Rev. bras. farmacogn. vol.27 número5

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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

Antinociceptive

activity

of

Cistanche

salsa

stolons,

growing

in

the

Republic

of

Kazakhstan

Elmira

B. Kartbaeva

a

_,

_Graciela

_R.

_Donald

b

_,

_Zuriyadda

_B.

_Sakipova

a

_,

_Liliya

_N.

_Ibragimova

a

_,

Elmira

N. Bekbolatova

a

_,

_Inna

_I.

_Ternynko

c

_,

_Patricia

_D.

_Fernandes

b

_,

_Fabio

_Boylan

d,∗

a_The_Module

«Pharmacist–Technologist»,AsfendiyarovKazakhNationalMedicalUniversity,Almaty,Kazakhstan

b_Laboratório_de_Farmacologia_da_Dor_e_da_Inflamac¸_ão,_Instituto_de_Ciências_Biomédicas,_Universidade_Federal_do_Rio_de_Janeiro,_Rio_de_Janeiro,_RJ,_Brazil

c_The_Chair_of_{Pharmaceutical}_Chemistry_and_{Pharmacognosy,}_Lugansk_State_Medical_University,_Rubezhnoe,_Stoiteley,_Ukraine

d_School_of_Pharmacy_and_{Pharmaceutical}_Sciences,_Trinity_Biomedical_Sciences_Institute,_Trinity_College_Dublin,_Dublin,_Ireland

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19April2017 Accepted17May2017

Availableonline2September2017

Keywords:

HerbaCistanche

HPLC-UV Echinacoside Acteoside Tubuloside Standardization

a

b

s

t

r

a

c

t

HerbaCistanche(Cistanchespecies)inTraditionalChineseMedicineisusedforthetreatmentofseveral

diseasesandsymptoms,toincludepain.Theobjectiveofthisstudywastoevaluatetheantinociceptive effectofthehydroethanolextractofCistanchesalsa(C.A.Mey.)Beck,Orobanchaceae,stolonsinanimal modelsofpain.ChemicalcompositionofHerbaCistanchewasanalyzedbyHPLC-UV.MiceSwiss Web-ster(25–30g,n=6)wereorallypre-treatedwithHerbaCistanche(10,30or100mg/kg)andevaluatedin theformalintestandinthecapsaicin-orglutamate-inducedlickingresponse.KazakhHerbaCistanche

iscomposedmainlybyphenylpropanoidglycosides,fromwhichechinacoside,acteosideandtubuloside Barethemainconstituents.WhenHerbaCistanchewasadministeredtomiceithadaneffectinboth phasesoftheformalintest(77%activityat30mg/kgforphase1and62%activityat100mg/kgforphase 2)suggestinganalgesicandanti-inflammatoryproperties.KazakhHerbaCistanchewasabletoreduce theanimalslickingtimeafterinjectionofglutamate(81%reductionat30mg/kg)andcapsaicin(81% reductionat100mg/kg).WeconcludethatphenolicspresentinthehydroethanolextractofC.salsacould beresponsibleforitspharmacologicalprofile.Inordertosourceagoodqualityrawmaterialfor Tradi-tionalChineseMedicinewerecommendedthisKazakhspeciestobestandardizedusingechinacoside andacteosideasmarkers.

Introduction

Cistanche salsa (C.A. Mey) Beck, Orobanchaceae, is a para-siticplantfromtheRepublicofKazakhstanwhere itis usedas industrialfeedstock(Sarsenbayevetal.,2011;Grudzinskayaand Gemedzhieva,2012).ThescientificvalueofthisherbinTraditional Chinese Medicine relates tothe treatment of kidney problems (varyingfrompaintoinsufficiency),impotence,femaleinfertility, morbidleucorrhea,profusemetrorrhagiaandsenileconstipation (Jiangsu New Medical College Dictionaryof TraditionalChinese Drugs,1977;ChineseMedicinalHerbal,1988).Thechemical com-positionofstolonsofotherspeciesofCistanchehasalreadybeen studied in detail by Chinese scientists. The following phenolic compoundswereidentified:echinacoside,tubuloside,acteoside,

∗ Correspondingauthor.

