severe hepatotoxicityin experimental animals and humans [13ndash15] In our workhepatotoxicity was reflected by a marked elevation of the lev-els of serummarker enzymes (AST ALT andALP) increasedMPOactivity and histopathologic alterationsThese enzymesin serum are useful quantitative markers of the extent andtype of hepatocellular damage High levels of AST indicatea loss of the functional integrity of the liver similar to theeffects seen in viral hepatitis cardiac infraction and muscleinjuryTheALT enzyme catalyzes the conversion of alanine topyruvate and glutamate and is released in a similar mannerTherefore ALT ismore specific to the liver and thus is a betterparameter for detecting liver injury [16ndash18]

Acetaminophen is converted to a toxic reactive inter-mediate called N-acetyl-p-benzoquinone imine (NAPQI)following metabolism by number of isozymes of cytochromeP-450 (CYPs) that is CYP 2E1 [19] CYP 1A2 [20] CYP2A6 [21] CYP 3A4 and CYP 2D6 [22] NAPQI could bebound covalently to cellular proteins including mitochon-drial proteins [23] and that in turn leads to mitochondrialdysfunction Mitochondrial respiration is inhibited whichresults in the formation of reactive oxygen species (ROS)and peroxynitrite in the mitochondria [24 25] The massiveproduction of reactive species (ROS) may lead to depletionof protective physiological moieties (glutathione and 120572-tocopherol) ensuing widespread propagation of the alkyla-tion as well as peroxidation causing damage to the macro-molecules in vital biomembranes [26] However reduced


0 1000 2000 30000

20

40

60

80

100

Activ

ity (

)

DPPH Thymus vulgaris

Concentrations (120583gmL)

y = 0029x + 9187

R2 = 0990

(a)

DPPH ascorbic acid

100

0 2 4 6 8

20

40

60

80

100


y = 1092x + 1989

R2 = 0995

Activ

ity (

)

(b)

0 1000 2000 3000 4000 50000

10

20

30

40

Inhi

bitio

n (

)

Lipidic peroxidation Thymus vulgaris

y = 0005x + 6489

R2 = 0992


(c)

Lipidic peroxidation ascorbic acid

0 50 100 1500

20

40

60

80

y = 0473x + 225

R2 = 0995


Inhi

bitio

n (

)

(d)

Figure 2 Antioxidant activity of the essential oil fromThymus vulgarisThe figure shows the percentage of neutralization of DPPH by (a) theessential oil of T vulgaris and (b) ascorbic acid in the DPPH assay (120583gmL) The inhibition of lipid peroxidation (LP) in the Fe2+ascorbatesystem induced by (c) the essential oil of T vulgaris and (d) ascorbic acid in the TBA assay is also shown

glutathione is one of the main defense mechanisms againstoxidative stress reducing peroxides and hydroperoxides [27]In addition the oxidative stress can induce a mitochon-drial membrane permeability transition and adenosine-51015840-triphosphate (ATP) depletion which results in membranepermeabilization membrane rupture and cell apoptosis [28ndash31]

We assessed the hepatoprotective effect of TEO inacetaminophen-induced hepatic damage in mice and theresults suggested that pretreatment with TEO could beprotecting the functional integrity of hepatocytes and thecellularmembrane fromdamage by toxic reactivemetabolitesproduced by acetaminophen biotransformation [32 33]

Inflammation also plays a central role during drug-induced acute hepatitis and products of arachidonic acidmetabolism have been extensively involved in inflammatoryprocesses [34] The histopathological analysis of the liversobtained from the TEO-pretreated group showed mild sinu-soidal congestion less inflammatory cell infiltration and

well-preserved hepatocytes with less of an area of necrosiscompared with the severe centrilobular necrosis observed inacetaminophen-treated mice These results suggest that theanti-inflammatory properties of TEO are partially involvedin the hepatoprotective effect of this essential oil Similarlyprevious studies have shown that extracts of plants protectthe liver from acetaminophen overdose suggesting thatthe hepatoprotective effect can be considered an expressionof the functional improvement of hepatocytes that resultsfrom accelerated cellular regeneration [35 36] Thus thecytoprotective effects of silymarin a natural product arealso mainly attributable to its antioxidant and free radical-scavenging properties Silymarin can interact directly withcell membrane components to prevent abnormalities in thecontent of the lipid fraction that is responsible for maintain-ing normal fluidity [37]

