w ww.e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
GABAergic
effect
of
valeric
acid
from
Valeriana
wallichii
in
amelioration
of
ICV
STZ
induced
dementia
in
rats
Shilpa
Vishwakarma,
Rohit
Goyal
∗,
Varun
Gupta,
Kanaya
Lal
Dhar
SchoolofPharmaceuticalSciences,ShooliniUniversity,Solan,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received17January2015 Accepted10February2016 Availableonline3May2016
Keywords:
GABA Alzheimer’s Valericacid Dementia Streptozotocin Memory
a
b
s
t
r
a
c
t
ValerianawallichiiDC.,Caprifoliaceae,isusedtohaveanti-ulcer,anti-spasmodic,anti-epileptic, mem-oryenhancer,anti-anxiety,anti-rheumatic,sedative,anti-asthmaticanddiureticactivities.V.wallichiiis reportedtocontainvalpotriates,valericacid,valerenicacid,valechlorine,valerianine,resinsand alka-loids.Valericacid,foundinV.wallichiiappearssimilarinstructuretotheneurotransmitterGABA.Valeric acidalsoactsasanNMDA-receptorantagonist.Theaimofpresentstudywastoinvestigatethe neuropro-tectiveeffectofV.wallichiicontainingvalericacidanditspossiblemechanismofactioninamelioration ofintracerebroventricularstreptozotocininducedneurodegenerationinWistarrats.TherhizomesofV. wallichiiwerepowderedcoarselyandextractedbypercolationmethodusingdichloromethane.Wistar rats(220–250g)ofeithersexweredividedinto5groups,comprising6animalseach.Valericacidwas isolatedfromplantextractandcharacterizedusingFT-IR.Picrotoxin(2mg/kg)wasusedasGABA-A antagonist.Intracerebroventricularstreptozotocinadministrationcausedsignificant(p<0.05)increase inescapelatency,retentiontransferlatencyonmorriswatermazeon17th,18th,19thand20thdayand ele-vatedplusmazeon19thand20thdayrespectively,ascomparedtonormaluntreatedrats.Treatmentwith
V.wallichiiextract100and200mg/kgandvalericacid20and40mg/kgsignificantlydecreasedtheescape latencyandretentiontransferlatency,ascomparedtointracerebroventricular-streptozotocingroup. Plantextractandvalericacidalsodecreasedtheleveloflipidperoxidationandrestoredglutathionelevel inratbrains.Administrationofpicrotoxinsignificantlyreversedtheeffectsproducedbyplantextractand valericacidinintracerebroventricular-streptozotocintreatedrats.Thefindingsmayconcludethatvaleric acidpresentinV.wallichiihassignificantGABAergiceffectinameliorationofexperimentaldementia.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Dementiaisaclinicalsyndromewhichisduetodegeneration ofneurons inbrain and spinalcord. It is associated with dete-riorationincognitiveabilityandcapacityforindependentliving (Fratiglionietal.,2007).Theprevalenceofneurodegenerative dis-orderslikeAlzheimer’sdiseases(AD)hasincreasedsignificantlyas globalpopulationage(ChenandZheng,2012).Currently,the num-berofdeathscausedbyADisseverelyhighandprojectedtorise by21.2millionin2025(Hirtzetal.,2007;Katzman,2008).The characteristicfeatureofADisvasculardementia,fronto-temporal dementia,andaggregationofamyloid,accumulationof neurofib-rillarytangles, lewisbody formationand neuronal death.There is the development of multiple cognitive deficits such as dis-turbancesinexecutivefunctioning,apraxia,agnosia,aphasiaand
∗ Correspondingauthor.
E-mail:[email protected](R.Goyal).
memoryimpairment(Fratiglionietal.,2007)thatinterfereswith
dailysocialandprofessionalbrainoutcomes.Themostcommon
symptomsofdementiaarepsychosis,aggression,insomnia, anxi-ety,depression,delirium,angerandsundowning(confusioninlate afternoonorearlyevening)(KullerandLopez, 2008).The treat-ment ofAD includesacetylcholinesteraseinhibitor likeTacrine, Donepezil,RivastigmineandGalantamine(Pokorski,2002). More-over,FoodandDrugAdministrationhaswarnednottouseatypical antipsychotics due to their increased mortalityrisk (Schneider etal.,2005).