E-mail:Fabio.Boylan@tcd.ie(F.Boylan).

besideslignans,iridoidsandacomplexpolysaccharide(Yongand Tu,2009;Zhangetal.,2003;Xieetal.,2005;Jiangetal.,2009;Sui etal.,2011;Liuetal.,2013;Zhouetal.,2014).

Shimodaetal.(2009)showedthatthisherbpossesses hypoc-holesterolemic effect while Yang et al. (2013) determined its hepatoprotective activity. Nan et al. (2013) reported the anti-inflammatoryactivityforitsextracts.Acomplexpolysaccharide previously isolatedfromthis plantshowedimmunomodulatory effects(Wangetal.,2009).

AlthoughthereisaconsiderableamountofCistanchestolonsin theRepublicofKazakhstan,thereisnopopularuseforthisplant bytheKazakhpopulationalthoughtheneighboursinChina exten-sivelyuseit.Duetothefactthatthereisaconsiderableproblem nowadaysinrelationtothesubstitution/adulterationofmedicinal plantsandtheincreasingneedtostandardizemedicinesfortheuse inTraditionalChineseMedicine,thisstudywasdesignedto ascer-tainthephenolicchemicalcompositionofthisrawplantmaterial growingintheneighbouringKazakhstanaswellastoassessitsuse

http://dx.doi.org/10.1016/j.bjp.2017.05.013

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asantinociceptivetopotentiallyeasepain–whichcharacterizes oneofitsmainuseinTraditionalChineseMedicine.

Materialsandmethods

Plantmaterial

Cistanchesalsa(C.A.Mey.)Beck,Orobanchaceae,stolonswere collectedatthedesertofMoinkum,VillageofBakanas,inJuly2014 inAlmaty.TheplantmaterialwasidentifiedbyDr.G.Sitpayeva fromtheInstituteof Botanyand Phytointroduction,Ministryof EducationandSciencesoftheRepublicofKazakhstanwhereitwas depositedunderthenumber01-04/306.

Chemicalanalysis

StolonsofC.salsa(2g)weresubmittedtoamicrowave extrac-tion.Initially thematerial was ground to 0.001–2.000mm and then placed in a hermetic vesseltobe extracted for 10min at 100–1100◦_C _with _ethanol _80% _(ratio _1:10). _The _hydroethanol extract(CSHE)wasanalyzedbyHPLC-MS.Crudeextractwas dis-solvedin methanol(9.6mg/ml) andfilteredthrough a0.45mm Teflonmembraneprior totheanalysis.A liquidchromatograph HP1100 Seriesmodel (company AgilentTechnologies, Inc.,CA, USA),equippedwithaflowingvacuumdegasser,afour-channel low-pressuregradient pump,and anautomaticinjector. Pheno-liccompoundswerechromatographicallyseparatedbyacolumn ZorbaxEclipseXDB-C18,2.1×50mm,filledwithoctadecylsilyl sil-icagelpolymer(1.8␮).Chromatographicanalysiswascarriedout withamobilephaseflowof0.2ml/min,eluentoperatingpressure of175–200kPa,columnoventemperatureof30◦_C,₂_ml_sample volume, gradient eluent feed mode: 0–36min10% A – 90% B; 36min–100%B(eluentA:methanol,eluentB:0.2%formicacid solution).DetectionwasperformedbyUVatthewavelengthsof 254,334,350,410,450and550nm.Comparisonoftheobtained retentiontimes,UVandmassspectrawiththoseofreference com-poundswasusedfortheidentificationofthechemicalcompounds intheextract.Quantitativeanalysiswasperformedbytheuseof standardverbascosideandechinacosideanalyzedunderthesame chromatographicconditions.Theircalibrationcurvesallowedfor thecalculationofthequantityofeachotherphenylpropanoid gly-cosideintheethanolextractofthis plant.Themethodwasnot validated.