Furthermore the free radical-initiated oxidation of cel-lular membrane lipids can lead to cellular necrosis andis now accepted to be important in various pathological


Table 2 Summary of IC50 values of thyme essential oil (TEO) andascorbic acid

DPPHIC50 (120583gmL) plusmn SD

Lipid peroxidationIC50 (120583gmL) plusmn SD

TEO 1377 plusmn 16970 8461 plusmn 77781

Ascorbic acid 440 plusmn 007928 6300 plusmn 33870

conditions High acetaminophen doses significantly elevatedreactive oxygen species levels ultimately depleting the levelsof superoxide dismutase (SOD) and GSH in liver tissue Thisoxidative stress contributes to the initiation and progressionof liver damage [33] Apoptosis is a form of cell death andhas deleterious consequences as observed in many diseasesincluding acquired immunodeficiency syndrome cancer andneurodegenerative disorders [38ndash40] Apoptosis is inducedby different stimuli such as oxidants xenobiotics glucocor-ticoids and irradiation [41] which converge to trigger a com-mon pathway of cell death activating proteases as caspase-3found only in cells undergoing apoptosis Protease caspase-3 can cleave and inactivate a nuclear protein poly (ADP-ribose) polymerase (PARP) an enzyme used for DNA repairMany bioactive substances exert their effect on apoptosisacting in cell cycle progression andor triggering apoptoticcell death However the effects of essential oils inducingor inhibiting apoptosis via mitochondrial stress and caspaseactivation are controversial [42ndash45] Thus various essentialoils have been shown to have antioxidant activity and havebeen used as antioxidant drugs inmany diseases [46ndash48]Theability of natural products to reduce acetaminophen-inducedlipid peroxidation could be as a result of their antioxidantconstituents [49] The mechanisms of TEO in reduction offree radical species and their effects on cells and tissuesdamage should be elucidated

5 Conclusion

TEO pretreatment improves the hepatotoxicity induced byacetaminophen in mice The effects of TEO partially involvethe antioxidative effect of this essential oil However furtherdetailed studies are required to investigate the mechanismby which TEO exerts its effects and determine the specificconstituents that are responsible for this action

Conflict of Interests

The authors declare that there is no conflict of interestregarding the publication of this paper

Acknowledgments

The authors thank Jailson Araujo Dantas and Celia ReginaMiranda for technical assistance This study was supportedby grants from the Coordenadoria de Aperfeicoamento dePessoal de Nıvel Superior (CAPES) Fundacao Araucariaand Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (CNPq) Brazil

References

[1] J S Gujral T R Knight A FarhoodM L Bajt andH JaeschkeldquoMode of cell death after acetaminophen overdose in miceapoptosis or oncotic necrosisrdquo Toxicological Sciences vol 67no 2 pp 322ndash328 2002

[2] D L Laskin and A M Pilaro ldquoPotential role of activatedmacrophages in acetaminophen hepatotoxicity I Isolationand characterization of activated macrophages from rat liverrdquoToxicology and Applied Pharmacology vol 86 no 2 pp 204ndash215 1986

[3] H Jaeschke and J R Mitchell ldquoNeutrophil accumulationexacerbates acetaminophen induced liver injuryrdquo The FASEBJournal vol 3 article A920 1989

[4] Y Cigremis H Turel K Adiguzel et al ldquoThe effects of acuteacetaminophen toxicity on hepatic mRNA expression of SODCAT GSH-Px and levels of peroxynitrite nitric oxide reducedglutathione and malondialdehyde in rabbitrdquo Molecular andCellular Biochemistry vol 323 no 1-2 pp 31ndash38 2009

