Rev. bras. farmacogn. vol.27 número3

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

Original

article

Anti-inflammatory

activity

and

chemical

composition

of

dichloromethane

extract

from

Piper

nigrum

and

P.

longum

on

permanent

focal

cerebral

ischemia

injury

in

rats

Bing

Wang

,

Yuanbin

Zhang

,

Jun

Huang

,

Lin

Dong

,

Tingting

Li

,

Xueyan

Fu

a_{SchoolofPharmacy,NingxiaMedicalUniversity,Yinchuan,China}

b_{SchoolofPharmacy,NanjingUniversityofChineseMedical,Nanjing,China}

c_{TraditionalChineseMedicineSchoolofNingxiaMedicalUniversity,Yinchuan,China}

d_{NingxiaEngineeringandTechnologyResearchCenterforModernizationofHuiMedicine,Yinchuan,China}

e_{KeyLaboratoryofHuiEthnicMedicineModernization,MinistryofEducation(NingxiaMedicalUniversity),Yinchuan,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29November2016 Accepted17February2017 Availableonline9March2017

Keywords:

Anti-inflammatory HPLC

Permanentmiddlecerebralarteryocclusion Piperine

a

b

s

t

r

a

c

t

Whitepepper(PipernigrumL.)andlongpepper(PiperlongumL.)belongtofamilyPiperaceaeandare commonlyusedashouseholdspicesandtraditionalmedicineworldwide,specificallyinChinaand South-eastAsia.InTraditionalChineseHuiMedicine,theseherbsarewidelyusedfortreatmentofstroke.Our presentstudyinvestigatedeffectsoftheseherbsoninflammationinratmodelwithcerebralischemia. Aftersubjectingtheratstopermanentmiddlecerebralarteryocclusion(pMCAO)for6h,atdosesof 100and200mg/kg,dichloromethanefractionfromwhitepepperandlongpepper,respectively,was intragastricallyadministeredonceadayforsevenconsecutivedays.Cerebralcorticalandhippocampal tissueswerecollectedaftersevendays.Superoxidedismutase,malonaldehyde,tumornecrosis factor-alpha(TNF-␣),interleukin-1beta(IL-1␤),andIL-6weremeasuredbyspectrophotometer.Phytochemical profileofdichloromethanefractionwasdeterminedthroughHPLC.Dichloromethanefraction exhib-itedanti-inflammatoryactivitybysuppressingexpressionorproductionofIL-1␤,IL-6,andTNF-␣.By contrast,dichloromethane fractionshowedactivity againstpMCAO injurybyreducingoxygen-free radicalsthroughincreasedsuperoxidedismutaseactivity anddecreasedmalonaldehydelevel.HPLC analysisrevealedpiperineasmajorcomponentofdichloromethanefraction.Theseresultsshowthat dichloromethanefractionprovidesprotectionagainstcerebralischemia.Thepossiblemechanismis relatedtoanti-inflammatoryactivityandreductioninoxygen-freeradicals.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

TraditionalChineseHuiMedicineisanintegrationoftraditional ChinesemedicineandArabmedicineandisfocusedonstroke treat-ment.Excellentachievementsinstroketreatmentwereattained bytheclassics,includingHuiHuiYaoFang,HuiYaoHerbal,and RuiZhuTangEmpiricalFormula(Liuetal.,2011;Lietal.,2013a,b,

2014;Zhangetal.,2014).Ourpreviousstudiesshowedthatin

tra-ditionalChineseHuimedicine,whitepepper(PipernigrumL.)and longpepper(P.longumL.)accountforhighestfrequenciesof56.4% (whitepepper)and42.6%(longpepper),respectively,compared withotherherbs prescribedfor stroketreatment.Furthermore,

∗ Correspondingauthor.

E-mail:[email protected](X.Fu).

1_{Theseauthorscontributedequallytothismanuscript.}

theseherbsare oftenusedin similarformula(Liet al.,2013b). Therefore,wespeculatedthatthesetwoherbsarethemain com-ponentsofstrokeprescriptions.

