Rev. bras. farmacogn. vol.26 número3

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

The

essential

oil

of

Artemisia

capillaris

protects

against

CCl

4

-induced

liver

injury

in

vivo

Qinghan

Gao

a,b,1

,

Xin

Zhao

a,1

,

Lei

Yin

a,1

,

Yuanbin

Zhang

a

,

Bing

Wang

a

,

Xiuli

Wu

a

,

Xinhui

Zhang

a

,

Xueyan

Fu

a,c,∗

,

Weihong

Sun

d,∗

a_School_of_Pharmacy,_Ningxia_Medical_University,_Yinchuan,_China b_School_of_Public_Health,_Ningxia_Medical_University,_Yinchuan,_China

c_Key_Laboratory_of_Hui_Ethnic_Medicine_{Modernization,}_Ministry_of_Education_(Ningxia_Medical_University),_Yinchuan,_China d_General_Hospital_of_Ningxia_Medical_University,_Yinchuan,_China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received13July2015 Accepted11January2016 Availableonline28January2016

Keywords: Essentialoil Carbontetrachloride Hepatoprotectiveeffects Invivo

H&E GC–MS

a

b

s

t

r

a

c

t

TostudythehepatoprotectiveeffectoftheessentialoilofArtemisiacapillarisThunb.,Asteraceae,on CCl4-inducedliverinjuryinmice,thelevelsofserumaspartateaminotransferaseandalanine amino-transferase,hepaticlevelsofreducedglutathione,activityofglutathioneperoxidase,andtheactivitiesof superoxidedismutaseandmalondialdehydewereassayed.AdministrationoftheessentialoilofA. capil-larisat100and50mg/kgtomicepriortoCCl4injectionwasshowntoconferstrongerinvivoprotective effectsandcouldobservablyantagonizetheCCl4-inducedincreaseintheserumalanineaminotransferase andaspartateaminotransferaseactivitiesandmalondialdehydelevelsaswellaspreventCCl4-induced decreaseintheantioxidantsuperoxidedismutaseactivity,glutathionelevelandglutathioneperoxidase activity(p<0.01).Theoilmainlycontained␤-citronellol,1,8-cineole,camphor,linalool,␣-pinene,␤ -pinene,thymolandmyrcene.ThisfindingdemonstratesthattheessentialoilofA.capillariscanprotect hepaticfunctionagainstCCl4-inducedliverinjuryinmice.

Introduction

Liver disease, a commondisorder caused by viral hepatitis, alcoholism, liver-toxic chemicals, unhealthy dietary habits and environmentalpollution,isaglobalconcern(Papayetal.,2009). However, medical treatment for this disease is often hard to administerandhasaconfinedeffect.TraditionalChineseherbal medicines,whichunderlienumerousprescriptionsusedtotreat liverdiseases,arestillwidelyusedbytheChinese(Zhaoetal.,2014).

Artemisia capillaris Thunb., Asteraceae,according to theBencao Gangmu,themostfamousrecordsofChineseTraditionalMedicine, hasbeenwidelyusedasamedicinetoclearheat,promotediuresis andremovejaundiceandhasalsobeenusedasaflavorin bever-ages,vegetables,andpastriesbecauseofitsparticularfragrance.A. capillarishasbeenregardedasatypeofChinesefolkmedicineand foodbyagrowingnumberofpeople.Therefore,therehavebeen

∗ Correspondingauthors.

E-mails:xueyanfu2661@163.com(X.Fu),tungbuo@163.com(W.Sun).

1_These_authors_equally_contributed_to_this_manuscript;_they_should_be_considered

firstauthor.

considerableeffortstodevelopusefulherbalmedicines,suchasA. capillaris,forthetreatmentofliverdisease.

Inrecentyears,herbalmedicineshavegainedmoreattention andpopularityforthetreatmentofliverdiseasebecauseoftheir safetyandefficacy(Dingetal.,2012).A.capillarishasbeenproven topossessgoodhepatoprotectiveactivitybasedonmodern phar-macological methods (Han et al.,2006).It is alsoanimportant medicinalmaterialinChinaand isa popularanti-inflammatory

(Chaetal.,2009a),choleretic(YoonandKim,2011),andanti-tumor

(Fengetal.,2013)herbalremedy.

Phytochemicalstudieshaverevealedanumberofvolatile essen-tialoils, coumarins, and flavonol glycosides aswell asa group ofunidentifiedaglyconesfromA.capillaris(Komiyaetal.,1976;

Yamaharaetal.,1989).TheessentialoilofA.capillaris(AEO)isone

ofthemainpharmacologicalactivecompoundsandconfers anti-inflammatory(Chaetal.,2009a)andanti-apoptoticproperties(Cha

etal.,2009b).However,asAEOisoneofthemaincompoundsof

A.capillaris,thepotentialhepatoprotectiveactivitiesofthemajor constituentsfromA.capillarisshouldbeexplored.

