Rev. bras. farmacogn. vol.26 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

Anticonvulsant

mechanism

of

saponins

fraction

from

adventitious

roots

of

Ficus

religiosa

:

possible

modulation

of

GABAergic,

calcium

and

sodium

channel

functions

Damanpreet

Singh

1

_,

_Rajesh

_Kumar

_Goel

∗

DepartmentofPharmaceuticalSciencesandDrugResearch,PunjabiUniversity,Patiala,Punjab,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8May2015 Accepted27October2015 Availableonline21July2016

Keywords:

BAYk-8644 Bicuculline Clonic

Convulsionsepilepsy

Ficusreligiosa

Veratridine

a

b

s

t

r

a

c

t

Inourpreviousstudies,quantifiedsaponins-richfractionfromadventitiousrootextractofFicusreligiosa L.,Moraceae,showedanticonvulsanteffectinacute,aswellaschronicmicemodelsofepilepsy.The presentstudywasdesignedtorevealputativeanticonvulsantmechanismofquantifiedsaponins-rich fractionusingtargetspecificanimalmodels.Theanticonvulsanteffectofquantifiedsaponins-rich frac-tionwasinitiallystudiedinmaximalelectroshockandpentylenetetrazoltestat1,2and4mg/kg;i.p.doses. Basedontheresultsofinitialanticonvulsanttesting,differentgroupsofmicewereinjectedwithvehicle orquantifiedsaponins-richfraction(4mg/kg;i.p.),30minpriortoaninjectionofN-methyl-d_-aspartic acid(100mg/kg;s.c.),bicuculline(5mg/kg;i.p.),strychninehydrochloride(2mg/kg;i.p.),BAYk-8644 (37.5␮g;i.c.v.),veratridine(500␮g/kg;i.p.)andtheconvulsiveepisodeswerestudied.Treatmentwiththe extract(1,2and4mg/kg)showedsignificantprotectioninmaximalelectroshockand pentylenetetrazol-inducedconvulsiontests,inadose-dependentmanner.Moreover,quantifiedsaponins-richfractionat 4mg/kgdoseshowedsignificantincreaseinlatencytoclonicconvulsions,decreaseinseizureseverity andincreaseinaveragewaveamplitudeinbicuculline,BAYk-8644andveratridinetests,respectively, ascomparedtovehiclecontrol.However,SRFtreatmentfailedtoabolishN-methyl-d-asparticacidand strychnine-inducedconvulsions,indicatedbyinsignificantchangeintheappearanceofturningbehavior andonsetoftonicextension,respectively,ascomparedtovehiclecontrol.Fromtheresultsofpresent study,itisconcludedthatquantifiedsaponins-richfractionsuppressmaximalelectroshock, pentylenete-trazol,bicuculline,BAYk-8644andveratridine-inducedconvulsions,indicatingitsGABAergic,Na+_and Ca2+_channel_modulatory_effects._Further_it_can_be_correlated_that_quantified_{saponins-rich}_fraction_causes deactivationofvoltage-gatedNa+_and_Ca2+_channels,_without_effecting_ligand-gated_Na+_and_Ca2+ chan-nels.Morestudiesarerequiredatmolecularlevelsusinginvitrotechniquestounderstandtheexact molecularinteractionsofquantifiedsaponins-richfractionwiththesepathways.

Introduction

Ficus religiosaL., Moraceae,is a medicinally importantplant ofthegenusFicus,and hasbeenextensivelyusedin traditional medicine for a wide range of ailments. Its different botanical partshavebeenusedfortheethnomedicaltreatmentofepilepsy. Many of its traditional uses have been validated in different experimentalstudiesthroughouttheglobe.Itsdifferentpartshave shown a variety of neurological effects including antiamnesic,

∗ Correspondingauthor.

E-mail:rkgoel@pbi.ac.in(R.K.Goel).