WhitepepperandlongpepperbelongtofamilyPiperaceaeand arewidelycultivated inChinaandSoutheast Asia.Theseplants arecommonlyusedashouseholdspices(suchasfoodadditives andcondiments)andtraditionalmedicinebymanypeople world-wide,specificallyinChinaandSoutheastAsia(Kimetal.,2012). These medicines present well-documented properties, such as anti-plateletaggregation,anti-atherogenic,antioxidant,and anti-inflammatoryactivities(Kimetal.,2012;Sonetal.,2012).Afew studies wereconductedon useof whitepepper and long pep-perforstroketreatment.However,theirpharmacologicaleffects andunderlyingmechanismsarepoorlyunderstood.Thus,studies shouldcenteronmechanismonhowwhitepepperandlong pep-perprotectthebrainagainstcerebralischemia.Residues(extracted bythesupercriticalfluidCO2)containethanolextracts,including

http://dx.doi.org/10.1016/j.bjp.2017.02.003

supercriticalfluidCO2 extractofwhitepepperandlongpepper, dichloromethanefraction(DF),ethylacetatefraction,butylalcohol fraction,andwater,whichwereusedinpermanentmiddle cere-bralarteryocclusion(pMCAO)ratmodelsduringourpreliminary experiment.WeobservedthatDFcausedlessirritationand signifi-cantlyimprovedneurologicalstatusoftherats.Thus,DFissafeand effectiveforstrokeprevention.However,studiesshouldidentify supportingroleofthiscompound.

Thisexperimentalstudyinvestigated effects ofDF on oxida-tivestressandinflammationinendovascularpMCAOratmodels ofembolicstroke.ChemicalcompositionofDFwassimultaneously determinedwithhighperformanceliquidchromatography(HPLC). This study aids in understanding intricate material basis and mechanismsofDFintreatmentofstrokeandprovidespotential strategiesfortreatingischemicstrokeusingnewdrugfrom Tradi-tionalChineseHuiMedicine.

Materialsandmethods

Drugsandchemicals

HPLCgrade methanol waspurchased fromFischer Scientific (ThermoElectron LLSIndia Pvt. Ltd.,Mumbai, India). Ultrapure waterwasobtainedfromMilliQsystem(MilliporeCorp.,Bedford, MA,USA).Thefollowingdrugsandchemicalswereused:ethanol, methanol,petroleumether,dichloromethane,ethylacetate,butyl alcohol,andchloralhydrate;thecompoundswereobtainedfrom DamaoChemicalCompany(Tianjin,China).DFwassuspendedin 0.5%(w/v)dimethylsulfoxide(DMSO)/0.5%(w/v)sodiumcarboxyl methylcellulose(Na-CMC).

Herbalpreparationandextraction

Fruitsofwhite pepper (Piper nigrumL.) andlong pepper (P. longumL.)werepurchased formAnhui TaiyuanChinese Herbal PharmacologyCo.,Ltd.(VoucherNo.,SCHM121215).Plantsamples wereauthenticatedbyLinDongofPharmacognosyDepartment, College of Pharmacy, Ningxia Medical University. A voucher specimenwasdeposited in thesame unit (Herbarium number, 20141220).

Ethanol(70%)(solidtoliquidmassratioof1:10)wasusedto extractresiduesofsupercriticalfluidCO2extractofwhitepepper andlongpepper(20kg)thrice.Extractingsolutionwasmixedfor rotatoryevaporationuntilalcoholictastewasnolongerdetected. Theyieldwascrudeextract,whichwasthensuccessively parti-tionedwithpetroleumetheranddichloromethane,ethylacetate, butylalcohol,andaqueousresidue.Theseextractivefractionswere thencombined,evaporatedtodryness,andstoredat4◦_Cuntil fur-theranalyses.

Experimentalanimals

Specificpathogen-freemaleSprague–Dawleyrats(260–320g; license no., SCXK (NING) 2012-0001) were purchased from theExperimental Animal Center of Ningxia Medical University (Ningxia,China).Ratswereindividuallyhousedinstandard lab-oratory cages in barrier environment with moderate humidity (55%±5%)and constant temperature (22±1◦_{C) were provided} withfoodandwateradlibitumunder12hlight–darkcycleprior theexperiments.ExperimentprotocolwasapprovedbytheEthics CommitteeofNingxiaMedicalUniversity,Ningxia(Ethicsapproval, 2015-156).

AnimalpMCAOmodel

pMCAOwasperformedusingapreviouslypublishedmethod

(Koizumietal.,1986;Kogure,1989;Luoetal.,2014)withminor

modification.Afterfooddeprivationfor12handwateradlibitum, animalswereanesthetizedwithchloralhydrate(7%,0.5ml/100g; intraperitonealinjection)andwereplacedinsupineposition(body temperaturewasmaintainedat37◦_{C usingfeedback-controlled} heatingpad;theratswerepositionedinstereotaxicframe).After performingmedianincisiononneckskin,therightprimarycarotid artery(commoncarotid artery(CCA),external artery(ECA),and internal carotid artery (ICA)) were careful isolated. In sham-operatedcontrolrats,rightECAwasligated;a4-0monofilament nylonthread(A4-2026;SunbioBiotech,China)withroundedtip coatedwithpoly-l_{-lysinewasinsertedfromECAintoICAupto}

5–10mm.In the remainingrats, rightMCA wasoccluded with rounded tip coated with poly-L-lysine of monofilament nylon thread(A4-2026), whichwasinserted fromECAintoICAupto a distance of 18–20mm. Nylon thread was cut off from and outsidethebloodvessels.Beforetheratswakeup,body temper-aturewasmaintainedatnormallimits(37±1◦_{C)usingaheating} pad.