ADischaracterizedbyaccumulationofamyloidbetapeptide
(A)asfibrillaryplaquesandsolubleoligomersinbrainregions (Coleand Vassar,2007).Tauproteinis alsoidentified asoneof themaincomponentofneurofibrillarytanglesinvarious neuro-logicaldiseases(GendronandPetrucelli,2009).Inmanyareasof
CNS,overactivationofNMDARandsubsequentinfluxorrelease
of excessive Ca2+ has been noted to be predominant form of
neurotoxicity.Ca2+overloadcouldultimatelyleadtoproduction ofreactiveoxygenspecies (ROS)and nitricoxide(NO) radicals,
http://dx.doi.org/10.1016/j.bjp.2016.02.008
mitochondrialdysfunction,neuronalexcitotoxicityandultimately neuronaldegeneration(MajdiandChen,2009).Recentresearches haveemergedwithevidencesthatsomepathogenicreceptorsare alteredwithlowerlevelsofGABAAR-subunits␣1,␣2,␣4,␦,and 2,mRNAinprefrontalcortexand␣1,␣5,and3mRNAsin hip-pocampuswereobservedinAD(RissmanandMobley,2011).
ValerianawallichiiDC.,Caprifioliaceae,isfoundabundantlyat
analtitudeof1300–3800mintemperateHimalayasfrom
Kash-mir toand between 1250and 1800min Khasi hills (Chauhan,
2006).Ithasgotconsiderablereputationforitstraditionalusein pain(VohoraandDandiya,1992),epilepsy,insomnia,neurosis, sci-atica(Nadkarni,1976;Marder et al., 2003).The plantiswidely usedinthetreatmentofanxietyanddepression (Panijel,1985; Ronet al.,2000).It hasbeenusedassedativein thetreatment of insomniaand restlessness (Leathwoodand Chauffard, 1985). Valerianaisreportedtohaveantidepressant,anxiolytic, antispas-modic,anti-inflammatoryandanticonvulsantactivities(Sahetal., 2010).Valericacid(VA)orpentanoicacid,achiefconstituentofV. wallichii(Kokateetal.,2007),isastraightchainalkylcarboxylic acidwith chemical formula C5H10O2, molecularweight 102.15 andmeltingpoint:34.5◦C.VAappearssimilarinstructuretothe
neurotransmitterGABAbut lacksaminefunctionalgroupwhich
contributesforthebiologicalactivitiesofGABA.Itisreportedas GABA-Aagonistandisaverygoodrelaxant.Italsoactsasan NMDA-receptorantagonistandshowedanti-epilepticeffect(Loebetal., 1990).GABA-mediatedpostsynapticinhibitionincultured mam-malianneuronswasobservedontreatmentwithVA.Itincreases availabilityofsynapticGABAand/orenhancespostsynapticGABA responses,andthusenhancesGABAergicactivity.Keepingthisin view,thepresentstudywashypothesizedtoinvestigate GABAer-giceffectofVAfromV.wallichiiinameliorationofexperimental dementiain intracerebroventricularstreptozotocinmodel inrat brain.
Materialsandmethods
Collectionandauthenticationofplantmaterials
TherhizomesofValerianawallichiiDC.,Caprifioliaceae,were col-lectedfromKufri,Shimla,HPanddulytaxonomicallyauthenticated byDr.R.Raina,SeniorScientist,Professor(MedicinalPlants),Dr. Y.S.ParmarUniversityofHorticultureandForestry,Nauni,Solan, HP,IndiaandthesamplewaslinkedtoUHF-Herbariumwithfield
booknumber12425.