In this study, the protective effect of AEO on carbon tetrachloride(CCl4)-inducedhepatotoxicitywasevaluatedby

bio-chemical methods, such as hepatic reduced glutathione (GSH),

http://dx.doi.org/10.1016/j.bjp.2016.01.001

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malondialdehyde(MDA)levels,superoxidedismutase(SOD),and glutathioneperoxidase(GSH-Px)activity,aswellastheactivities ofaspartateaminotransferase(AST)andalanineaminotransferase (ALT)intheserum.TheextentofCCl4-inducedliverinjurywasalso

analyzed through histopathological observations, accompanied withphytochemicalanalysisbyGC–MStoidentifytheconstituents ofAEO.

Materialsandmethods

Drugsandchemicals

The essential oil of Artemisia capillaris Thunb., Asteraceae, wasobtainedfromKangshenNaturaloilsCo.,Ltd(Jishui,Jiangxi Province,China).CCl4waspurchasedformDamaoChemical

Com-pany (Tianjin, China). Bifendate tablets were purchased from Yunpeng,ShanxiPharmaceuticalCo.,Ltd.(no.A130602).The diag-nostickitsusedforthedeterminationofALT,AST,SOD,MDA,GSH andGSH-PxwereobtainedfromtheNanjingJianchengInstituteof Biotechnology(Nanjing,China).

AEOanalysis

AEOwasanalyzedby gaschromatography–mass spectrome-try(GC/MS)usingaShimadzuQP2010plusGCwithRxi-5SilMS (30m×0.25mm; 0.25␮mfilm thickness). The GC/MS was run under thefollowing conditions: a fused-silica capillary column with heliumat 1.60ml/min and an injector at 250◦_C. _The _GC oventemperaturewasinitiallyheldat40◦_C_for₅_min._Thereafter, thetemperaturewasraisedwithagradientof3◦_C_min−1_until_it

reached230◦_C._This_temperature_was_held_for₃_min._Finally,_the temperaturewasthenraisedwitha gradientof1◦_C_min−1 _until

260◦_C_and_again_held_for₁₀_min._Mass_spectra_were_taken_at₇₀_eV from33to500Da.Identificationofthecompoundswasbasedon thecomparisonofthemassspectraldataonacomputermatched withNIST(similarityindex>85%)andthosedescribedinthe lit-erature(Sylvestre etal., 2006; Chenget al., 2008; Ait-Ouazzou

etal.,2012;ArgyropoulouandSkaltsa,2012; ´Cavaretal.,2012;Ben

Mansouretal.,2013;Chenetal.,2013;GouveiaandCastilho,2013;

MuruganandMallavarapu,2013;Singhetal.,2013;Bagherietal.,

2014;DaSilvaetal.,2014;Desaietal.,2014;Pandeyetal.,2014;

Qietal.,2014;Ratheretal.,2014;Sadgroveetal.,2014;Sereshti

etal.,2014;Taoetal.,2014;Tianetal.,2014).Theidentification

ofcompoundswasperformedaccordingtotheirretentionindices relativetoC8–C24n-alkanesandMS.

Animals

MaleICRmiceat6–8weeksofage(18–22g)werepurchased fromtheMedicineExperimentAnimalCenterofNingxiaMedicine University.Theanimalswereallowedtoacclimatizefortwodays toanimalroomconditionsandweremaintainedonastandard pel-letdietandwateradlibitum.Thefoodwaswithdrawnontheday beforetheexperiment,buttheanimalswereallowedfreeaccess towater.AllanimalswerecaredforincompliancewiththeGuide fortheCareandUseofLaboratoryAnimals(1996).The experimen-talprocedureswereapprovedbyourinstitutionalanimalresearch ethicscommittee(approvalnumber:SYXK(NING)2011-0001).

Rodentmodeldesign

Theanimalswererandomlydividedintofivegroupsoffifteen miceeach.Thecontrolgroupand modelgroupmicewere gav-agedwithsesameoilfor6days.Positivecontrolgroupmicewere gavagedwithbifendatetablets(BT,10mg/kg)for6days.The exper-imentalgroupsweretreatedwith100mg/kgand50mg/kgAEO

dissolvedinsesameoilfor6days.Onday6,thecontrolgroupwas treatedwithsesameoil,andalloftheothergroupsweretreated withasingledoseof0.2%CCl4insesameoil(10ml/kg)by

intraperi-tonealinjection.Themicewerethenfastedfreeofwater,andblood samples werecollected from the retrobulbarvessels; collected bloodwascentrifugedat3000×gfor10mintoseparatetheserum. Cervicaldislocationwasperformedimmediatelyafterwithdrawal ofblood,andliversampleswerepromptlyremoved.Onepartof theliversamplewasimmediatelystoredat−20◦_C_until_analysis, andanotherpartwasexcisedandfixedina10%formalinsolution; theremainingtissueswerestoredat−80◦_C_for_{histopathological} analysis(Wangetal.,2008;Hsuetal.,2009;Nieetal.,2015).