1_Present_address:_Regulatory_Research_Centre,_CSIR_–_Institute_of_Himalayan

BioresourceTechnology,Palampur,HimachalPradesh,India.

acetylcholinesterase inhibitory, parasympathetic modulatory, antianxietyand reversalofreserpine-inducedbehavioral effects (Singhetal.,2011a).Apartfromthesepharmacologicaleffects,it hasalsobeenstudiedforitsanticonvulsantpotentialindifferent experimentalstudies.

In ourprevious study, the crude fruit extract of F. religiosa

haveshownanticonvulsantactivity,whichwasfoundtobedue tomodulationofserotonergicfunctionsofthebrain(Singhand Goel, 2009; Goel and Singh, 2013).The flavonoid-rich fraction ofthefruitextract incombinationwithphenytoinalsoshowed protection in kindling mice model, along with attenuation of associated cognitive and behavioral impairments (Singh et al., 2014a). Its leaf extract wasstudied in acute animal models of convulsion,butwasfoundtobeineffective(Singhetal.,2011b). Inarecentstudythecrudebarkextractoftheplantalsoshowed

http://dx.doi.org/10.1016/j.bjp.2015.10.007

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protectioninacuteanimalmodelsofconvulsions,whichwasfound tobeduetoGABAaminotransferaseinhibitoryactivityofits bioac-tivemetabolites(Singhetal.,2014b).Thecrudeadventitiousroot extractofF.religiosahasalsobeenstudiedforitsanticonvulsant activity(Patiletal.,2011).Whenpartitioned,onlythesaponins-rich fraction(SRF)oftheextractretainedanticonvulsantactivity,rest allotherfractionswerefoundtoineffective.Thestudyindicated saponinspresentintheadventitiousrootextracttoberesponsible foritsactivity(Singhetal.,2012).SRFpersealsoprevented behav-ioralimpairmentsassociatedwithkindlinginmice,butfailedto preventcognitivedeficit(Singhetal.,2013).

Thesaponinsareatypeofnaturallyoccurringsurface-active gly-cosides,whicharegenerallyproducedbyplantswithanexception ofsomelowermarineanimalsandbacteria(Francisetal.,2002). Thetherapeutic role ofsaponins hasbeensuggested inseveral central as wellas peripheral pathological conditions like, neu-rodegeneration, epilepsy, cognitive impairments, hypertension, atherosclerosis, inflammation, allergic reactions, cancer, hyper-glycemiaand manymore(Radadetal.,2004;Nah etal.,2007). Thesaponinsisolatedfromotherplantshavebeenfoundto inter-actwithallthepathologicalprocessesinvolvedinepilepsy.They showedGABAergicagonist(Kimuraetal.,1994;Kimetal.,2001; Choietal.,2003),glutamatergicantagonist(KimandRhim,2004; Pengetal.,2009),glycinergicagonist(Nohetal.,2003;Kimetal., 2004),Ca2+₍_Zhong_et_al.,_1995;_Kim_et_al.,₂₀₀₈₎_and_Na+_channel blockadeeffects(Liuetal.,2001;Kimetal.,2005;Chindoetal., 2009).Duetowidespectrumofneuronalpathwayinteractionby saponins,thepresentstudywasenvisagedtounderstandthe puta-tiveanticonvulsantmechanismofSRFofadventitiousrootextract ofF.religiosa,byusinganimalmodelsofepilepsyinvolving pri-marilymodulationofcalcium,sodium,glutamateandGABAergic pathways.

Methods

Plantmaterial,extraction,fractionationandquantification

SRFwaspreparedandquantifiedasdescribedinourprevious study(Singhetal.,2012).Briefly,theadventitious rootsofFicus religiosawerecollected,cleaned,shade-dried,powderedand sub-jectedtorepetitiveextractionwith50%ethanolin apercolator. Afterdryingthecombinedpercolate,theresultantextractwas dis-persedinwaterandfractionatedwithhexane,chloroform,ethyl acetateandbutanol.Thebutanolfractionwasprecipitatedandthe totalsaponinscontentwasdeterminedintheprecipitatescollected usingcolorimetricmethodwithvanillin-sulphuricacidsystem,as discussedinourpreviousstudy.Thedoseswerepreparedfreshly beforeuseandwasinjectedintraperitoneally(i.p.).Thedosesof SRFwereselectedbasedontheresultsofourpreviousstudy(Singh etal.,2012)andwasadministeredat1,2and4mg/kg.

