Rev. bras. farmacogn. vol.27 número5

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

Original

Article

Sedative

and

muscle

relaxant

activities

of

diterpenoids

from

Phlomidoschema

parviflorum

Abdur

Rauf

_,

_Umar

_Farooq

_,

_Ajmal

_Khan

_,

_Taibi

_Ben

_Hadda

_,

_Sadia

_Naz

_,

_Aliya

_Ibrar

_,

Noor

Jehan

_,

_José

_P.

_{Cerón-Carrasco}

_,

_Helena

_den

_Haan

_,

_Jorge

_{Pe ˜na-García}

_,

Horacio

Pérez-Sánchez

_,

_Haroon

_Khan

_,

_Mohamed

_Fawzy

_Ramadan

_,

_Tareq

_Abu-Izneid

_,

Saud

Bawazeer

a_{DepartmentofChemistry,UniversityofSwabi,KhyberPakhtunkhwa,Pakistan}

b_{DepartmentofChemistry,Comsat,InstituteofInformationTechnology,Abbotabad,Pakistan}

c_{MaterialsChemistryLaboratory,DepartmentofChemistry,MohammedFirstUniversity,Oujda,Morocco}

d_{BioinformaticsandHighPerformanceComputingResearchGroup,UniversidadCatólicaSanAntoniodeMurcia,Murcia,Spain} e_{DepartmentofPharmacy,AbdulWaliKhanUniversity,Mardan,Pakistan}

f_{DepartmentofBiochemistry,FacultyofAgriculture,ZagazigUniversity,Zagazig,Egypt}

g_{DepartmentofPharmaceuticalChemistry,FacultyofPharmacy,UmmAl-QuraUniversity,Makkah,SaudiArabia}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3March2017 Accepted25July2017 Availableonline17August2017

Keywords:

Rosanediterpenoids Stachysrosane Sedative

Skeletonmusclerelaxantactivity Moleculardocking

a

b

s

t

r

a

c

t

Phlomidoschemaparviflorum(Benth.)Vved.(Basionym:StachysparvifloraBenth.)Lamiaceae,have sig-nificancemedicinalimportanceasitisusedinnumberofhealthdisordersincludingdiarrhea,fever, soremouthandthroat,internalbleeding,weaknessesoftheliverandheartgenital tumors, sclero-sisofthespleen,inflammatorytumorsandcancerousulcers.Thepresentcontributiondealswiththe sedativeandmusclerelaxantlikeeffectsofditerpenoidstriviallynamedstachysrosaneand stachys-rosane,isolatedfromtheethylacetatesolublefractionofP.parviflorum.Bothcompounds(at5,10 and15mg/kg,i.p)wereassessedfortheirinvivosedativeandmusclerelaxantactivityinopenfield andinclinedplanetest,respectively.Thegeometriesofbothcompoundswereoptimizedwithdensity functionaltheory.Themoleculardockingofbothcompoundswereperformedwithreceptorgamma aminobutyricacid.Bothcompoundsshowedmarkedactivityinadosedependentmanner.The dock-ingstudiesshowedthatbothcompoundsinteractstronglywithimportantresiduesinreceptorgamma aminobutyricacid.Thereporteddatademonstratethatbothcompoundsexhibitedsignificant seda-tive andmusclerelaxant-like effectsinanimal models, whichopensa doorfornovel therapeutic applications.

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

Introduction

Phlomidoschemaparviflorum(Benth.)Vved.(Basionym:Stachys parvifloraBenth.)belongstotheLamiaceae(mint)familythat dis-tributedinthetropicalandtemperateregionsofPakistan(Ahmad etal.,2006).Plantsofthisgenushavebeenextensivelyapplied totreatmentofwidepanelofhealthdisordersincludingdiarrhea, fever,soremouthandthroat,internalbleeding,weaknessesofthe liverandheart(Holimanetal.,1996),genitaltumors,sclerosisof

∗ Correspondingauthors.

E-mails:abdurrauf@uoswabi.edu.pk(A.Rauf),umarf@ciit.net.pk(U.Farooq),

ajmalkhan@ciit.net.pk(A.Khan).

thespleen,inflammatoryandcancerousulcers(Skaltsaetal.,1999). ThespeciesofthegenusStachyslavandulifoliashowedsedativeand anxiolyticlikeeffectsinanimalstudies(Rabbanietal.,2003,2005). PhytochemicalinvestigationsofStachysspecieshaveshownthe occurrenceofflavonoids,diterpenes,phenylethanoidglycosides, alkaloids,andsaponins(Khanavietal.,2005;Ahmadetal.,2006).