Measurementofthebiochemicalparametersintheserum

Liverinjurywasassessedbyestimatingtheenzymaticactivities ofserumALTand ASTusingthecorrespondingcommercialkits accordingtotheinstructionsforthekits(Nanjing,JiangsuProvince, China).Theenzymaticactivitieswereexpressedasunitsperliter (U/l).

MeasurementofMDA,SOD,GSHandGSH-Pxinliverhomogenates

Livertissueswerehomogenizedwithcoldphysiologicalsaline ata1:9ratio(w/v,liver:saline).Thehomogenateswerecentrifuged (2500×gfor 10min) tocollectthe supernatantsfor the subse-quentdeterminations.Liverdamagewasassessedaccordingtothe hepaticmeasurementsoftheMDAandGSHlevelsaswellasthe SODandGSH-Pxactivities.Alloftheseweredetermined follow-ingtheinstructionsonthekit(Nanjing,JiangsuProvince,China). TheresultsforMDAandGSHwereexpressedasnmolpermg pro-tein(nmol/mgprot),andtheactivitiesofSODandGSH-Pxwere expressedasUpermgprotein(U/mgprot).

Histopathologicalanalysis

Portionsoffreshlyobtainedliverwerefixedina10%buffered paraformaldehyde phosphate solution. The sample was then embeddedin paraffin,slicedinto3–5␮msections, stainedwith hematoxylinandeosin(H&E)accordingtoastandardprocedure, andfinallyanalyzedbylightmicroscopy(Tianetal.,2012).

Statisticalanalysis

Theresultswereexpressedasthemean±standarddeviation (SD).TheresultswereanalyzedusingthestatisticalprogramSPSS Statistics,version19.0.Thedataweresubjectedtoananalysisof variance(ANOVA, p<0.05)followed byDunnett’stest and Dun-nett’sT3testtodeterminethestatisticallysignificantdifferences

betweenthevaluesofvariousexperimentalgroups.Asignificant differencewasconsideredatalevelofp<0.05.

Resultsanddiscussion

ConstituentsofAEO

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Table1

ChemicalcompositionofessentialoilfromArtemisiacapillarisThun.(AEO).

Compound Rta _RIcalcb _RIlitc _%d

␣-Pinene 11.133 928 933 7.21

Camphene 11.340 943 946 1.52

Sabinene 13.269 968 969 1.09

␤-Pinene 13.640 975 974 3.99

Myrcene 14.313 987 988 2.02

Iso-cineole 15.612 1012 1012 1.27

p-Cymene 16.025 1020 1020 2.28

Limonene 16.273 1025 1024 2.00

1,8-Cineole 16.424 1028 1026 13.09

Artemisiaketone 17.965 1057 1056 1.44

Terpinolene 19.226 1081 1086 1.30

Linalool 20.112 1098 1095 11.33

trans-Pinocarveol 17.965 1135 1135 1.44

Camphor 22.215 1140 1141 12.59

Terpinen-4-ol 23.998 1176 1174 1.21

␣-Terpineol 24.744 1191 1186 1.89

Dodecane 25.173 1199 1200 2.44

␤-Citronellol 26.407 1226 1211 16.23

Thymol 29.356 1288 1289 3.22

Longipinene 34.330 1401 1402 2.16

(E)-Caryophyllene 34.813 1413 1417 1.73

a_Rt_–_retention_time_(min)_form_a_linear_temperature_program. b_RIcalc_–_retention_index_calculated_for_each_compound. c _RIlit_–_retention_index_obtain_from_published_literatures. d _%_–_relative_abundances_from_the_peak_area_integration.

(7.21%),␤-pinene(3.99%),thymol(3.22%), andmyrcene(2.02%). Thevariationinthechemicalcompositionmayberelatedtothe environmentalconditionsthattheplantwasexposedto,suchas mineralwater,sunlight,thestageofdevelopmentandnutrition.

Aspreviouslysuggested,fewstudieshavereportedthe hepato-protectiveeffectsof someofthesemajorcompounds.However, therearefindingsthatindicatethatmajorcompoundspossessan anti-inflammatoryeffect,suchasthymol(Bragaetal.,2006), euca-lyptol,limonene,andlinalool(KuandLin,2013).␤-Myrceneand 1,8-cineolewerealsofoundtoeliminateTCDD-inducedoxidative stressinrats(Ciftcietal.,2011).