Experimentalgroups

PipernigrumandP.longumsampleweighs24g,andDFyield is3.95%. NormalhumandailydoseofDFis (24×3.95%)g/60kg bodyweight.Accordingtotheformuladrat=dhuman×(6–7), nor-maldoseofDFformiceshouldbe94.80–101.06mg/kg/day.Inthe presentstudy,weselected200and100mg/kg/dayashighandlow dosagesfortherats,respectively.Toinvestigateneuroprotective effectsofDF,weusedratpMCAOmodel.Fifty-oneratswere ran-domlydividedintofivegroups,namely,theshamgroup,model group,andDFtreatmentgroups.ForDFLDandDFHDgroup,doses of100and200mg/kgbodyweight,respectively,weredissolvedin 0.5%DMSO/0.5%Na-CMC(1ml/100g).Allratswereintragastrically administeredwiththemedicineevery6honedayafterinducing ischemia.Intragastricaladministrationcontinuedforsevendays. Sevendaysafterreperfusion,allratswereeuthanized;cerebral cor-ticalandhippocampaltissueswerequicklyisolated,immediately frozen,andstoredat−80◦_{Cuntilfurtheruse.}

Biochemicalanalysis

Malondialdehyde (MDA) level and total superoxide dismu-tase (SOD)activityofischemiccorticesand hippocampuswere measuredusingacommerciallyavailablekit(NanjingJiancheng BioengineeringInstitute,China)accordingtomethodofLuoetal.

(2014).Assays wereconductedper manufacturer’sinstructions.

Absorbanceoftestsolutionwasmeasuredat532nmforMDAand 550nmforSOD.

Enzyme-linkedimmunosorbentassay(ELISA)

The proteins were isolated from ischemic cortices and hippocampus.Hippocampaltissueswerehomogenizedby ultra-sonication,andhomogenatewascentrifugedfor30minat1000×g at 4◦_{C. In culture supernatants, cytokine interleukin-1␤} (IL-1␤), IL-6, and tumor necrosis factor-␣ (TNF-␣) levels were protein-quantifiedbyELISAkits(BIOSWAMP)permanufacturer’s protocol.

ChemicalcompositionsofDFbyHPLC

China Pharmacopeia (2015) frequently uses HPLC equipped

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Cortical Hippocampus

Cortical Hippocampus

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10

Sham Model DFLD DFHD

8

6

4

2

0

600

400

200

0

MD

A (nmol/mg tissue)

SOD (U/mg tissue)

A

B

Fig.1. DFtreatmentattenuatedoxidativestressaftercerebralischemicinjury.(A) Malondialdehyde(MDA)level.(B)Superoxidedismutase(SOD)activity.Thevalues areexpressedasthemean±SD(n=9–13).*_p<0.05,**_{p<0.01comparedwithmodel}

group;#_p<0.05,and##_{p<0.01comparedwithshamgroup.}

detectionofmaincompoundsinDF(Commission,2015).Analysis wasperformedonanAgilentTechnologies1260HPLCsystemfitted withAgilentSB-18column(length=250mm,diameter=4.6mm, andpackagingsize=5mm).Columntemperaturewassetat25◦_C. Chromatogramwasmonitoredat343nm.

Statisticalanalysis

Dataarepresentedasmeanvalues±standarddeviation(SD). Statisticalanalysiswasconductedusingone-wayANOVA,followed byDunnett’stestusingSPSSVersion19.0.Differenceswithp<0.05 wereconsideredstatisticallysignificant.

Table1

After7daysofabsolutenumbersandpercentages(%)ofanimalsoperated,animal survival,andmortalitydefinedintosham,modelandDFgroups.

Groups Animals operated

After7days inanimal survival(%)

Animal mortality(%)

Total 51 42(82.35) 17.76

Sham 13 13(100.00) 0.00

Model 15 11(73.33) 26.67

DFLD 12 9(75.00) 25.00

DFHD 11 9(81.82) 18.18

Results

MortalityofpMCAOanimals

Among 51animals withMCAO,an 82.35% survival ratewas observed on seven-day period (Table 1). This finding includes 100.00% survival rate of sham-operated animals. Results also showedthatmortalityofmodelanimals(26.67%)washigherthan thatoftreatmentgroups;lowestdeathratewasobservedforthe DFHDgroup(18.18%).