Animals

MaleSwissalbinomice,weighing20–30gobtainedfromCCS Haryana Agricultural University, Hisar, were employed in the presentstudy. Theanimalswerehousedin standard cages and maintainedatroomtemperaturewithnaturaldayandnightcycles. Theanimalswereallowedfreeaccesstofood(standardlaboratory rodent’schow)andwaterduringthestudyperiod.Allthe exper-imentswereconductedbetween9amand4pm.Allprocedures werecarriedoutaccordingtotheguidelinesofCommitteeforthe PurposeofControlandSupervisiononExperiments onAnimals (CPCSEA),IndiaandapprovedbytheInstitutionalAnimalEthical Committee(no.:107/99/CPCSEA-2009-4.1).

Drugsandchemicals

Pentylenetetrazol(PTZ)(dissolvedinnormalsaline),strychnine hydrochloride(dissolvedinnormalsaline),bicuculline(dissolved inminimum0.1NHCl,volumewasmakeupwithnormalsaline, pHwasadjusted to6.65withNaOH), N-methyl-d-aspartic acid

(NMDA)(dissolved in normal saline)and veratridine (dissolved inminimum0.1NHCl,volumewasmakeupwithnormalsaline, pH was adjusted to 6.65 with NaOH) were purchased from Sigma–Aldrich (St. Louis, MO). Reference drugs, diazepam and phenytoinwereobtained locallyfrom Jackson LaboratoriesLtd. (Amritsar, India) and Cadila Laboratories (Ahmedabad, India), respectively.

Maximalelectroshock(MES)-inducedconvulsions

Miceindifferentgroupswereinjectedwiththevaryingdoses ofSRF(1,2and4mg/kg;i.p.),vehicle(10ml/kg;i.p.)andphenytoin (25mg/kg;i.p.).After30minofthesetreatments,allthegroups weredeliveredacalibrated(throughacurrentcalibrator[Rolex, Ambala,India])transauricularelectroshockof56mAfor0.2susing aconvulsiometer(Rolex,Ambala,India),viaapairofcrocodileear clips.Thedurationoftonichindlimbextension(seconds)wasnoted andwascomparedwiththatofvehiclecontrol(Swinyardetal., 1952).

PTZ-inducedconvulsions

PTZatadoseof75mg/kgwasgiventofivedifferentgroupsof micepretreated30minpriorwiththevaryingdosesofSRF(1,2and 4mg/kg;i.p.),vehicle(10ml/kg;i.p.)anddiazepam(5mg/kg;i.p.). Latencytoclonicconvulsions(min)wasnotedandwascompared withthatofvehiclecontrol.

NMDA-inducedconvulsions

Miceintwodifferentgroupsweretreatedwitheithervehicle (10ml/kg)orSRF(4mg/kg,i.p.)30minprior toasubcutaneous injectionofNMDA(100mg/kg).Thereafter,themicewereobserved fortheappearanceofturningbehaviorfornext30min.Turning behaviorwascharacterizedastwoconsecutive360cycles com-pletedby thesameanimal (Bhutada et al.,2010).Thetest was performedtodeterminetheroleofglutamatergicprocessesforthe anticonvulsanteffectofSRF.

Bicuculline-inducedconvulsions

Bicuculline wasadministered(5mg/kg;i.p.)in twodifferent groupsofmice,30minaftertreatmentwithvehicle(10ml/kg;i.p.) orSRF(4mg/kg;i.p.).Themicewereobservedfortheappearance ofclonic–tonicseizuresanddeathforaperiodof30minafter bicu-cullineinjection.Antagonismofbicucullineseizureswasdefinedas theabsence/delayofclonic–tonicseizuresfor30min(Irifuneetal., 2003).ThetestwasperformedtoexaminetheGABAergiceffectsof SRF.