[5] W M Lee and G Ostapowicz ldquoAcetaminophen pathology andclinical presentation of hepatotoxicityrdquo in Drug-Induced LiverDisease N Kaplowitz and L D DeLeve Eds pp 327ndash344Marcel Dekker New York NY USA 2003

[6] D J Daferera P A Tarantilis and M G Polissiou ldquoCharac-terization of essential oils from Lamiaceae species by Fouriertransform Raman spectroscopyrdquo Journal of Agricultural andFood Chemistry vol 50 no 20 pp 5503ndash5507 2002

[7] J Javanmardi A Khalighi A Kashi H P Bais and JM Vivanco ldquoChemical characterization of basil (Ocimumbasilicum L) found in local accessions and used in traditionalmedicines in Iranrdquo Journal of Agricultural and Food Chemistryvol 50 no 21 pp 5878ndash5883 2002

[8] R Baranauskiene P R Venskutonis P Viskelis and EDambrauskiene ldquoInfluence of nitrogen fertilizers on the yieldand composition of thyme (Thymus vulgaris)rdquo Journal of Agri-cultural and Food Chemistry vol 51 no 26 pp 7751ndash7758 2003

[9] F C Fachini-Queiroz R Kummer C F Estevao-Silva et alldquoConstituents of Thymus vulgaris L Essential Oil on theinflammatory responserdquo Evid Based Complement Alternat Medvol 2012 Article ID 657026 2012

[10] D A Pearson E N Frankel R Aeschbach and J B GermanldquoInhibition of endothelial cell-mediated oxidation of low-density lipoprotein by rosemary and plant phenolicsrdquo Journalof Agricultural and Food Chemistry vol 45 no 3 pp 578ndash5821997

[11] N Dasgupta and B De ldquoAntioxidant activity of Piper betle Lleaf extract in vitrordquo Food Chemistry vol 88 no 2 pp 219ndash2242004

[12] J C Espın C Soler-Rivas and H J Wichers ldquoCharacterizationof the total free radical scavenger capacity of vegetable oilsand oil fractions using 22-diphenyl-1-picrylhydrazyl radicalrdquoJournal of Agricultural and Food Chemistry vol 48 no 3 pp648ndash656 2000

[13] Y Ito E R Abril N W Bethea and R S McCuskeyldquoEthanol binging enhances hepatic microvascular responses toacetaminophen inmicerdquoMicrocirculation vol 11 no 7 pp 625ndash632 2004

[14] H-J Hwang M-J Kwon I-H Kim and T-J Nam ldquoChemo-protective effects of a protein from the red algae Porphyrayezoensis on acetaminophen-induced liver injury in ratsrdquo Phy-totherapy Research vol 22 no 9 pp 1149ndash1153 2008


[15] Y-L Wu D-M Piao X-H Han and J-X Nan ldquoProtectiveeffects of salidroside against acetaminophen-induced toxicity inmicerdquo Biological and Pharmaceutical Bulletin vol 31 no 8 pp1523ndash1529 2008

[16] S K Mitra M V Venkataranganna R Sundaram and SGopumadhavan ldquoProtective effect of HD-03 a herbal formu-lation against various hepatotoxic agents in ratsrdquo Journal ofEthnopharmacology vol 63 no 3 pp 181ndash186 1998

[17] R Sallie J M Tredger and R Williams ldquoDrugs and theliver Part 1 testing liver functionrdquo Biopharmaceutics and DrugDisposition vol 12 no 4 pp 251ndash259 1991

[18] E M Williamson D T Okpako and F J Evans SelectionPreparation and Pharmacological Evaluation of Plant MaterialJohn Wiley amp Sons Chichester UK 1996

[19] E Tanaka M Terada and S Misawa ldquoCytochrome P450 2E1its clinical and toxicological rolerdquo Journal of Clinical PharmacyandTherapeutics vol 25 no 3 pp 165ndash175 2000

[20] K Venkatakrishnan ldquoHuman cytochromes p450 mediatingphenacetin 0-deethylation in vitro validation of the highaffinity component as an index of CYP1A2 activityrdquo Journal ofPharmaceutical Sciences vol 87 no 12 pp 1502ndash1507 1998