Animals

SwissWebstermice(20–25g,twomonthsold)wereusedin this study(donatedby InstitutoVitalBrazil,Niterói, RJ, Brazil). Theanimalswerekeptinstandardconditions(light-darkcycleof 12h,22±2◦_C_and_70–80%_humidity._Food_and_water_ad_libitum_). Animalsreceivedonlywaterinordertoavoidfoodinterference withsubstanceabsorption12hpriortotheonsetof the exper-iments. Acclimatization to the laboratory conditions happened foratleast1hbeforeeachtest.Allprotocolswereconductedin accordancewiththeGuidelinesonEthicalStandardsfor Investiga-tionofExperimentalPaininAnimalsandfollowedtheprinciples andguidelines adoptedbytheNationalCouncil fortheControl ofAnimalExperimentation(CONCEA),approvedbythe Biomedi-calScienceInstitute/UFRJ,EthicalCommitteeforAnimalResearch, and received the number DFBCICB015-04/16. All experimental protocolswereperformed duringthelightphase. Animal num-berspergroupwerekeptataminimumandaccordingtorules fromCONCEA.Attheendofeachexperimentmicewerekilledby ketamine/xylazineoverdose.

Treatments

In this study CSHE was evaluated at 10, 30 and 100mg/kg. The extract wasdissolvedin dimethyl sulfoxide (DMSO, Fisher Biotech)inordertoprepareastock solutionat 100mg/ml.PBS wasusedasdiluentforthepreparationofthedifferentdoses. Solu-tionscontaining10,30and100mg/kgofthehydroethanolextract of C. salsawere prepared.The standard drugs used were mor-phine2.5mg/kg(Merck,dilutedinphosphatebuffersaline(PBS)), acetylsalicylicacid200mg/kg(SigmaAldrich,dissolvedwith5Mof sodiumhydroxide(NaOH)in0.9%saline)andcapsazepine10nMol perpaw. SalineplusDMSO(atthesameconcentrationasinthe highesttreatmentwithextract)wasgiventothenegativecontrol group.Alltreatments(testedextractandstandards)were admin-isteredbyoralroute.Theonlyexceptionwascapsazepinewhich wasadministeredbyintraplantarinjection.

Formalin-inducedacutepain

Asolutionof2.5%formalin(37%formaldehyde)wasinjected (20␮l)intheplantarregionoftherighthindpawofmice60min aftertreatment(hydroethanolextractofC.salsaoracetylsalicylic acid200mg/kgormorphine2.5mg/kg)(Matheusetal.,2005).The animalswereindividuallyplacedinatransparentglasschamber andthedurationoftimeinsecondsthattheyspentlickingtheirpaw afterinjectionofformalinwasrecordedandanalyzedovertwo sep-arateperiods,earlyphase-neurogenicpain(0–5minafterinjection) andlatephase-inflammatorypain(15–30minafterinjection).

Nociceptioninducedbycapsaicin

ThistestwasbasedonthemethoddescribedbySakuradaetal. withsomemodifications(Sakuradaetal.,1992).Capsaicin(20␮l) C18H27NO3 (Galena, Campinas, SP) wasinjected in the plantar

regionoftherighthindpawofthemice(1.6␮g/paw)onehour aftertreatment(hydroethanol extractofC.salsaorcapsazepine 10nMol/paw). The animals wereindividually placed in a glass chamberandpaw-lickingduration(s)wasrecordedbetween0and 5minafterthecapsaicininjectionandthenanalyzed.

Glutamate-inducednociception

InthemethoddescribedbyBeirithetal.(2002),glutamate solu-tioninPBS(20␮l)(l-glutamicacid,Sigma–Aldrich,3.7ng/paw)was injectedintheplantarregionoftherighthindpawofthemiceone houraftertreatment(hydroethanolextractofC.salsaormorphine 2.5mg/kg).Theanimalswereplacedindividuallyinaglass cham-berandpaw-lickingduration(seconds)wasrecordedbetween0 and15minaftertheglutamateinjectionandthenanalyzed.