VariouschemicalconstituentslikestachyssaponinA, stachys-saponinB,parviflorosidesAaswellasparviflorosidesBhasbeen isolated fromP.parviflorum (Ahmad et al.,2006, 2008). Anew rosane-type diterpenoid from P. parviflorum has been isolated (Farooqetal.,2014).

Unfortunately,literaturesurveyrevealedthatlimitedworkhas beendonesofaronP.parviflorum.Consideringitsmedicinal impor-tance, the present study is based on the sedative and muscle

http://dx.doi.org/10.1016/j.bjp.2017.07.003

relaxanteffectsof compounds1and 2isolatedfromP. parviflo-rumintwoanimalprotocols.Thecomputationalstudiesofthese compoundswerecarriedout,inordertoknowthemolecular mech-anismatatomiclevel.

Materialsandmethods

Generalexperimentalprocedure

Column chromatography was performed using silica gel (70–230mesh,E-Merck)andsilicagel(230–400mesh,E-Merck). AnalyticalTLCwasperformedonprecoatedplates(silicagel60 F254). Optical rotations were recorded using a Jasco-DIP-360-digitalpolarimeter.UVandIRspectrawererecordedwithUV-240 and JASCO-320-A spectrophotometers, respectively. EI-MS and HR-EI-MSwere recordedona double-focusingvarian MAT-312 spectrometer. The 1_{H and} 13_{C NMR spectra were recorded on} a Bruker AMX-400 spectrometer, while 2D-NMR spectra were recorded on Bruker AMX-500MHz NMR spectrometer. Chemi-cal shifts in parts per million (ı) relative to tetramethylsilane as aninternal standard, and scalar couplings (J)werereported inHz.

Plantmaterial

The whole plant Phlomidoschema parviflorum (Benth.) Vved. (Basionym: Stachys parviflora Benth.), Lamiaceae, was collected fromAbbottabad(Pakistan)inMarch2012.Theplantwasidentified byDr.ManzoorAhmad(Taxonomist,DepartmentofBotany, Post-GraduateCollege,Abbottabad).Avoucherspecimen(No.14230) hasalsobeensubmittedintheherbariumofthesamedepartment.

Extractionandisolation

Theair-driedwholeplant(9kg)wasgrindedandextractedwith methanolatroomtemperature.Theextractwasevaporatedona rotaryevaporatorunderreducedpressuretoobtainacrudeextract. Theresultingmethanolextract(2500g)wassuspendedinwater andsuccessivelypartitionedtoprovidehexane(110g),chloroform (80g),ethylacetate(35g),n-butanol(95g),andaqueousextract (150g).

The ethyl acetate fraction was further subjected to column chromatographyoverasilicagelcolumnusinghexanewitha gra-dientof ethyl acetate upto 100% followed by methanol. Eight sub-fractions were collected through column chromatography. Sub-fraction7–8wassubjectedtocolumnchromatography and elutedwithEtOAc/hexane35:65topurifycompound1(20mg). Thecombinedlesspolarfractions5–6ofthisseparationwere re-subjectedtocolumnchromatography,whichresultedcompound

2 (17mg) at EtOAc/hexane 30:70 polarity. The purity of com-poundswascheckedonTLCandHPTLCplates. Thestructureof thesecompoundswasrecentlyreportedbyourgroup(Farooqetal., 2015).

11 12

17 15

16 16

15 17 12

13 14 8 9 11 1 2 3

4 10

20 5

6

19 18

7 14

13 8 9 10 1 2 3

4 5 20 7

6 18 19_CO

2H CO₂Me

H H

H

H

1 2

Animals

NMRImice(20–25g)wereusedinvariousexperiments.Animals werefedwithstandardlaboratoryfoodandwateradlibitum. Ani-malswerekeptunderstandardconditionoftemperatureandlight. Beforethestartofexperimentanimalswereacclimatizedwith lab-oratoryconditions.Allexperimentalprotocolswereapprovedfrom ethicalcommittee foranimal studiesof University ofPeshawar underreferencenumber14009(PUP)keepinginmindthe inter-nationalguidelineofanimalstudies.