Measurementofthebiochemicalparametersintheserum

CCl4-inducedhepaticinjury isa commonlyused

experimen-talanimalmodel(JohnstonandKroening,1998).Apreviousstudy reportedthatCCl4administrationisassociatedwithactivationby

thecytochromesystem(e.g.,CYP2E1)toformtrichloromethyl

radi-cals(CCl3•).ItisgenerallythoughtthatCCl4 toxicityisduetoa

reactiveintermediatethatisgeneratedbyitsreductivemetabolism. Thishighlyreactiveintermediateisknowntoinducelipid peroxida-tion,oxidativestress,hepaticnecrosisandapoptosis(Weberetal.,

2003).

Inthisassay,theresultsofthehepatoprotectiveeffectsofAEOon theenzymaticactivitiesofserumALTandASTareshowninFig.1A andB,respectively.AnintraperitonealinjectionofCCl4 induced

anotableincreaseinALTandASTactivitiesincomparison with untreatedcontrolmice(p<0.01);theincreasedserumactivitiesof ALTandASTenzymesinCCl4treatedmiceconfirmedthehepatic

damage.Conversely,pretreatmentwithAEOatdosesof100mg/kg and50mg/kg,aswellasbifendatetablets,obviouslyreversedthe toxin-inducedincreaseintheALTandASTlevelscomparedwith toxinmodelgroup2(p<0.01).Whenthedosereached100mg/kg, theresultswereasgoodasBT.

ALTandASTareimportantmetabolicenzymesoftheliver.After CCl4administration,theserumALTandASTactivitiesinmicewere

evaluated.These enzymesnormallyexist in thecytoplasm, but upon liverinjury, theycanenter thecirculatory systemdueto

toxicity-mediatedalteredpermeabilityofthecellularmembrane

(WillsandAsha,2006).Inapreviousstudy,itwasreportedthat

administrationofCCl4inmicecausedincreasedALTandAST

activ-ities.

OurdatashowedthattheliverdamageinducedbyCCl4

ele-vateslivermarkerenzymes.Elevatedactivitiesofserumenzymes, ALTandASTareindicativeofcellularleakageandthelossofthe functionalintegrityofthecellmembraneintheliver,while pre-treatmentwithAEOeffectivelydecreasedtheamountofASTand ALTleakage.

MeasurementofMDA,SOD,GSHandGSH-Pxinliverhomogenates

ThelevelsofMDAsuggestenhancedperoxidationleadingto tissuedamageandfailureoftheantioxidant-defensemechanisms topreventtheformationofexcessivefreeradicals(Chengetal.,

2013; Pareeket al., 2013).However, AEO at 50 and 100mg/kg

couldmarkedlypreventtheincreaseinMDAformation(Fig.1C), whichclearlydemonstratedtheabilityofAEOtorelievelipid per-oxidation.MDAiswellknowntobethemostabundantindividual aldehyderesultingfromlipidperoxidationandiscommonlyused asanindicatoroflivertissuedamageinvolvingaseriesof oxida-tivechainreactions(Huangetal.,2012).Inthepresentresearch, micetreatedwithCCl4showedastrikingincreaseinMDAlevels

compared tountreatednormal mice(p<0.01).SODand GSH-Px

arethemajorenzymesthatplayanimportantroleinthe elim-ination of toxic metabolites, which are the major cause of the liver pathology caused by CCl4 (Cengiz et al., 2013; Xia et al.,

2013).Here,administrationofCCl4tomicesharplydecreasedthe

SODandGSH-Pxactivitiesinmouselivertissues,asevidencedby theinhibitionoftheirenzymaticactivities.However,thedecrease in theseenzymatic activities wassignificantlyelevated by pre-treatmentwithAEO,suggestingthattheycouldprotectthethree antioxidantenzymesinCCl4-damagedlivers.Theprotectiveeffect

of AEO waspotent in terms of the SOD and GSH-Px activities, whichismostlikelyrelatedtotheeliminationoftoxic metabo-lites(Fig.1DandF).SODisamanganese-containingenzymeinthe mitochondriaandconvertsthedismutationofsuperoxideanions into hydrogen peroxide (H2O2) (Reiter et al., 2000).GSH-Px is

animportant enzymethat catalyzesthereduction ofH2O2 and

hydroperoxidesandterminatesthechainreactionoflipid peroxi-dationbyremovinglipidhydroperoxidesfromthecellmembrane (Aietal.,2013).Similarly,GSHisanimportantwater-phase antiox-idantandanessentialcofactorforantioxidantenzymes;itprotects themitochondriaagainstendogenousoxygenradicals(Hanetal., 2006).TheendogenousantioxidantGSHperoxidaseinhibits oxida-tive stressby quickly removing superoxideradicals (Chaudiere et al.,1999) and playsanimportant role inclearing intracellu-lar hydrogenperoxide and lipid peroxides. Therefore, thelevel ofGSHinthebodyisconsideredtobeanimportantindicatorof antioxidativecapacity(Campoetal.,2001;Iwamotoetal.,2002).