Statisticalanalysis

The chemical data is presented as mean±SD of five exper-iments. Allpharmacological experimentalgroups consistedof a minimumofsixmice.Analysisofone-wayvariance(ANOVA) fol-lowed by Dunnett’s test allowed thevisualization of statistical significancebetweengroupsusingGraphPadPrism5.0software.p valueswereconsideredsignificantwhentheywerelessthan0.05 (p<0.05).

Results

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0 0.8

0.6

0.4

0.2

0.0

5 10 15 20 25 30 35 Time[min]

Intens

Salsaside cistanoside A

P

oliumoside

isoacteoside

cistanoside C

tub

uloside A

2'

-acetylacteoside

echinacoside

tubuloside B acteoside

Fig.1.ChromatogramoftheCSHE.

Table1

IdentificationofphenylpropanoidglycosidesinHerbaCistanche. Peak Retentiontime(min) Content(mg/g) Compound

1. 10.9 0.65±0.01 Salsaside

2. 15.6 9.44±0.01 Acteoside

3. 17.1 5.68±0.01 Poliumoside

4. 17.7 10.98±0.01 Echinacoside 5. 17.9 0.63±0.01 CistanosideA 6. 18.9 0.08±0.01 Isoacteoside 7. 19.3 2.29±0.01 CistanosideC

8. 19.7 7.94±0.01 TubulosideB

9. 20.5 0.73±0.01 TubulosideA

10. 21.1 1.11±0.01 2′_{-Acetylacteoside}

thephenoliccompoundstogetherwiththeirrespectiveretention timesinthechromatogram.

EffectofCSHEonformalin-inducedacutepain

Intheformalin-inducedacutepaintest,theCSHEat10,30and 100mg/kgwereabletodecreasepaw-lickinginthefirstphaseof thetestknownastheneurogenicpainphase,thesereductionswere 60,77and58%,respectively.Inaddition,theywerealsoeffective inreducinginflammatorypaininducedduringthesecondphase ofthetest,thepercentageinhibitionofinflammatorypainwere 42, 46and 62%,respectively (Fig.2).Morphine(2.5mg/kg)and

acetylsalicylicacid(200mg/kg)presented thefollowingresults: 55%/33%and31%/54%forthefirstandsecondphases,respectively.

EffectofCSHEonglutamate-inducednociception

TheCSHEreducedthelickinginducedbyglutamateatthethree dosestested,10,30and100mg/kgby76,81and53%,respectively (Fig.3).Morphineat2.5%resultedin76%reduction.

EffectofCSHEoncapsaicin-inducednociception

InordertoverifyiftheCSHEwouldaffectnociceptionthrough TRPV1receptors,itwastestedinamodelofpaininducedby cap-saicin.Theantinociceptiveeffectthroughthismodelwasobserved fortheCSHEinthethreetesteddoses,by76,79and81%, respec-tively(Fig.4).Capsazepine10nMol/pawresultedin60%reduction.

Discussion

Atotal oftenphenylpropanoidglucosideswereidentifiedin theC.salsa(growinginKazakhstan)hydroethanolextract. Echi-nacoside (10.98mg/g), acteoside (9.44mg/g) and tubuloside B (7.94mg/g)werethemajorcompoundsidentified.Theobtained datawascorrelatedwiththeavailableliteratureforspeciesof Cis-tanchegrowingindifferentregionsofAsia(Zhouetal.,2014;Xie etal.,2005;Jiangetal.,2009).Echinacosideandacteosidearethe

vehicle F1morphine ASA

salsa 10mgsalsa 30mg_{salsa 100mg}vehicle F2morphine ASA

salsa 10mgsalsa 30mg_{salsa 100mg}

0 20 40 60 80

0 50 100 150 200

phase I phase II

*

_*

*

Licking (seconds)