Animals
Wistar Albino rats weighing 200–250g of either sex were
obtained from animal house (Reg No. 1541/PO/a/11/CPCSEA),
ShooliniUniversity,Solan,HP,India.Theanimalswereallowedto acclimatizetotheenvironmentfor7dayspriortotheexperimental session.Theanimalsweredividedintodifferentgroupsconsistof sixanimals(n=6)ineachgroup.Thestudywasconductedasper theguidelinesofCommitteeforthepurposeofcontroland supervi-sionofexperimentsonanimals(CPCSEA).Theexperimentalstudy wasdulybeenapprovedbyInstitutionalAnimalEthical
Commit-tee(IAEC)videprotocolno.IACE/SU-PHARM/2/020.Theanimals
weremaintainedatstandardfoodpelletdietfromAshirwad Indus-tries,Chandigarh,India,wateradlibitum,temperature25±2◦C andhumidity45±5%.
Extractionofplantmaterial
Driedpowdered rhizomes of V. wallichii (500g) were taken
andextracted bypercolationmethodusing dichloromethaneas
solvent.The powdered material wasmoistenedwith150ml of
dichloromethaneand allowedtostandforapproximately4hin
percolator.Aftercompletionof4h,200mlofdichloromethanewas againaddedtoformashallowlayerabovethemass,andthe mix-turewasallowedtomacerateinclosedpercolatorfor 24h.The outletofpercolatorwasopenedafter24handtheliquidcontained thereinwasallowedtodripslowly.Additionaldichloromethane wasaddeduntilthepercolatemeasuresaboutthree-quartersof requiredvolumeoffinishedproduct.Aftercompletionof
extrac-tion, solvent was then recovered and dried in rotary vacuum
evaporator.
Isolationofvalericacid
TheextractofV.wallichiiwastreatedwithmethanolandsodium bicarbonate,whichproduceseffervescence.Thenextractsolution wascentrifuged,supernatantwasseparatedandthesolidmasswas dissolvedinwater.It wascompletelydissolvedinwaterbystir continuouslyanddilutehydrochloricacidwasaddeddropwiseto maintainpH2.Theextractionwasdonethreetofourtimeswith methylenechlorideinaseparatingfunnel,concentratedthe solu-tionandthusobtainedproductwassubjectedtocharacterization (Houghton,1988;Barnes,2002;Gruenwald,2004).
Fingerprintanalysisofvalericacid
The fingerprint analysis of isolated VA in reference to
standard samplewasdoneusingAgilentTechnologiesCary630
Fourier transform infrared spectrophotometry (FTIR) at range
4000–650cm−1.
Intracerebroventricular(ICV)administrationofstreptozotocin (STZ)fordementia
Rats were anaesthesized using ketamine hydrochloride
(70mg/kgi.p.)andxylazine(5mg/kg,i.p.).Theheadoftheanimal was positioned in stereotaxic apparatus and a midline sagittal incisionwasmadeinthescalp.Twoholesweredrilledthroughthe skullforplacementofinfusioncannulaintolateralcerebral ven-tricleswiththecoordinates:0.8mmposteriortobregma;1.5mm lateraltosaggitalsuture;3.6mmventrallyfromthesurfaceofthe brainandthetoothbarwassetat0mm.AHamilton®syringeof
10lwasattachedthroughaskullholetoastereotaxicapparatus andpistonofthesyringewasloweredmanuallyintoeachlateral ventricle.STZ(3mg/kgintwodivideddoses)dissolvedincitrate buffer,pH4.4,wasslowlyinfusedthroughintracerebroventricular route bilaterally (Tiwari et al., 2009).After ICVadministration, thecutskinwassuturedandthepostoperativecareweredulybe
madebyapplyingpovidone–iodinesolutiononwound.
Thebehavioralassessmentsweredoneusingmorriswatermaze teston17th,18th,19thand 20th dayandelevatedplusmazeon 19th and20thday.Oncompletionofprotocol,theanimalswere sacrificedandbrainwasisolated.