Strychnine-inducedconvulsions

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BAYk-8644-inducedconvulsions

BAYk-8644wasusedtoinduceconvulsionsasdescribedby

VonVoigtlanderetal.(1987)withslightmodifications.Afreehand

i.c.v.injection ofBAY k-8644 (37.5␮g/10␮l) wasmade in con-sciousmouse,30minaftervehicle(10ml/kg;i.p.)orSRF(4mg/kg;

i.p.)treatmentbyvisuallocationmethoddescribedbyHaleyand McCormick(1957).Briefly,eachanimalwasfirmlygripedbytautly pullingthelooseskinbehindthehead,amidlinewasdrawnusing amarkerthroughtheanteriorbaseoftheearsandtheinjection wasmade2mmoneithersidetheline.Acrossbetweentheleft eye–rightearandrighteye–leftearwasdrawntolocatethe mid-pointtodrawmidline.Theinjectionsweremadewithhypodermic needleof0.4mmexternaldiameterattachedtoa10␮lHamilton syringe(Hamilton,Bonaduz,Switzerland).Theneedlewascovered withapolypropylenetube,leaving3mmofthetipregion,soasto insertthisportionthroughtheskullintothebrainofmouse.The syringewasheldatanangleofaround45degreetotheskull,with thebevelofneedlefacingup,pointinginthedirectionofthetail. Inducedseizureswereratedaccordingtoascaledevisedasstage1, scratchingandtwistingoftheforelimbs(score1);stage2,rearing andwalking(score2);stage3,intermittentclonicjerksoflimbs withtonicflexionofforelimbsandtailflexion;stage4,head bob-bingwithcomplexgroomingactions(lickingoffurandscratching); stage5,jumping,squeakingandtonicextensionofhindlimbs;stage 6,barrelrolling(KaurandGoel,2011).Theoccurrenceofseizure signsandlatencywererecordedfor1h.Thetestwasperformedto investigatetheroleofCa2+_channels.

Veratridine-inducedseizure

Veratridine seizures in mice were induced by the methods describedbyOtoometal.(2006)andOtoomandSequeira(2011), with slight modifications. Two different groups of mice were treatedwithvehicle(10ml/kg;i.p.)orSRF(4mg/kg;i.p.),30min prior to an intraperitoneal injection of veratridine (500␮g/kg). After15minofveratridineadministration,electroencephalogram (EEG)recordingwasperformedusingaDigitalPolygraph(PC-2004, MedicaidSystem,Chandigarh,India)bythemethoddescribedby

OtoomandSequeira(2011),butusingsurfaceelectrodes.Average waveamplitude(␮V/min)andwavepatterninvehiclecontrol ani-malswerecomparedwiththatofnormal(naïve)andSRFtreated animals.

Statisticalanalysis

Alltheresultswereexpressedasmean±SEM.Thesignificance ofdifferencebetweenmeanswasdeterminedusingone-way anal-ysisof variance (ANOVA)followed byTukey’s test in MES,PTZ and veratridine tests. Unpaired Student’s t-test was employed todeterminesignificanceofdifferencebetweenmeaninNMDA, bicuculline,strychnineandBAYk-8644-inducedconvulsiontests. Statisticalanalysisforthepercentagemortalityandpercentageof animalsshowingturningbehavior pergroupwasperformedby Chi-squaretest.Theresultswereregardedassignificantatp<0.05.