*

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Glutamate

vehicle morphine

salsa 10mg salsa 30mgsalsa 100mg 0

20 40 60

*

Licking (seconds) *

Fig.3.EffectofCSHEonglutamate-inducednociceptioninmice.Animalswere pre-treatedwiththeCSHE10mg/kg,30mg/kgor100mg/kg(p.o.),morphine2.5mg/kg (p.o.)orvehicle(DMSO).Resultsarepresentedasmean±S.D.(n=6).One-way ANOVAwithDunnett’smultiplecomparisonwith vehicle,post-test(*p<0.05). GraphPadPrismversion5.1.

Control

salsa 10mg/kg salsa 30mg/kg salsa 100mg/kg

Capsazepine 10n Mol

0 10 20 30 40

50 Capsaicin

licking (seconds)

Fig.4.EffectofCSHEoncapsaicin-inducednociceptioninmice.Animalswere pre-treatedwithCSHE10mg/kg,30mg/kgor100mg/kg(p.o.)orvehicle(DMSO).Results arepresentedasmean±S.D.(n=6).One-wayANOVAwithDunnett’smultiple com-parisonwithvehicle,post-test(*p<0.05).GraphPadPrismversion5.1.

maincomponents,whichformthebasisforstandardizationofthe officinalspecies C.deserticola and C.tubulosa,listedin the Chi-nesePharmacopoeiain2005.Weproposethatthesecompounds couldalsobeusedforthestandardizationofC.salsastolonsfrom Kazakhstan,astheyarethemajoronesinthestudiedhydroethanol extract.

Accordingtothepharmacologicalresultsofthisstudyitis possi-bletosuggestthattheCSHEisaneffectiveagentagainstneurogenic painand inflammationasobservedin theformalintest. Tothe bestofourknowledgeC.salsahasneverbeentestedinrelation toitsantinociceptiveactions.Astudyfrom2002withC.deserticola (Linetal.,2002)hasshownthatthisplantpresented antinocicep-tiveandanti-inflammatoryactivitieswhenassayedinmodelssuch astotalwrithing,formalinandpawoedema.Althoughtherewas noattempttoinvestigatethemainconstituents intheextracts thatwereactive,theydeemedthebutanolextractandthewater layeras theactive ones.Not surprisingly these solvents are at thescaleofpolaritythatmatchesthephenylpropanoidglycosides foundinC.salsa.Moreover,Linandco-workersestablishedthat

theantinociceptiveactionofC.deserticolaextractswasnotdueto theactionofthecompoundintheopiatereceptororrelatedtothe immunesystem.Inourstudy,thephenylpropanoid-richextract showedactivityinbothphasesoftheformalintest.Theactivity observedwasduein facttothepharmacologicaltarget investi-gatedandnottoanypossiblemotoralterationasevidencedbythe rotarodtest.Formalintestresultsindicatepotentialforneurogenic painaswellaspaininducedbyinflammatorymediators.Initially wedecidedtoinvestigatetheneurogenicpainroute.Because simi-larcompoundsfromotherspeciespreviouslystudieddidnotshow anyopiatereceptoractivity,wedecidedtotestothermodels,such asGlutamateandcapsaicinmodels.