Fig.1EshowedthatCCl4treatmentinducedasignificantdecrease

inthelevelofGSHinliverhomogenatescomparedtocontrollivers. ThetreatmentofmicewithAEOat50and100mg/kgsignificantly increasedthehepaticGSHcontentcomparedwiththeCCl4-treated

group.Thisfindingindicatedthatthefreeradicalsreleasedinthe liverwereeffectivelyscavengedduringAEOtreatment.Allofthese data suggest that thehepatoprotective effects of AEO onCCl4

-inducedliverdamageinmiceareassociatedwithitscapacityfor freeradicalscavengingandantioxidation.

Histopathologicalexaminationofmouselivers

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Bifendatatum 100 mg/kg

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Fig.1.EffectsoforalpretreatmentwithAEOonCCl4-inducedhepaticinjury.Theresultsofthebiochemicalparameters(ALT,AST)andhepaticlevels(MDA,SOD,GSH

andGSH-Px)arerepresentedasthemean±SD(n=15).ThemicewerepretreatedwithAEO(100,50mg/kg)oncedailyfor6consecutivedays.Thecontrolmiceweregiven sesameoil.DifferencesbetweenthegroupsweredeterminedbyANOVAfollowedbyDunnett’stest.*p<0.05,**p<0.01comparedtothemodelgroup,and#_p_<_0.05,##_p_<_0.01

comparedtothecontrolgroup.

hepaticcellswithawell-preservedcytoplasmandlegiblenucleus, hepatocytesthatwereradiallyarrangedaroundthecentralvein, andawell-definedsinusoidalline(Fig.2A).CCl4causeddamageto

thehepaticarchitectureandproducedhistologicalchanges,suchas severehepatocellulardegenerationandnecrosisaroundthecentral vein,sinusoidaldilatation,lossofcellularboundaries, inflamma-torycell infiltration, and cytoplasmic vacuolation,all of which confirmedthesuccessfulestablishment ofliverinjury (Fig.2B). In contrast, CCl4-intoxicated mice pretreated with BT showed

nearnormalization of liver tissues withno significant changes inhepatocytes(Fig.2C).The liverdamagewasnoteworthyand dose-dependentlyreducedbypretreatmentwithAEOatdifferent doses,asindicatedbythesignificantreductioninthenumberof ballooning-degeneratedhepatocytes and significantlydecreased

necrotic area. In the grouppretreated with a low doseof AEO (Fig.2E),theliversectionsshowedmoderatehypertrophyof hepa-tocyteswitharelativelyintactcentralvein,ashrunkensinusoidal areaandreducedinflammatorycells.Theadministrationofahigh doseofAEO(Fig.2D)inducedanear-normalappearance, suggest-ingthatAEOatadoseof100mg/kgwasmoreeffectivecompared tothedoseof50mg/kgandthatAEOcouldprotecttheliverfrom acuteCCl4-inducedhepaticdamage.Thiswasingoodagreement

withtheresultsfromtheserumbiochemicalmarkers.

Insummary,theresultsfromthisstudyclearlydemonstratethat AEOiseffectiveforthepreventionofCCl4-inducedhepaticinjury

inmice.Thisstudyindicatesthepotentialforthedevelopmentof AEOasapotentialhepatoprotectiveagentincasesofCCl4induced

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Fig.2.EffectsofAEOonthehistologicalchangesoftheliverafterCCl4treatmentinmice(originalmagnification400×):(A)controlgroup;(B)CCl4-intoxicatedgroup(model

group);(C)bifendatatum(10mg/kg)+CCl4;(D)AEO(100mg/kg)+CCl4;(E)AEO(50mg/kg)+CCl4.

Authorcontributions

QG,LYandXZcontributedtoperformingthelaboratorywork. YZandXZcontributedtotheestimationofthechemical compo-sition.BWcontributedtotheanalysisofthedata.LYwrotethe manuscript.QGandXWcontributedtothecriticalreadingofthe manuscript.XFandWSdesignedthestudy,supervisedthe labora-toryworkandcontributedtothecriticalreadingofthemanuscript. Alloftheauthorshavereadthefinalmanuscriptandapprovedits submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (Project No. 2013BAI05B01).