[21] W Chen L L Koenigs S J Thompson et al ldquoOxidationof acetaminophen to its toxic quinone imine and nontoxiccatechol metabolites by baculovirus-expressed and purifiedhuman cytochromes P450 2E1 and 2A6rdquo Chemical Research inToxicology vol 11 no 4 pp 295ndash301 1998

[22] H Dong R L Haining K E Thummel A E Rettie andS D Nelson ldquoInvolvement of human cytochrome P450 2D6in the bioactivation of Acetaminophenrdquo Drug Metabolism andDisposition vol 28 no 12 pp 1397ndash1400 2000

[23] S D Nelson ldquoMolecular mechanisms of the hepatotoxicitycaused by acetaminophenrdquo Seminars in Liver Disease vol 10no 4 pp 267ndash278 1990

[24] J S Beckman T W Beckman J Chen P A Marshall andB A Freeman ldquoApparent hydroxyl radical production byperoxynitrite implications for endothelial injury from nitricoxide and superoxiderdquo Proceedings of the National Academy ofSciences of the United States of America vol 87 no 4 pp 1620ndash1624 1990

[25] T R Knight A Kurtz M L Bajt J A Hinson and H JaeschkeldquoVascular and hepatocellular peroxynitrite formation duringacetaminophen toxicity role of mitochondrial oxidant stressrdquoToxicological Sciences vol 62 no 2 pp 212ndash220 2001

[26] W N Aldridge ldquoMechanisms of toxicity New concepts arerequired in toxicologyrdquo Trends in Pharmacological Sciences vol2 pp 228ndash231 1981

[27] M B Gutierrez B S Miguel C Villares M J Tunon and JGonzales-Callego ldquoOxidative Stress Induced by Cremphor ELis not accompanied by changes in NF-KB activation or iNOSexpressionrdquo Toxicol vol 222 pp 125ndash131 2006

[28] T Qian B Herman and J J Lemasters ldquoThe mitochondrialpermeability transition mediates both necrotic and apoptoticdeath of hepatocytes exposed to Br-A23187rdquo Toxicology andApplied Pharmacology vol 154 no 2 pp 117ndash125 1999

[29] J-S Kim L He and J J Lemasters ldquoMitochondrial permeabil-ity transition a common pathway to necrosis and apoptosisrdquoBiochemical andBiophysical ResearchCommunications vol 304no 3 pp 463ndash470 2003

[30] J-S Kim T Qian and J J Lemasters ldquoMitochondrial perme-ability transition in the switch from necrotic to apoptotic celldeath in ischemic rat hepatocytesrdquo Gastroenterology vol 124no 2 pp 494ndash503 2003

[31] K Kon J-S Kim H Jaeschke and J J Lemasters ldquoMitochon-drial permeability transition in acetaminophen-induced necro-sis and apoptosis of cultured mouse hepatocytesrdquo Hepatologyvol 40 no 5 pp 1170ndash1179 2004

[32] W A Pryor ldquoOxy-radicals and related species their formationlifetimes and reactionsrdquo Annual Review of Physiology vol 48pp 657ndash667 1986

[33] R Van de Straat J De Vries and N P E Vermeulen ldquoRoleof hepatic microsomal and purified cytochrome P-450 in one-electron reduction of two quinone imines and concomitantreduction ofmolecular oxygenrdquoBiochemical Pharmacology vol36 no 5 pp 613ndash619 1987

[34] V Perez-Alvarez R A Bobadilla-Lugo P Muriel L Favari andC Villanueva-Lopez ldquoEffects of leukotriene synthesis inhibi-tion on acute liver damage induced by carbon tetrachloriderdquoPharmacology vol 47 no 5 pp 330ndash336 1993

[35] S Sadasivan P G Latha J M Sasikumar S Rajashekaran SShyamal and V J Shine ldquoHepatoprotective studies onHedyotiscorymbosa (L) Lamrdquo Journal of Ethnopharmacology vol 106no 2 pp 245ndash249 2006