Sedativepropertiesinopenfieldtest

Theapparatususedforthis activitywasconsistedofanarea ofwhitewood(150-cmdiameter)enclosedbystainlesssteelwalls anddividedinfoursquaresbyblacklines.Theopenfieldwasplaced insidealightandsound-attenuatedroom.Animalswere acclima-tizedunderredlight (40Wredbulb)60minbeforethestartof experimentinlaboratorywithfoodandwateravailableadlibitum. Animalsweredividedinvariousgroups(n=6)andwere admin-istered withnormal saline(negativecontrol,10ml/kg,i.p.), test compounds(5,10and15mg/kg,i.p)andbromazepam(0.5mg/kg,

i.p.).After30min,eachanimalwasplacedinthecenterofthebox andthenumbersoflinescrossedwerecountedforeachmouse (Raufetal.,2017a,b).Toassessprocessofadjustmenttothe nov-eltyoftheground,micewereexposedtotheapparatusfor5min ontwosuccessivedays.

Inclinedplanetest

Theplaneusedinthisprocedurewasconsistingoftwo play-woodboards.Bothboardswereconnectedwitheachotherinsuch awaythatoneboardformthebaseandotherisfixwiththebase at 65◦_{.Different groups (n=6) weretreated withbromazepam}

(1mg/kg,i.p.),normalsaline(10ml/kg,i.p.)andtestcompounds (5,10and15mg/kg,i.p).After30,60and90minoftreatmentthe animalswereplacedontheupperpartoftheinclinedplanefor30s tohangorfall(Raufetal.,2017a,b).

Statisticalanalysis

Resultsareexpressedasmean±S.E.M.One-wayANOVAwas usedforcomparisontestofsignificantdifferencesamonggroups followedbyDunnet’smultiplecomparisonposttest.Alevelof sig-nificance(p<0.05or0.01)wasconsideredforeachtest.

Molecularmodeling

Molecularmodelingexperimentshavebeenperformedonboth compounds.Theirgeometrieswerefullyoptimizedwithinthe den-sityfunctionaltheory(DFT)frameworkattheB3LYP/6-31G(d)level (NewmanandCragg,2012;Malamietal.,2014).Vibrational cal-culations weresubsequently performed toascertainthe nature ofeverylocalizedstructure:astablestructure correspondstoa minimuminthepotentialenergysurfaceandconsequentlyall fre-quenciesarereal.Partialatomicchargeswerenextcomputedby usingtheMerz-Singh-KollmanESPprotocol(NewmanandCragg, 2012),tobeusedduringdockingsimulations.Requiredquantum chemicalcalculationswereperformedwiththeGaussian09suite ofprograms(Malamietal.,2014).

100

30 min 60 min 90 min

30 min 60 min 90 min 75

50

25

0

100

75

50

25

0

5 10

Bromazepam

mg/kg

%animal falling in 30 s %animal falling in 30 s

mg/kg

Bromazepam

15 5 10 15

*

* *

* * *

* *

* * * * *

*

* * *

* *

* *

* * * * *

* * *

1

2

Fig.1. Effectofisolatedcompounds1and2fromP.parviflorumonmusclecoordinationininclinedplane,barrepresentsthepercenttimespentinsecondsbywhichmice slideofftheinclinedplane,30,60and90minaftertreatmentwithnormalsaline(10ml/kg),compounds(5,10and15mg/kg)orbromazepam(1mg/kg).*p<0.05,***p<0.001, allwithrespecttocontrol.

thepotentialenergy surface withthe geneticalgorithm imple-mentedintheLeadFinderdockingprogram.Fortherecords,the full-atommodelofGABAAusedindockingcalculationswas pre-paredfromtherawPDBstructure 4COF.Watermoleculeswere removedandtheionizationstatesoftheaminoacidswereassessed usingProtonate3DfunctionoftheMOEsoftwarepackage (Chemi-calComputingGroup,LLC).Thesameprotocolwasusedforadding missinghydrogenatomsinthePDBfiles,whereallchargedside chainswereconsideredintheirdefaultprotonationstatesat neu-tralpH.Proteinatomicpartialchargeswerederivedthroughthe AMBER99 forcefield (Newman and Cragg, 2012), implemented inMOE. The sizeof thegridbox forliganddocking wassetto 120 ˚AineachdirectionfromthegeometriccenteroftheAquaporin model.ThedGscoreproducedbyLeadFinderwastakenasthe pre-dictedvalueoftheligandbindingenergy.Scoringfunctionfrom LeadFinderconsidersLennard–Jonesterm(LJ),metalinteractions, solvationterm,hydrogenbonds(H-bonds),andelectrostatic inter-actions,internalenergyoftheligand,contributionstoentropydue toligandtorsionsandasolvationpenaltyterm.Inthisblinddocking approachmultipledockingrunsstartedaroundgeometriccenters ofallresidueswithintheselectedthreshold.