Drugadministration
The plantextract of V. wallichiiwasused in thedoserange from50to400mg/kginvariousinvestigationsusingrats(Subhan etal.,2009).Therefore,100and200mg/kgwereselectedasits submaximal dosetoinvestigate thebiological potentialagainst
experimentally induced neurodegeneration. The dosage of VA
selectedwas20and40mg/kgduetoitsprotectiveactionagainst variousexperimentaldiseasedconditionsinrats(Loebetal.,1990). V.wallichiiextract100and200mg/kg,p.o.(suspendedin1%CMC solution)andvalericacid20and40mg/kg,i.p.(suspendedin1%
fromday3rdto21stdaysoncedaily.Picrotoxin(2mg/kg,i.p., sus-pendedin1%Tween80solution)wasgivenasGABA-Aantagonist (Sattigeri etal.,2012)30minbeforevalericacidadministration daily.
Morriswatermazetest
Themorriswatermazetestwasconductedforassessmentof
contextual/spatialmemoryinacylindricalpool120cmindiameter and60cmindepth,filledwithwater(maintainedat28±2◦Cand madeopaqueusingnon-toxicpaint)toadepthof40cm.Acircular platformof9cmdiameterand38cmheightwasplacedinwater tank2cmbelowthewaterlevelformazeacquisitiontest.Thepool isdividedinfourequalquadrantsandaplatformislocatedinthe centeroftargetquadrant.Theplatformremainsinthesame posi-tionduringthetraining.Thepoolwasplacedinalargeroom,the visiblecueswereprovidedfordevelopmentofspecialmemoryand
remainunchangedthroughoutthestudy.Animalswerereceived
fourdailyconsecutivetrainingsessionsfrom17thdayto20thday. Ineachtrial,animalwasdroppedinwaterfacingitsheadtoward thewall,subjectedtoswimandthelatencytofindtheplatform
wasrecordeduptothemaximumof2min.Theanimalsreached
totheplatformwereallowedtoremaintherefor20sbeforenext trial.Thetimetakenbyarattoreachtheplatformwasrecordedas escapelatency(EL)fromwater(Sachdevaetal.,2014;Yangetal., 2014).
Elevatedplusmazetest
Anelevatedplus-mazewasusedtotestthecognitivefunction ofanimals.Elevatedplus-mazeconsistsoftwooppositeopenarms (50cm×10cm),crossedwithtwoclosedarmsofthesame dimen-sionswith40cmhighwalls.Thearmswereconnectedbyacentral square(10cm×10cm)andtheapparatuswaselevated70cmfrom thefloor.Onday19thand20th,ratswereplacedindividuallyatone endoftheopenarm,facingawayfromthecentralsquare.Thetime takenforeachrattomovefromtheopenarmandenterintooneof theclosedarmswasrecordedas“initialtransferlatency”(ITL).The animalwasallowedtoexplorethemazefor30safterrecordingITL andreturnstoitshomecage.Then,24hafterITL,theratwasplaced againontheopenarmandtheretentiontransferlatency(RTL)was noted(Sachdevaetal.,2014).
Tissuebiochemicalestimationsforoxidativestress
Onday21st,animalsweresacrificedandtheirbrainsquickly removed,washedwithice-coldsalineandstoredat−80◦C.Brain
tissue samples(striatum and cortex regions)were thawed and
homogenizedwithtentimes(w/v)ice-cold 0.1mol/l phosphate
buffer(pH7.4).Thehomogenateswerecentrifugedat15,000×gfor 20minandsupernatantwasthenusedtoforassessmentoflevelof lipidperoxidationandglutathioneasamarkerforoxidativestress.
Estimationoflipidperoxidation
Thereactionmixtureconsistsof0.1mloftissuehomogenate, 0.2mlof8.1%sodiumdodecylsulphate(SDS),1.5mlof20%acetic acidand1.5mlof0.8%aqueoussolutionofthiobarbituricacid(TBA). ThepHof20%aceticacidwasadjustedwith1NNaOHto3.5.The mixturewasfinallymadeupto4mlwithdistilledwaterandheated at95◦Cfor60minonanoilbath.Aftercoolingundertapwater,1ml ofdistilledwaterand5mlofmixtureofbutanolandpyridine(15:1, v/v)wasaddedandthemixturewasshakenvigorouslyona vor-texmixer.Thencentrifugationat2800×gfor5minwasgivenand absorbanceoforganiclayer(upperlayer)wasmeasuredat532nm
usingUVvisiblespectrophotometer.Theleveloflipidperoxidation wasexpressedasnM/mgtissue(Ohkawaetal.,1979).