Results

EffectonMES-inducedconvulsions

Treatment with SRF showed significant (p<0.001) dose-dependentdecreaseinthedurationoftonichindlimbextension at 1mg/kg (7.8±0.86s), 2mg/kg (4.1±1.2s), and 4mg/kg (1.4±0.8s),ascomparedtovehiclecontrol,withmaximum pro-tection observedat 4mg/kg. The anticonvulsantactivity of SRF

20

18

16

14

12

10

8

6

4

2

0

Vehicle

P<.001 P<.001

Phenytoin

Dur

ation of

THLE (seconds)

SRF 1 SRF 2 SRF 4

Fig.1. Effectdifferentdosesofsaponins-richfractionfromtheadventitiousroot extractofFicusreligiosaonthedurationofMES-inducedtonichindlimbextension. THLE,tonichindlimbextension;SRF1,2and4,saponins-richfraction1,2and 4mg/kg,respectively.

16

Vehicle Diazepam N/C

Con

vulsion latency (min)

SRF 1 SRF 2 SRF 4

14

12

10

8

6

4

2

0

P<.017 P<.001

P<.001

Fig.2. Effectdifferentdosesofsaponins-richfractionfromtheadventitiousroot extractofFicusreligiosaonthelatencytoPTZ-inducedconvulsions.N/C,no convul-sions;SRF1,2and4,saponins-richfraction1,2and4mg/kg,respectively.

at 4mg/kg was found to be comparable to phenytoin treated (1.2±0.8s)group(Fig.1).

EffectonPTZ-inducedconvulsions

InPTZ-inducedconvulsionstest,treatmentwithSRFat1mg/kg (p=0.017),2mg/kg(p<0.001)and4mg/kg(p<0.001)significantly increasedthelatencytoclonicconvulsions,ascomparedto vehi-clecontrolat5.58±0.14min,9.54±0.64minand12.28±1.12min, respectively.Similarly,asinMEStest,SRFshoweddose-dependent anticonvulsanteffect.SRFwasfoundtobelesseffectiveasthatof standarddiazepam,asitstreatmentonlydelayedtheclonic con-vulsions,wereasdiazepampretreatmentcompletelyabolishedthe inductionofPTZconvulsions(Fig.2).

EffectonNMDA-inducedconvulsions

TreatmentwithSRFshowednoprotectionagainstconvulsions inducedbyNMDA,indicatedbyinsignificantchangeintheonset andoccurrenceofturningbehaviorinmiceascomparedtovehicle control(Fig.3AandB).

Effectonbicuculline-inducedconvulsions

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8 NS

Vehicle SRF 4 Vehicle SRF 4

NS

A

B

Onset of tur

ning beha

vior (min)

% animals sho

wing tur

ning beha

vior

120

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0 7

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Fig.3. Effectsofsaponins-richfractionfromtheadventitiousrootextractofFicusreligiosaonNMDA-inducedconvulsions.(A)Effectofsaponins-richfractionontheonset ofNMDA-inducedturningbehavior.(B)Effectofsaponins-richfractiononthepercentageofanimalsshowingNMDA-inducedturningbehavior;NS,notsignificant;SRF4, saponins-richfraction4mg/kg.

5

A

B

Vehicle SRF 4 Vehicle

% mor

tality

Con

SRF 4

P<.001 120 NS

100

80

60

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20

0 4.5

4

3.5

3

2.5

2

1.5

1

0.5

0

Fig.4.Effectsofsaponins-richfractionfromtheadventitiousrootextractofFicusreligiosaonbicuculline-inducedconvulsions.(A)Effectofsaponins-richfractiononthe latencytoconvulsionsinducedbybicuculline;(B)Effectofsaponins-richfractiononthepercentagemortality;NS,notsignificant;SRF4,saponins-richfraction4mg/kg.

3.5 120

100

80

60

40

20

0 3

2.5

2

1.5

1

0.5

Vehicle SRF 4 Vehicle

NS NS

A

B

Con

vulsion latency (min) % mor

tality

SRF 4

Fig.5.Effectsofsaponins-richfractionfromtheadventitiousrootextractofFicusreligiosaonstrychnine-inducedconvulsions.(A)Effectofsaponins-richfractiononthe latencytoconvulsionsinducedbystrychnine.(B)Effectofsaponins-richfractiononthepercentagemortality;NS,notsignificant;SRF4,saponins-richfraction4mg/kg.