Results

Sedativeeffectoftestcompounds(1and2)

Thecompoundscauseddose-dependenteffectonmobilityat varioustestdoses.Pretreatmentofbothcompoundshad signifi-cantreductioninmobilityat10and15mg/kg.Theoveralleffectof compound1and2wasalmostequallysignificant.Thelocomotion decreasedbybromazepamwasmostsignificantthanthetestdoses ofisolatedcompounds(Table1).

Musclerelaxanteffectoftestcompounds

Theresultsofmusclerelaxanteffectoftestcompounds(1and

2)ininclinedplanetestarepresentedinFig.1.Bothcompounds elicitedsignificantskeletonmusclerelaxantlikeeffectinadose dependentmanner.Themaximumactivitywasnotedat15mg/kg after60minoftreatment.Compounds1and2producedalmost similarrelaxationofmiceskeleton musclebutless pronounced thenstandarddrug(bromazepam).

Table1

Effectofisolatedcompounds1and2fromP.parvifloruminopenfieldtest (locomo-tiveactivity).

Sample Dose No.oflinescrossed Control 5ml/kg 130±3.95 Bromazepam 0.5mg/kg 9±0.55c

Compound1

5mg/kg 100±5.03

10mg/kg 82±4.34a

15mg/kg 74±3.60b

Compound2

5mg/kg 101±5.67

10mg/kg 88±4.67a

15mg/kg 79±4.45a

Valuesrepresentthenumberoflinescrossedbyanimalinbox,30minafter treat-mentwithnormalsaline(10ml/kg,control),compounds(5,10and15mg/kg)or bromazepam(0.5mg/kg).Datapresentedasmean±S.E.M.(n=6).

a_p<0.05. b_p<0.01.

c _{p<0.001,allcomparedwithcontrol.}

Molecularmodeling

Thebromazepamand compounds1and 2werefirstdocked intotheGABAAreceptor,whichactsasthehostentity.Theposes withthehighestvaluefortheprotein–ligandinteractionenergy werenextselectedforaqualitativeanalysis.InFigs.2–4results obtainedfromblinddockingforbromazepam,compound 1and compound2areshown.Thesefigurespointedoutthatmain sta-bilizinginteractionforbromazepanareduetopi–piinteractions between Phe200C, Tyr62B and aromatic scaffold of the ligand, hydrophobicinteractionswithsameresidueTyr62Bandhydrogen bondwithGln64B.Suchlargenumberofprotein–ligand interac-tionsexplainswhybromazepaminteractssostronglywithGABAA. Regarding docking results for compound 1 and 2, we can see that in both cases they bind to the protein in the same area asbromazepam,throughhydrophobicinteractionswithresidues Phe200C,Asp43BandTyr62B,andhydrogenbondswithAsn41B and Thr176B (compound 1, Fig. 3) and Tyr205C (compound 2,

Phe200C

Tyr62B

Tyr62B

Gln64B H₂B

HO

O

O NH

H H N

N

N

Fig.2. 2Drepresentationofmainprotein–ligandinteractionsestablishedbetween bromazepamandGABAA.Greenlinerepresentshydrophobicinteractions,black

dashedlineshowshydrogenbondsandgreendashedlinescorrespondtopi–pi interactions.

H

Phe200C Asp43B

Tyr62B

Ala201C Asn41B

Thr176B H

H

O

O O

O

H

H N

Fig.3. 2Drepresentationofmainprotein–ligandinteractionsestablishedbetween compound1andGABAA.Greenlinerepresentshydrophobicinteractions,andblack

dashedlinesshowhydrogenbondinteractions.

Asp43B Phe200C

Tyr62B

Tyr205C H

H

H O O

O

Fig.4. 2Drepresentationofmainprotein–ligandinteractionsestablishedbetween compound2andGABAA.Greenlinerepresentshydrophobicinteractions,while

blackdashedlineshowshydrogenbondinteractions.