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Cortical

A

B

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Hippocampus

Cortical Hippocampus

Cortical Hippocampus

Sham Model DFLD DFHD

IL-1 beta (pg/mg protein)

IL-6 (pg/mg protein)

TNF-alpha (pg/mg protein)

40

30

20

10

0

150

100

50

0

250

200

150

100

50

0

Fig.2. DFtreatmentattenuatedinflammationinducedbycerebralischemicinjury. (A)Interleukins-1␤(IL-1␤)level,(B)interleukins-6(IL-6)level,and(C)tumor necro-sisfactor-␣(TNF-␣)level.Thevaluesareexpressedasthemean±SD(n=9–13).

*_p<0.05,**_{p<0.01comparedwithmodelgroup;}#_p<0.05,and##_{p<0.01compared}

mAU

1200

1000

800

600

400

200

0

10 20 30

Piperine

40 50 60 min

Fig.3. HPLCchromatogramsofDFfromPipernigrumandP.longumrecordedat343nm.

Biochemicalanalysis

MDA content of (Fig. 1A) and SOD activity (Fig. 1B) were measuredtoinvestigatepotentialeffectsofDFonendogenous anti-oxidantsystemincorticalandhippocampaltissues.Resultsshowed significantlyincreasedMDAlevelsinthemodelgroupcompared withsham-operatedcontrols(p<0.01).DF-treatedratsshowed sig-nificantlyreducedMDAcontentcompared withmodelgroupin adose-dependentmanner.Atotalof200mg/kgDFreducedMDA contentby46.57%atcorticaltissuesand34.29%atthehippocampus (bothp<0.01).Conversely,themodelgroupexhibitedsignificantly reducedSODactivity,which wassignificantlyattenuatedbyDF. Comparedwiththemodelgroup,DF(dosesof200mg/kg)increased SODlevelsby61.11%atcorticesand34.22%atthehippocampus (bothp<0.01).SOD activityin DF-treatedgroups increasedin a dose-dependentmanner.

ELISAassay

Relative protein levels of IL-1␤, IL-6, and TNF-␣ were sig-nificantly elevated in the animal model group (Fig.2A−C). All proteinlevelsincreasedinthemodelgroupcomparedwiththose inshamgroup(p<0.05).Dose-dependenttreatmentwithDF(100 and200mg/kg)reducedlevelsofIL-1␤,IL-6,andTNF-␣inserum with respect to the model group. DF HD (dose of 200mg/kg) significantly reduced IL-1␤, IL-6, and TNF-␣ levels in rat pre-frontalcortex and hippocampus. IL-1␤ decreased by41.21% at cortices(p<0.01)and by 44.69% atthehippocampus(p<0.01); IL-6decreasedby16.22%atcorticalarea(p<0.01)andby16.67% at the hippocampus (p<0.01); TNF-␣ decreased by 15.73% at cortices(p<0.01) and by14.22% at thehippocampus (p<0.01), respectively.

AnalysisofchemicalconstituentofDFbyHPLC

Inthisstudy,DFfromP.nigrumandP.longumwasdetermined usingHPLCchromatograms,whicharepresentedinFig.3. Compar-ingretentiontime(RT)inFig.3,RTofstandardcompoundpiperine wassimilartothatofthemaincompound.Thus,piperineisthe majorconstituentof studiedherbs.Asdeterminedby HPLC,DF contained30.18%piperine.

Discussionandconclusions

Ischemicstrokeisoneofthesignificantcausesofmorbidityand mortalityworldwide.pMCAOisawell-characterizedmodelandis extensivelyusedtostudymechanismsofcerebralischemicinjury

(Ishratetal.,2010;Changetal.,2013;Taoetal.,2015;Zouetal.,

2016).Researchshowsthat changesinoxidativepropertiesand inflammationtobraininjuryplayvitalrolesinpathogenesisand exertdeleteriouseffectsonprogressionoftissuedamage(Yeetal.,

2016;Zhangetal.,2016).Therefore,reducingoxidativecapacity

and inhibitinginflammatory responseare importantin treating cerebralinjuryandpromotingbrainrepair.