Glutamateisanexcitatoryneurotransmitterthathasan impor-tantroleinmodulatingpainthroughouttheperipheralandcentral nervoussystem.Thisactionismediatedbyligand-gatedionotropic glutamatereceptors(iGluRs)andmetabotropicglutamate recep-tors. The iGluRs can be divided into N-methyl-d_-aspartate (NMDA)and␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA)(Kolber,2015).Researchhasshownthatwhen antag-onizingNMDAand AMPAreceptorswithketamineandkainate, respectively,anantinociceptioneffectisobserved.However, antag-oniststargetingthesereceptorssofarhaveinducedasubstantial adverseeffectandforthisreasonanewresearchfocusonmGluRs hopingthatmedicinemediatedthroughthisreceptorwouldcause lesssideeffecthasbeencarriedout(Palazzoetal.,2014).Theresults oftestingtheCSHE showedthatthistreatmentwaseffectivein reducingpaininducedbyglutamate.However,itisstillunknown ifthiseffectismediatedthroughionotropicand/ormetabotropic receptors.Morphineisoneofthedrugsavailablethathasaneffect onglutamatergictransmission(Deyamaetal.,2007).Itisreported intheliteraturethatglutamatereceptoractivationincreasesTRPV1 responses(Szteynetal.,2015).Forthatreason,itispossiblethat theeffectobservedforCSHEcouldbeduetoactivityonTRPV1and notnecessarilythroughadirectresponseonglutamatereceptors.

Thenextstepofthisresearchwastocarryoutapaw-lickingtest usingcapsaicinasthealgesicagent,whichisaTRPV1receptor ago-nist.TheresultsshowedthatindeedCSHEdecreasedpainthrough TRPV1receptor.

InthecaseofCSHE,almostinallthetestedmethodologies,we couldobservethatthedoseof 30mg/kgachievedbetterresults than100mg/kg.Thiscanbeduetothesaturationofthesolution leadingtoprecipitatedcompoundsandeffectivelylessamountof drugsbeingbioavailable.

Confirming the established chemical composition for the

Kazakh extract CSHE was an important step because the

major constituents present in it have already had their

antinociceptive/anti-inflammatory actions somehow confirmed. For example,echinacosidewasestablished asoneof theactive principlesresponsiblefortheantinociceptiveactionofEchinaceae (Hostettmann,2003).Also,apreviousstudybySchapovaland co-workers(Schapovaletal.,1998)pointedoutacteosideasoneofthe mainactiveprinciplesinanethanolextractpreparedwith Stachy-tarphetacayennensis asassessed by pawoedemaand hotplate models.Backhouseandco-workers(Backhouseetal.,2008)also deemedacteosidetobetheactive principleof Buddlejaglobosa usingseveralmodelsofpainassessment,includingformalintest. Newformulationsarenowbeingdevelopedusingthestateofthe artknowledgetoincreasestabilityandprolongtheantinociceptive actionofacteoside(Isacchietal.,2016).

Conclusion

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experimentalmodelsperformedherein.Takingintoconsideration thatontheterritoryofKazakhstanthegenusCistancheis repre-sentedmainlybyspeciesC.salsa,wecanrecommenditsharvesting andstandardizationusingechinacosideandacteosideasstandard compounds,tosourceagoodqualityrawmaterialforTraditional ChineseMedicine.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authors’contributions

EBKexecuted thecollectionof theplant, preparation ofthe extractandanalysisofthechemical resultsaswellasthe writ-ingofthepaper;GRD,executedallthepharmacologicaltests;ZBS, designedthestudyinconjunctionwithFB;LNIandENB, partici-patedinthepharmacopoeialdesignofthestudyfortheinclusion oftheobtainedresultsintheKazakhpharmacopoeia;IIT,executed theHPLCanalyses;PDF,designedthepharmacologicalstudyand FB,organizedtheteamtogetherinordertowritethispaper.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Theauthors fromBrazilthank Mr.AlanMinho for technical assistanceandtheInstitutoVitalBrazilfordonationsofanimals used.TheyalsowanttoacknowledgethegrantsfromCNPqand Fundac¸ão Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro. The authors from Irelandwish to acknowl-edgetheHighEducationAuthority’sProgrammeforResearchin Third-LevelInstitutions Cycle 5’sfunding support for TBSI and toreinforcetheimportanceofSFIprogrammeISCA-Brazil(grant no.SFI/13/ISCA/2843)thatcontributedtothecollaborativework betweenBraziland Ireland.The authorsfromKazakhstanwant toacknowledgeKazNMUforthefinancialsupportallowedtothe executionofthiswork.

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