However,nosignificantprotectionagainstmortalitywasobserved aftertreatmentwithSRFasthatofvehiclecontrol(Fig.4AandB).

Effectonstrychnine-inducedconvulsions

TreatmentwithSRFshowednoprotectionagainstconvulsions inducedby strychnine,indicated by insignificantchangein the latencytoconvulsions,ascomparedvehiclecontrol.Moreover,no significantprotectionwasobservedonthepercentagemortalityas thatofcontrol(Fig.5AandB).

EffectonBAYk-8644-inducedconvulsions

BAY k-8644, a Ca2+ _channel _agonist_{administration} _in _mice resultedinconvulsionswithalatencyofaround7.28±0.27min

in vehicle control group. Treatment with SRF, significantly (p<0.001) increased the latency to BAY k-8644-induced con-vulsions to 15.85±0.89min. SRF treatment also significantly (p<0.001)reducedtheseizureseverityscoreto1.83±0.6,as com-paredtovehiclecontrol(5.33±0.21)group(Fig.6AandB).

Effectonveratridine-inducedseizure

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18

_A

_B

P<.001

Vehicle

Con

Seizure se

ver

ity score

SRF 4 Vehicle SRF 4

P<.001

16

14

12

10

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6

4

2

0

6

5

4

3

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1

0

Fig.6.Effectsofsaponins-richfractionfromtheadventitiousrootextractofFicusreligiosaonBAYk-8644-inducedconvulsions.(A)Effectofsaponins-richfractiononthe latencytoconvulsionsinducedbyBAYk-8644;(B)Effectofsaponins-richfractionontheseizureseverityscore;SRF4,saponins-richfraction4mg/kg.

90

80

70

60

50

40

30

20

10

0

P<.001 P<.001

Naïve Vehicle

A

ver

age amplitude (

µ

V/min)

SRF 4

Fig.7.Effectsofsaponins-richfractionfromtheadventitiousrootextractofFicus

religiosaonveratridine-inducedchangesinwaveamplitude.SRF4,saponins-rich

fraction4mg/kg.

A

B

C

50 µV

2 sec. 0.2 sec.

Fig.8.Effectsofsaponins-richfractionfromtheadventitiousrootextractofFicus

religiosaonveratridine-inducedchangesinwavepatternduringseizuresphase.(A)

Naïvegroup;(B)vehiclecontrolgroup;(C)saponins-richfractiontreatedgroup (4mg/kg).

Discussion

It is important tostudy the mechanism of a novel anticon-vulsant component, as it might act on well-recognized targets innovelwaysand/orbynovelcombinationsofactionson well-recognizedtargets.In somecases,suchapracticehad revealed entirelynewantiepileptictargetsliketopiramateandlamotrigine (Rogawski,2006a).Hence,tostudytheanticonvulsantmechanism of SRF, thepresent study wascarried out. Treatmentwith SRF

resultedinsuppressionofMESandPTZ-inducedconvulsionsina dose-dependentmanner.Theresultsofthisinitialscreeningare inlinewiththeresultsofourpreviousstudy(Singhetal.,2012). SRFtreatmentresultedinmarkedsuppressionofBAYk-8644and veratridine-inducedconvulsions, and showed partialprotection againstbicuculline-inducedconvulsion.Howeveritwasfoundto betotallyineffectiveagainststrychnineandNMDA-induced con-vulsions.Since,MESandPTZtestsareconsideredtobethe“gold standards”inearlystagesofdrugtesting(Rogawski,2006b),hence initialconfirmationoftheanticonvulsanteffectofSRFwascarried inthesetestsatalldoses.Theothertests(NMDA,strychnine, bicu-culline,BAYk-8644andveratridine-inducedconvulsiontests)were performedtoinvestigateitsputativeanticonvulsantmechanism andeffectindifferentacuteseizuremodelsofvariedphenotype. Asinglemosteffectivedosewasusedtoreducethenumberof animalsrequiredinthestudy,duetoethicalconstraints.