Discussion

Thepresentstudydemonstratedamarkedsedativeand mus-clerelaxanteffectsofditerpenoidsisolatedfromP.parviflorumin twoanimalmodels.Theopenfieldtestisoneofthesimplestand commonlyusedscreeningtoolsfortheassessmentofsedativelike potential(Uddinetal.,2014;Raufetal.,2017a,b).Additionally,it canbeusedtoevaluateexplorationand anxietybehaviors.The numberofcentralsquareentriesandthedurationoftimespent in thecentral squarearemeasuresof exploratorybehaviorand anxiety.A highfrequency/duration ofthese behaviorsindicates highexploratorybehaviorandlowanxietylevelsandviceversa. Theisolatedcompounds(1and2)leadtoasignificantlyreduction ofthemovementofmiceintheopenfieldtestapparatus.These evidenceshintthesedativelikenatureofbothcompounds. Simi-larly,themusclerelaxantlikepropertyofcompounds(1and2)also wastestedintheinclinedplanetestthatiscommonlyusedassay (ElliotandWhite,2001).Itwasinitiallydesignedtodifferentiating betweenneurolepticsfromothercentrallyactivedrugs(Randall etal.,1961),butlateron,themethodwasusedformuscle relax-antlikeeffectoftestarticles(RivlinandTator,1977).Theresults showedmarkedskeletonmusclerelaxantlikeeffects ofthetest compounds.

The bromazepam, which demonstrated outstandingsedative andmusclerelaxanteffects,wasusedhereasstandardcompound toprovideameaningfulbiologicalconclusions.Indeed,the ben-zodiazepines(BDZs)exhibitmarkedsedativeandmusclerelaxant effects mostlyattributedtointerferewiththeactionof gamma aminobutyricacid(GABAA)(DaillyandBourin,2008),whichmakes bromazepamanexcellentcompoundtoprovidearelativeactivity order.Thesignificantsedativeandmusclerelaxantlikeeffectsof compoundsinourstudycouldbeattributedtointerferewiththe actionofGABAA likeBDZ.However,furtherstudiesarerequired toascertainitsmechanismforsedativeandmusclerelaxant-like effectsofthesecompoundsforleadcompoundsinthetherapeutic classwithbetterefficacyandsafety.

Tyr62Bandaromaticscaffoldoftheligand,hydrophobic interac-tionswithsameresidueTyr62BandhydrogenbondwithGln64B. Such largenumber ofprotein–ligand interactions explainswhy bromazepaminteractssostronglywithGABAA.Regarding com-pounds1and2,ourmolecularmodelingapproachdoesnotfindso manyinteractionsforthesecompoundsaswithbromazepambut thereishighshapecomplementarityofthetwocompoundswith thebindingsite.Therefore,thehydrogenbonds,hydrophobicity andshapecomplementarityallowustodelineatethe experimen-tallyobservedaffinityofGABAAtothesecompounds.

Conclusion

We can conclude that compounds 1 and 2 isolated from

P.parviflorum possess significant sedativeand skeleton muscle relaxant-likeeffectsinanimalmodels.Ourstudyconfirmedthefolk usesoftheplantassedativebyprovidingpharmacologicalrationale ofisolated compoundsfrom theplant.Results arefully consis-tentwiththeperformedmolecularmodelingcalculations,which demonstratethemolecularmechanismatatomiclevel.According tothecomputationalsimulations,bothcompounds1and2 estab-lishedsimilarinteractionswithGABAAthanbromazepamthough withlessaffinitygiventhelowernumberofprotein–ligand inter-actions.The reported resultsnot only help in rationalizingthe observedselectivityofstachysrosaneand stachysrosanetoward GABAAbutalsocouldbeusedtoguidethedesignofmoreselective compoundsinfurtherexperimentaland/ortheoreticalstudies.

Author’scontribution

AR,UFandAKsupervisedthewholeproject.SZandAIinvolved intheisolationofcompounds,JP,HDH,JPandHPperformed com-putationalstudyandHKperformedbiologicalscreening.NJ,TBH, MFR,TAandSBmakefurthereditingandcorrectionsinthisMSand finalizeforsubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WeacknowledgeHigherEducationCommission(HEC)of Pak-istanandCOMSATSforfinancialsupport.Thisworkwassupported bytheFundación Séneca–AgenciadeCienciayTecnologíadela RegióndeMurcia(Spain)underProject19419/PI/14-1andbythe CentrodeAltoRendimientodelaRegióndeMurcia(Spain)under projectCFE-CAR-23/15.

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