References

Ai,G.,Liu,Q.,Hua,W.,Huang,Z.,Wang,D.,2013.Hepatoprotectiveevaluationof thetotalflavonoidsextractedfromflowersofAbelmoschusmanihot(L.)Medic:

invitroandinvivostudies.J.Ethnopharmacol.146,794–802.

Ait-Ouazzou,A.,Lorán,S.,Arakrak,A.,Laglaoui,A.,Rota,C.,Herrera,A.,Pagán,R., Conchello,P.,2012.Evaluationofthechemicalcompositionandantimicrobial activityofMenthapulegium,Juniperusphoenicea,andCyperuslongusessential oilsfromMorocco.FoodRes.Int.45,313–319.

Argyropoulou,C.,Skaltsa,H.,2012.Identificationofessentialoilcomponentsof Mar-rubiumthessalumBoiss.&Heldr.growingwildinGreece.Nat.Prod.Rep.26, 593–599.

Bagheri,H.,AbdulManap,M.Y.B.,Solati,Z.,2014.Responsesurfacemethodology appliedtosupercriticalcarbondioxideextractionofPipernigrumL.essential oil.LWT–FoodSci.Technol.57,149–155.

BenMansour,M.,Balti,R.,Rabaoui,L.,Bougatef,A.,Guerfel,M.,2013.Chemical compositionangiotensinI-convertingenzyme(ACE)inhibitory,antioxidantand antimicrobialactivitiesoftheessentialoilfromsouthTunisianAjugapseudoiva

Rob.Lamiaceae.ProcessBiochem.48,723–729.

Braga,P.C.,DalSasso,M.,Culici,M.,Bianchi,T.,Bordoni,L.,Marabini,L.,2006. Anti-inflammatoryactivityofthymol:inhibitoryeffectonthereleaseofhuman neutrophilelastase.Pharmacology77,130–136.

Ćavar,S.,Maksimovi ć,M.,Vidic,D.,Pari ć,A.,2012.Chemicalcompositionand antiox-idantandantimicrobialactivityofessentialoilofArtemisiaannuaL.fromBosnia. Ind.CropsProd.37,479–485.

Cengiz,N.,Kavak,S.,Guzel,A.,Ozbek,H.,Bektas,H.,Him,A.,Erdo˘gan,E.,Balahoro˘glu, R.,2013.InvestigationofthehepatoprotectiveeffectsofSesame(Sesamum indicumL.)incarbontetrachloride-inducedlivertoxicity.J.Membr.Biol.246, 1–6.

Cha,J.D.,Moon,S.E.,Kim,H.Y.,Lee,J.C.,Lee,K.Y.,2009a.Theessentialoilisolated fromArtemisiacapillarispreventsLPS-inducedproductionofNOandPGE(2)by inhibitingMAPK-mediatedpathwaysinRAW2647macrophages.Immunol. Invest.38,483–497.

Cha,J.D.,Moon,S.E.,Kim,H.Y.,Cha,I.H.,Lee,K.Y.,2009b.EssentialoilofArtemisia capillarisinducesapoptosisinKBcellsviamitochondrialstressandcaspase activationmediatedbyMAPK-stimulatedsignalingpathway.J.FoodSci.74, T75–T81.

Chen,Y.,Zhou,C.,Ge,Z.,Liu,Y.,Liu,Y.,Feng,W.,Li,S.,Chen,G.,Wei,T.,2013. Compo-sitionandpotentialanticanceractivitiesofessentialoilsobtainedfrommyrrh andfrankincense.Oncol.Lett.6,1140–1146.

Cheng,N.,Ren,N.,Gao,H.,Lei,X.,Zheng,J.,Cao,W.,2013.Antioxidantand hepato-protectiveeffectsofSchisandrachinensispollenextractonCCl4-inducedacute

liverdamageinmice.FoodChem.Toxicol.55,234–240.

Cheng,S.S.,Liu,J.Y.,Lin,C.Y.,Hsui,Y.R.,Lu,M.C.,Wu,W.J.,Chang,S.T.,2008. Ter-minatingredimportedfireantsusingCinnamomumosmophloeumleafessential oil.Bioresour.Technol.99,889–893.

Ciftci,O.,Ozdemir,I.,Tanyildizi,S.,Yildiz,S.,Oguzturk,H.,2011.Antioxidativeeffects ofcurcumin,beta-myrceneand1,8-cineoleagainst 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced oxidative stress in rats liver. Toxicol. Ind. Health 27, 447–453.