[36] N Tabassum S Chattervedi S S Aggrawal and N AhmedldquoHepatoprotective studies on Phyllanthus niruri on paracetamolinduced liver cell damage in albino micerdquo JK Practitioner vol12 no 4 pp 211ndash212 2005

[37] P Muriel and M Mourelle ldquoPrevention by silymarin of mem-brane alterations in acute CCl4 liver damagerdquo Journal of AppliedToxicology vol 10 no 4 pp 275ndash279 1990

[38] C B Thompson ldquoApoptosis in the pathogenesis and treatmentof diseaserdquo Science vol 267 no 5203 pp 1456ndash1462 1995

[39] D K Ingawale S K Mandlik and S R Naik ldquoModels ofhepatotoxicity and the underlying cellular biochemical andimmunological mechanism(s) a critical discussionrdquo Environ-mental Toxicology and Pharmacology vol 37 no 1 pp 118ndash1332013

[40] D X -Tan R Hardeland L C Manchester A Galano andR J Reiter ldquoCyclic-hidroxymelatonin (C3HOM) a potentantioxidant scavanges free radical and suppresses oxidativereactionsrdquo Current Medicinal Chemistry 2013

[41] C M Payne C Bernstein and H Bernstein ldquoApoptosisoverview emphasizing the role of oxidative stress DNA damageand signal-transduction pathwaysrdquo Leukemia and Lymphomavol 19 no 1-2 pp 43ndash93 1995

[42] J-D Cha S-E Moon H-Y Kim I-H Cha and K-Y LeeldquoEssential oil of artemisia capillaris induces apoptosis in KBcells via mitochondrial stress and caspase activation mediatedby MAPK-stimulated signaling pathwayrdquo Journal of Food Sci-ence vol 74 no 9 pp T75ndashT81 2009

[43] J Usta S Kreydiyyeh K Knio P Barnabe Y Bou-Moughlabayand S Dagher ldquoLinalool decreases HepG2 viability by inhibit-ing mitochondrial complexes I and II increasing reactiveoxygen species and decreasing ATP and GSH levelsrdquo Chemico-Biological Interactions vol 180 no 1 pp 39ndash46 2009

[44] H-N Koo S-H Hong C-Y Kim et al ldquoInhibitory effect ofapoptosis in human astrocytes CCF-STTG1 cells by lemon oilrdquoPharmacological Research vol 45 no 6 pp 469ndash473 2002

[45] J-D Cha Y-H Kim and J-Y Kim ldquoEssential oil and 18-cineole fromArtemisia lavandulaefolia induces apoptosis in KBCells via mitochondrial stress and caspase activationrdquo FoodScience and Biotechnology vol 19 no 1 pp 185ndash191 2010

[46] A K Grover and S E Samson ldquoAntioxidants and vision healthfacts and fictionrdquoMolecular and Cellular Biochemistry 2013


[47] E Sendra M M Viuda A B D Nasser et al ldquoChemicalcomposition and antioxidant and anti-Listeria activities ofessential oils obtained from some Egyptian plantsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 16 pp 9063ndash90702010

[48] W-C Zeng R-X Zhu L-R Jia H Gao Y Zheng and QSun ldquoChemical composition antimicrobial and antioxidantactivities of essential oil from Gnaphlium affinerdquo Food andChemical Toxicology vol 49 no 6 pp 1322ndash1328 2011

[49] M Anoush M A Eghbal F Fathiazad H Hamzeiy andN S Kouzehkonani ldquoThe protective effects of garlic extractagainst acetaminophen-induced oxidative stress and Glu-tathione depletionrdquo Pakistan Journal of Biological Sciences vol12 no 10 pp 765ndash771 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Essential oils extracted from fresh leaves and flowerscan be used as aroma additives in food pharmaceuticalsand cosmetics [7] The leaves of thyme (Thymus vulgaris)can be used fresh or dried as a spice Thyme also possessesvarious beneficial effects including antiseptic carminativeantimicrobial and antioxidative effects [8] Recently in ourlaboratory it was showed that constituents thymol andcarvacrol of Thymus vulgaris L essential oil present effectson the inflammatory response [9] Besides essential oils andphenolic compounds such as thymol in thyme have antiox-idative properties and may have hepatoprotective properties[8 10] To our knowledge scarce information is availableabout the effects of Thymus vulgaris essential oil (TEO) inexperimental hepatotoxicity models Therefore the presentstudy investigated the hepatoprotective effect of TEO onacetaminophen-induced hepatic damage in mice