Someofthepreviousinvitro studiesrevealedthat saponins isolatedfromginsengandotherFicusspeciescausesblockadeof voltagedependentNa+_channels,_hence_decreases_neuronal exci-tation (Liu et al., 2001; Kim et al., 2005; Chindo et al., 2009). Hence,tostudy theeffectof SRFonNa+ _channels, veratridine-inducedseizuremodelwasused.Veratridineisacommonlyused experimentaltoolinvariouselectrochemicalstudies,itopensNa+ channels during sustained membrane depolarization by inhibi-tinginactivation,leadingtoNa+_influx,_secondarily_it_causes_Ca2+ influx,increasedpumpactivityandinturnexocytosis,leadingto experimentalseizures in rodents(Ulbricht,1998; Otoom etal., 2006).Inthepresentstudy,administrationofveratridinein vehi-clecontrolanimalsresultedinseizures,indicatedbycharacteristic EEG changes, these changes are in line with a previous study (Otoom and Sequeira,2011).Several clinically used antiepilep-ticdrugs actingthroughblockadeofNa+ _channels,_like_valproic acid,phenytoinandcarbamazepinehavebeenfoundtosuppress veratridine-inducedabnormalneuronalexcitationinexperimental studies(OtoomandAlkadhi,2000a,b).Sinceinourstudy, pretreat-mentwithSRF,abolishedtheveratridine-inducedseizureactivity indicated by normalization of EEGpattern, indicating itseffect mightbeduetoNa+_channel_blockade_activity.

Panaxatriol saponins, the main constituents extracted from

Panax notoginseng have beenreported topossess aninhibitory

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byBAYk-8644,whichinduceconvulsionsthroughactivationof voltage-gatedCa2+ _channels._SRF_pretreatment_inhibited_BAY k-8644-inducedconvulsion,therebysuggestingitseffecttobedueto inhibitionofCa2+_channels.

Severalpreclinicalstudiesindicatingthemodulationof GABAer-gic functionsby plantisolated saponins have beenreported in literature.SaponinsisolatedfromPanaxginsengshowedGABAA receptorbindingintheratbrain(Kimuraetal.,1994;Kimetal., 2001).ThesesaponinsalsoincreasedtheGABA-mediatedinward peakcurrentinXenopusoocytes(Choietal.,2003).Effectiveness ofSRFininitialanticonvulsantscreeninginPTZ(GABAAreceptor antagonist)testindicateditsGABAergicmodulatoryeffect.Since severalotheranticonvulsantmoleculesactingthroughother mech-anismshavealsoshownactivityagainstPTZconvulsions,henceto furtherconfirmtheGABAergiceffectofSRF,itseffectwasstudiedin bicuculline(GABAAreceptorantagonist)test.TreatmentwithSRF resultedinsuppressionofbicuculline-inducedconvulsions, indi-catedbyincreaseinlatencytoclonicconvulsions,buthoweverit wasfoundtobeineffectiveinreducingmortality.Furtherstudies arerequiredtounderstandtheexactGABAergicmodulatoryeffects ofSRF.

InhibitionofNMDA-inducedexcitatoryresponsesbyplant iso-latedsaponinshasalsobeenwelldocumentedinseveralstudies. SaponinsisolatedfromginsenghaveshowninhibitionofNMDA receptor-mediatedepilepticdischargesinculturedhippocampal neurons(KimandRhim,2004).GinsenosideRb3haveshown neu-roprotectiveroleonhippocampalneurons,throughfacilitationof Ca2+_-dependent_deactivation_of_NMDA_receptors₍_Peng_et_al.,₂₀₀₉_). Clinicallyuseddrugslikefelbamateandlacosamideactthrough inhibitionofNMDAreceptor(Rogawski,2006).Therefore,tostudy theeffectofSRFonNMDA-mediatedexcitatoryresponses, NMDA-inducedconvulsionstestwasused.HoweverSRFfailedtosuppress NMDA-induced convulsions, indicating lack of NMDA receptor interactionsofSRF.