Da Silva,J.K.R.,Pinto, L.C.,Burbano,R.M.R., Montenegro,R.C., Guimarães,E.F., Andrade,E.H.A.,Maia,J.G.,2014.EssentialoilsofAmazonPiperspeciesandtheir cytotoxicantifungal,antioxidantandanti-cholinesteraseactivities.Ind.Crop. Prod.58,55–60.

Desai,M.A.,Parikh,J.,De,A.K.,2014.Modellingandoptimizationstudieson extrac-tionoflemongrassoilfromCymbopogonflexuosus(Steud.).Wats.Chem.Eng. Res.Des92,793–803.

Ding,R.B.,Tian,K.,Huang,L.L.,He,C.W.,Jiang,Y.,Wang,Y.T.,Wan,J.B.,2012.Herbal medicinesforthepreventionofalcoholicliverdisease:areview.J. Ethnophar-macol.144,457–465.

Feng,G.,Wang,X.,You,C.,Cheng,X.,Han,Z.,Zong,L.,Zhou,C.,Zhang,M.,2013. AntiproliferativepotentialofArtemisiacapillarispolysaccharideagainsthuman nasopharyngealcarcinomacells.Carbohydr.Polym.92,1040–1045.

Gouveia,S.C.,Castilho,P.C.,2013.ArtemisiaannuaL.:essentialoilandacetoneextract compositionandantioxidantcapacity.Ind.Crop.Prod.45,170–181. Guo,F.Q.,Liang,Y.Z.,Xu,C.J.,Li,X.N.,Huang,L.F.,2004.Analyzingofthevolatile

chemicalconstituentsinArtemisiacapillarisherbabyGC–MSandcorrelative chemometricresolutionmethods.J.Pharm.Biomed.Anal.35,469–478. Han,K.H.,Jeon,Y.J.,Athukorala,Y.,Choi,K.D.,Kim,C.J.,Cho,J.K.,Sekikawa,M.,

Fukushima,M.,Lee,C.H.,2006.AwaterextractofArtemisiacapillarisprevents 2,2′_{-azobis(2-amidinopropane)}_{dihydrochloride-induced}_liver_damage_in_rats._J.

Med.Food9,342–347.

Hsu,Y.W.,Tsai,C.F.,Chen,W.K.,Lu,F.J.,2009.Protectiveeffectsofseabuckthorn (HippophaerhamnoidesL.)seedoilagainstcarbontetrachloride-induced hepa-totoxicityinmice.FoodChem.Toxicol.47,2281–2288.

Huang,Q.,Zhang,S.,Zheng,L.,He,M.,Huang,R.,Lin,X.,2012.Hepatoprotective effectsoftotalsaponinsisolatedfromTaraphochlamysaffinisagainstcarbon tetrachlorideinducedliverinjuryinrats.FoodChem.Toxicol.50,713–718. Johnston,D.E.,Kroening,C.,1998.Mechanismofearlycarbontetrachloridetoxicity

inculturedrathepatocytes.Pharmacol.Toxicol.83,231–239.

Komiya,T.,Naruse,Y.,Oshio,H.,1976.StudiesonInchinkoII.Studiesonthe com-poundsrelatedtocapillarisinandflavonoids.YakugakuZasshi96,855–862. Ku,C.M.,Lin,J.Y.,2013.Anti-inflammatoryeffectsof27selectedterpenoid

com-poundstestedthroughmodulatingTh1/Th2cytokinesecretionprofilesusing murineprimarysplenocytes.FoodChem.141,1104–1113.

(6)

Nie,Y.,Ren,D.,Lu,X.,Sun,Y.,Yang,X.,2015.Differentialprotectiveeffectsof polyphenolextractsfromapplepeelsandfleshesagainstacuteCCl(4)-induced liverdamageinmice.FoodFunct.6,513–524.

Pandey,V.,Verma,R.S.,Chauhan,A.,Tiwari,R.,2014.Compositionalvariationin theleafflowerandstemessentialoilsofHyssop(HyssopusofficinalisL.)from Western-Himalaya.J.Herb.Med.4,89–95.

Papay,J.I.,Clines,D.,Rafi,R.,Yuen,N.,Britt,S.D.,Walsh,J.S.,Hunt,C.M.,2009. Drug-inducedliverinjuryfollowingpositivedrugrechallenge.Regul.Toxicol.Pharm. 54,84–90.

Pareek,A.,Godavarthi,A.,Issarani,R.,Nagori,B.P.,2013.Antioxidantand hepato-protectiveactivityofFagoniaschweinfurthii(Hadidi)Hadidiextractincarbon tetrachlorideinducedhepatotoxicityinHepG2celllineandrats.J. Ethnophar-macol.150,973–981.