2 Methods

21 Extraction of Essential Oil The fresh leaves of Thymusvulgaris L were collected from the Proffessor Irenice SilvaMedicinal Plant Garden on the campus of the StateUniversityof Maringa Parana Brazil The leaves were identified andauthenticated by botanist Maria Aparecida Sert A voucherspecimenwas deposited in theHerbarium of the Departmentof Botany State University of Maringa (number 11329)The essential oil was obtained by hydrodistillation using aClevenger-type apparatus Approximately 556 g of the leaveswas subjected to steam distillation for 2 h The oil was driedover sodium sulfate and stored in an amber flask at 4∘C TheTEO yield was 176 vw

22 Essential Oil Analysis

221 Gas Chromatography-Mass Spectrometry (GC-MS)Gas chromatographic (GC) analysis was performed witha Thermo Electron Corporation Focus GC model underthe following conditions DB-5 capillary column (30m times032mm 050mm) column temperature 60∘C (1min) to180∘C at 3∘Cmin injector temperature 220∘C detector tem-perature 220∘C split ratio 1 10 carrier gas He flow rate10mLmin The volume injected 1 120583L diluted in chloroform(1 10) The GCMS analysis was performed in a Quadrupolemass spectrometer (Thermo Electron Corporation DSQ IImodel) operating at 70V Identification of the individualcomponents was based on comparison of their GC retentionindices (RI) on apolar columns and comparison with massspectra of authentic standard purchased from Sigma-Aldrichliterature data

222 NuclearMagnetic Resonance (NMR) 1H (30006MHz)and 13C NMR (7545MHz) spectra were recorded in deuter-ated chloroform (CDCl

3) solution in aMercury-300BB spec-

trometer with 120575 (ppm) and spectra referred to CDCl3(120575 727

for 1H and 7700 for 13C) as internal standard

23 Animals Male Balbc mice weighing 24 plusmn 2 g wereprovided by the Central Animal House of the State University

of Maringa The animals were housed at 22 plusmn 2∘C under a1212 h lightdark cycle Prior to the experiments the animalsfasted overnight with water provided ad libitum The experi-mental protocols were approved by the Ethical Committee inAnimal Experimentation of the State University of Maringa(CEAEUEM 1262010)

24 Treatment of Animals The experimental animals weredivided into six groups of five animals each Firstly eachgroup received orally during seven days the following treat-ment Group I the mice did not receive any treatmentIn Group II the mice received TEO vehicle (saline thatcontained 01 Tween 80) In Groups IIIndashV the mice werepretreated with TEO at doses of 125 250 and 500mgkgrespectively In Group VI the mice were pretreated with thestandard drug silymarin (200mgkg) After this time theanimals fasted for 8 h and then received oral acetaminophenon the seventh day at a dose of 250mgkg in Groups IIndashVIGroup I orally received saline that contained 01 Tween 80(APAP vehicle) After 12 h the mice were anesthetized withhalothane and blood was collected for the determinationof serum AST ALT and alkaline phosphatase (ALP) Thelivers were then used to determine myeloperoxidase (MPO)enzyme activity and for histological analysis

25 Determination of Serum ALT AST and ALP LevelsBlood samples were collected and centrifuged at 3000timesg for15min at 4∘C Serum ALT AST and ALP levels were thenmeasured using the Analyze Gold enzymatic test kit