Theroleofglycinergicreceptormechanisminthepathogenesis ofclinicalandexperimentalepilepsieshasbeenwellestablished.In differentstudies,theglycinergicdrugshaveshownsuppressionof epilepticdischarge(Laubeetal.,2002;ChattipakornandMcMahon, 2003).Ginsenosideshaveshownenhancementofglycine-induced inwardpeakcurrentinhumanglycinereceptorchannelactivity expressedinXenopusoocytes(Nohetal.,2003).Theyhavealsobeen foundtoantagonizeNMDAreceptorsthroughtheglycine modu-latorysiteinratculturedhippocampalneurons(Kimetal.,2004). Theseliteraturefindingssuggesttheglycinergiceffectsofsaponins. HencetostudytheglycinergicmodulationofSRF,itseffectwas studiedagainstconvulsionsinducedbystrychnine(glycine recep-tor antagonist). Since, the SRF was found to be ineffective in suppressingstrychnine-induced convulsions, thereby indicating lackofeffectivenessasdirectinteractionwithglycinergicpathway. Theresultsof thepresentstudy showedthatSRFtreatment inhibitedMES-inducedconvulsionsandsuppressedBAYk-8644, veratridineandGABAAreceptorantagonists(PTZand bicuculline)-induced convulsions at tested doses, indicating it to be acting throughmultiplemechanismsasGABAagonistaswellasNa+_and Ca2+ _channels_inhibitor. _Several_clinically _used _drugs _have _also beenfoundtobeactingthroughmultiplemechanisms(Köhling, 2002).Interestingly, SRFshowedprotectionin BAYk-8644 and veratridine tests,which actthrough activationof voltage-gated Ca2+_and_Na+_channels,_{respectively,}_but_was_found_to_be ineffec-tiveagainstconvulsionsinducedbyNMDA(receptor-operatedion channels)receptoragonist,NMDA.Thesimilaranticonvulsant pro-fileisshownbyethosuximide,aclinicallyusedantiepileptic(Turski etal.,1990;OtoomandSequeira,2011).Theseresultsindicated theeffectivenessofSRFinblockadeofvoltage-gatedionchannels andineffectivenesstoward ligand-gatedNa+ _and_Ca2+_channels. Since,veratridineactbydepolarizationofexcitablecells,thereby

preventinginactivationofNa+_channels,_leading_to_an_increase_in influxofNa+_and_Ca2+₍_Jordán_et_al.,₂₀₀₀_),_therefore_it_is_not_exactly clearthattheanticonvulsanteffectofSRFisduetoblockadeofNa+ orCa2+_channels_or_both,_as_it_has_also_shown_protection_against seizuresinducedbyBAYk-8644.As,ethosuximidewhichshows anticonvulsanteffectthroughtheblockadeofCa2+_channels_also suppressveratridine-inducedseizures.Morestudiesatmolecular levelsarerequiredtounderstandtheseeffectsaccurately.

Conclusion

TheresultsofpresentstudyconcludedthatSRFexhibitits anti-convulsanteffectthroughmodulationofGABAergic,Na+_and_Ca2+ channel functions.Effectiveness ofSRFin BAY k-8644and ver-atridinetestsindicateditsvoltage-gatedNa+ _and_Ca2+_channels inhibitoryeffectsbutnotligand-gatedNa+_and_Ca2+_channels._More studiesarerequiredatmolecularlevelstounderstandtheexact molecularinteractionsofSRFwiththesepathways.

Ethicaldisclosures

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

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contribution

Thefirstauthorconductedtheexperiments,andsecondauthor plannedand supervised the study.Both theauthors wrotethe manuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WearethankfultotheUniversityGrantCommission,NewDelhi, India,forprovidingfinancialassistance[VideF.No.:34-130/2008 (SR)]for theprojectand projectfellowship toMr.Damanpreet Singh.

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