Qi,X.-L.,Li,T.-T.,Wei,Z.-F.,Guo,N.,Luo,M.,Wang,W.,Zu,Y.-G.,Fu,Y.-J.,Peng,X., 2014.Solvent-freemicrowaveextractionofessentialoilfrompigeonpealeaves [Cajanuscajan(L.)Millsp.]andevaluationofitsantimicrobialactivity.Ind.Crop Prod.58,322–328.

Rather,M.A.,Dar,B.A.,Shah,W.A.,Prabhakar,A.,Bindu,K.,Banday,J.A.,Qurishi,M.A., 2014.ComprehensiveGC–FIDGC–MSandFT-IRspectroscopicanalysisofthe volatilearomaconstituentsofArtemisiaindicaandArtemisiavestitaessential oils.Arab.J.Chem.,http://dx.doi.org/10.1016/j.arabjc.2014.05.017.

Reiter,R.,Tan,D.-X.,Osuna,C.,Gitto,E.,2000.Actionsofmelatonininthereduction ofoxidativestress.J.Biomed.Sci.7,444–458.

Sadgrove, N.J.,Goncalves-Martins, M.,Jones, G.L.,2014. Chemogeography and antimicrobialactivityofessentialoilsfromGeijeraparvifloraandGeijera sali-cifolia(Rutaceae):twotraditionalAustralianmedicinalplants.Phytochemistry 104,60–71.

Sereshti,H.,Heidari,R.,Samadi,S.,2014.Determinationofvolatilecomponents ofsaffronbyoptimisedultrasound-assistedextractionintandemwith dis-persiveliquid–liquidmicroextractionfollowedbygaschromatography–mass spectrometry.FoodChem.143,499–505.

Singh,S.,Kapoor,I.P.S.,Singh,G.,Schuff,C.,DeLampasona,M.P.,Catalan,C.A.N., 2013.Chemistryantioxidantandantimicrobialpotentialsofwhitepepper(Piper nigrumL.)essentialoilandoleoresins.Proc.Natl.Acad.Sci.IndiaB83,357–366.

Sylvestre,M.,Pichette,A.,Longtin,A.,Nagau,F.,Legault,J.,2006.Essentialoilanalysis andanticanceractivityofleafessentialoilofCrotonflavensL.fromGuadeloupe. J.Ethnopharmacol.103,99–102.

Tao,N.,Jia,L.,Zhou,H.,2014.Anti-fungalactivityofCitrusreticulataBlanco essen-tialoilagainstPenicilliumitalicumandPenicilliumdigitatum.FoodChem.153, 265–271.

Tian,J.,Zeng,X.,Zhang,S.,Wang,Y.,Zhang,P.,Lü,A.,Peng,X.,2014.Regional varia-tionincomponentsandantioxidantandantifungalactivitiesofPerillafrutescens

essentialoilsinChina.Ind.CropProd.59,69–79.

Tian,L.,Shi,X.,Yu,L.,Zhu,J.,Ma,R.,Yang,X.,2012.Chemicalcompositionand hepatoprotectiveeffectsofpolyphenol-richextractfromHouttuyniacordatatea. J.Agric.FoodChem.60,4641–4648.

Wang,N.,Li,P.,Wang,Y.,Peng,W.,Wu,Z.,Tan,S.,Liang,S.,Shen,X.,Su,W., 2008.HepatoprotectiveeffectofHypericumjaponicumextractanditsfractions. J.Ethnopharmacol.116,1–6.

Weber,L.W.,Boll,M.,Stampfl,A.,2003.Hepatotoxicityandmechanismofactionof haloalkanes:carbontetrachlorideasatoxicologicalmodel.Crit.Rev.Toxicol.33, 105–136.

Wills,P.J.,Asha,V.V.,2006.ProtectiveeffectofLygodiumflexuosum(L)Sw.extract againstcarbontetrachloride-inducedacuteliverinjuryinrats.J. Ethnopharma-col.108,320–326.

Xia,D.Z.,Zhang,P.H.,Fu,Y.,Yu,W.F.,Ju,M.T.,2013.Hepatoprotectiveactivityof puerarinagainstcarbontetrachloride-inducedinjuriesinrats:arandomized controlledtrial.FoodChem.Toxicol.59,90–95.

Yamahara, J., Kobayashi, G., Matsuda, H., Katayama, T., Fujimura, H., 1989. Theeffect ofscoparone a coumarinderivative isolated from theChinese crudedrug Artemisiaecapillarisflos,ontheheart.Chem.Pharm. Bull.37, 1297–1299.

Yoon,M.,Kim,M.Y.,2011.Theanti-angiogenicherbalcompositionOb-XfromMorus albaMelissaofficinalis,andArtemisiacapillarisregulatesobesityingenetically obeseob/obmice.Pharm.Biol.49,614–619.