SODis a keyantioxidant enzymein mitochondria(Ye etal., 2016).MDAisa product oflipid peroxidationandis an oxida-tivestress marker (Ahmed et al., 2014; Shu et al., 2016; Yang etal.,2016).SODand MDAare extensivelyusedin experimen-talstudiesoncerebralischemia.Previousstudiesprovedincreased resistancetoischemicinsultsofratsover-expressingSODandwith reducedMDA(Zhangetal.,2013,2016;Yangetal.,2016;Yeetal., 2016).OxidativecapacityisquantifiedbymeasuringlevelsofSOD andMDA.Inourpresentstudy,SOD activitylevelssignificantly decreased,whereasMDAlevelsincreasedafteronsetofcerebral ischemia.Theseresultsrevealedthatoxidativestressoccurs fol-lowingfocalcerebralischemicinjuryin rats.DFtreatmentwith dosesof100and200mg/kgsignificantlyinhibitedincreased lev-elsofMDAandpromotedproductionofcellularantioxidantsSOD inbraintissueofratsafterpMCAOinjury.Thus,wepresumethat DFplaysafundamentalroleasantioxidantagainstfocalcerebral ischemiathroughameliorationofoxidativestress.

In addition, oxidative stressis closely associated todamage causedby excessiveproductionof inflammatorycytokines(Yao

etal.,2011;Yangetal.,2016);oxidativestressresultsinincreased

productionofpro-inflammatorycytokines(Yangetal.,2016).Post ischemicinflammationiscloselyrelatedtobraininjury(Lietal.,

2012;Kaoet al.,2013).Microglialcellsof brainserve asmajor

inflammatoryincellpopulationsincentralnervoussystem; acti-vatedmicrogliareleaseawiderangeofsolublepro-inflammatory cytokines,suchasIL-1␤,IL-6,and TNF-␣,whichstimulate neu-rodegeneration(Xiongetal.,2009;Kaoetal.,2013).Thus,IL-1␤, IL-6,andTNF-␣playimportantrolesinprotectionagainst inflam-matorydamageofischemiainjury(Yaoetal.,2011;Singhetal.,

2013;Yangetal.,2013;Zhangetal.,2013;Luoetal.,2014).The

cytokines,IL-1␤,IL-6,andTNF-␣,increasedinthemodelgroup. Bycontrast,DFtreatmentsignificantlyreducedactivitiesofIL-1␤, IL-6,andTNF-␣inthecortexandhippocampus.Resultsshowed thattheseinflammatorymoleculesareinvolvedinpermanentfocal cerebralischemicinjury.DFtreatmentpreventsinjurybyreducing releaseofIL-1␤,IL-6,andTNF-␣.

Inthisstudy,weusedHPLCmethodtodeterminemajor com-positionofDF;itsprimarycomponentispiperine.Recentresearch suggested that piperine is a chemical trans-trans isomer of 1-piperoylpiperidine,analkaloidpresentinpepperfruits.Piperine wasestablishedasanantioxidant,anti-inflammatory(Singhand

Chopra,2013;Huetal.,2015),andcognitiveenhancer(Banjietal.,

2013).Astudyrecentlydiscoveredthatpiperineplaysamajorrole inoxidativestress(freeradicaldamage)andanti-inflammationby inhibitingactivityofNF-␬B, reducingexpressions ofIL-1␤,IL-6, TNF-␣,andMDAandenhancingSODactivity(Qingetal.,2014;

Danielaetal.,2016;Haoetal.,2016;Samraetal.,2016;Scottetal.,

2016).Piperinealsoreducesbraindamageinducedbyd_-galactose

(Banjietal.,2013).AsmajorcomponentofMu-Xiang-You-Fang(in

HuiHuiYaoFang),piperinealsoplaysanimportantrolein treat-mentofcerebralischemic-reperfusioninjury(Zhaoetal.,2016). Thus,piperineisimportantintreatingbraindamageofratswith permanentfocalcerebralischemiainjuryandhaspotentialusein developmentofnewdrugs.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthe publicationof patientdata.

Right to privacy and informed consent. The authors have obtainedthewritteninformedconsentofthepatientsorsubjects mentionedinthearticle.Thecorrespondingauthorisinpossession ofthisdocument.

Author’contributions

BWandYZcontributedincollectingplantsamplesand conduct-ingthelaboratorywork.JHcontributedtotheanalysisofthedata. LDandTLcontributedtothecriticalreadingofthemanuscript. XFdesignedthestudy,supervisedthelaboratoryworkand con-tributedtothecriticalreadingofthemanuscript.Alloftheauthors havereadthefinalmanuscriptandapproveditssubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Thiswork was supportedform the NationalNature Science FoundationofChinabygrants(ProjectNo.81360666).

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