26 Determination of MPO Activity The livers were used todetermine MPO enzyme activity in the homogenate super-natant of the liver sections which were placed in potassiumphosphate buffer that contained hexadecyltrimethylammo-nium bromide in a Potter homogenizer The homogenatewas stirred in a vortex and centrifuged Ten microliters ofthe supernatant was added to each well in triplicate in a96-well microplate Two hundred microliters of the buffersolution that contained 167mg O-dianisidine dihydrochlo-ride (Sigma) 90mL double-distilled water 10mL potassiumphosphate buffer and 50120583L of 1 H

2O2

was added Theenzymatic reaction was stopped by the addition of sodiumacetate Enzyme activity was determined by absorbancemeasured at 460 nm using a Spectra Max Plus microplatespectrophotometer

27 Histopathological Analysis The livers were washed in09 (wv) sodium chloride solution and placed in 10neutral buffered formalin for fixation Subsequently the liverswere processed to paraffin embedded and sectioned in semis-erial at a 6 120583m thickness on a Leica rotary microtome (LeicaMicrosystems Gladesville New SouthWales Australia) Thesections were stained with hematoxylin and eosin to evaluatetissue morphology using light microscopy (Olympus BX-41Tokyo Japan) The graded lesions were subjectively classifiedas absent mild moderate or severe according to lesion area






3 Results






50



50= 8461 plusmn

7778 120583gmL) The IC50


4 Discussion









(a)

(d)

(c)(b)

(f)(e)





0 1000 2000 30000

20

40

60

80

100

Activ

ity (

)



y = 0029x + 9187

R2 = 0990

(a)

DPPH ascorbic acid

100

0 2 4 6 8

20

40

60

80

100


y = 1092x + 1989

R2 = 0995

Activ

ity (

)

(b)

0 1000 2000 3000 4000 50000

10

20

30

40

Inhi

bitio

n (

)


y = 0005x + 6489

R2 = 0992


(c)


0 50 100 1500

20

40

60

80

y = 0473x + 225

R2 = 0995


Inhi

bitio

n (

)

(d)














5 Conclusion




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results






50



50= 8461 plusmn

7778 120583gmL) The IC50


4 Discussion









(a)

(d)

(c)(b)

(f)(e)





0 1000 2000 30000

20

40

60

80

100

Activ

ity (

)



y = 0029x + 9187

R2 = 0990

(a)

DPPH ascorbic acid

100

0 2 4 6 8

20

40

60

80

100


y = 1092x + 1989

R2 = 0995

Activ

ity (

)

(b)

0 1000 2000 3000 4000 50000

10

20

30

40

Inhi

bitio

n (

)


y = 0005x + 6489

R2 = 0992


(c)


0 50 100 1500

20

40

60

80

y = 0473x + 225

R2 = 0995


Inhi

bitio

n (

)

(d)














5 Conclusion




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























(a)

(d)

(c)(b)

(f)(e)





0 1000 2000 30000

20

40

60

80

100

Activ

ity (

)



y = 0029x + 9187

R2 = 0990

(a)

DPPH ascorbic acid

100

0 2 4 6 8

20

40

60

80

100


y = 1092x + 1989

R2 = 0995

Activ

ity (

)

(b)

0 1000 2000 3000 4000 50000

10

20

30

40

Inhi

bitio

n (

)


y = 0005x + 6489

R2 = 0992


(c)


0 50 100 1500

20

40

60

80

y = 0473x + 225

R2 = 0995


Inhi

bitio

n (

)

(d)














5 Conclusion




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1000 2000 30000

20

40

60

80

100

Activ

ity (

)



y = 0029x + 9187

R2 = 0990

(a)

DPPH ascorbic acid

100

0 2 4 6 8

20

40

60

80

100


y = 1092x + 1989

R2 = 0995

Activ

ity (

)

(b)

0 1000 2000 3000 4000 50000

10

20

30

40

Inhi

bitio

n (

)


y = 0005x + 6489

R2 = 0992


(c)


0 50 100 1500

20

40

60

80

y = 0473x + 225

R2 = 0995


Inhi

bitio

n (

)

(d)














5 Conclusion




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























5 Conclusion




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article hepatoprotective effect of pretreatment with ...mhz) spectra were recorded in...

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