Estimationofreducedglutathione(GSH)
Thetissuehomogenatewasaddedwithequalvolumeof20%
trichloroacetic acid(TCA) containing1mM EDTAto precipitate thetissue proteins.Themixturewasallowedtostandfor5min prior to centrifugation for 10minat 1000×g. The supernatant (200l)wasmixedwith1.8mlofEllman’sreagent(5,5′-dithio bis-2-nitrobenzoicacid)(0.1mM)preparedin0.3Mphosphatebuffer with1%ofsodiumcitratesolution).Theabsorbanceofsolutionwas
estimatedat412nm.ThelevelofGSHwasexpressedasM/mg
tissue(Ellman,1959).
Statisticalanalysis
Alltheresultswere expressedasmean±standard deviation
(SD)andanalyzedbyone-wayandtwo-wayANOVAfollowedby
Bonferroni’smulti-comparisontestasposthoctestusing biostati-calsoftwareGraphpadprism(version5).p<0.05wasconsidered tobestatisticallysignificant.
Results
Fingerprintanalysisofvalericacid
Fingerprintanalysisshowedprominentpeaksat1737cm−1due tostretchingfrequencyofC Oofcarboxylic,at2858,2929cm−1 duetoC HstretchingfrequencyofC Handaprominentpeakat 12cm−1duetostretchingofC OHofCOOH,alongwithexpected peaksinthefingerprintregionindirectionofstretchingfrequency of(CH2)nandofCH3[e.g.1465cm−1](supplementarymaterial).
Effectofdrugtreatmentsonbehavioralparametersonmorris watermaze
Theacquisition of memoryin normal animalswasfoundto
beincreasedasreflectedbyprogressiveandsignificant(p<0.05) decreaseinescapelatenciesofanimalsfrom17thto19thdays. ICV-STZadministrationcausedsignificant(p<0.05)increaseinescape latencyon18th,19thand20thday,respectively,ascomparedto normaluntreatedrats.TreatmentwithV.wallichiiextract(100and 200mg/kg)andVA(20and40mg/kg)significantlydecreasedthe timeofescapelatency,ascomparedtoICV-STZcontrolrats fol-lowedon17th,18th,19thand20thday.Administrationofpicrotoxin significantly(p<0.05)abolishedtheresponseofVA40mg/kgand showedincreasedescapelatency,ascomparedtoVA40.TheFvalue foreachvariationfactorlikeinteraction,drugandtimewerefound tobe44.95,453.3and3138respectively(Fig.1).
Effectofdrugtreatmentsonbehavioralparametersonelevated plusmaze:
No significant change has been observed on elevated plus
mazeduringassessmentofITLafterinductionwithSTZ.ICVSTZ (3mg/kg)producedsignificant(p<.05)increaseinretention trans-fer latency,as compared to initialtransfer latency and normal control.TreatmentwithV.wallichiiextract100and200mg/kg,VA 20and40mg/kgproducedsignificant(p<0.05)decreasein reten-tiontransferlatency,ascomparedtoICVSTZcontrol.Theeffect producedbyVA-40(highdose)wassignificant(p<0.05)in improv-ingmemory,ascomparedtoV.wallichiiextract.Theeffectproduced
0 20 40 60 80
Escape latency (sec)
a
b b b
17th day
b b c
∗
a
b b b b
18th day
c
a b
19th day 20th day ∗∗
b b
b b c
Normal Sham control ICV-STZ control VW Ext.(200) VW Ext.(100) Valeric acid (40) Valeric acid (20) VA (40)+Picro(2)
a b b
b b c
Fig.1.Effectofdrugtreatmentsonbehavioralparametersonmorriswatermaze:results:mean±SD;(n=6);analyzedbytwowayANOVAfollowedbyBonferroni’stest whereasap<0.05vsNC,bp<0.05vsICVSTZ,andcp<0.05vsVA(40);*p<0.05vsnormalon17thdayand**p<0.05vsnormalon18thday.
a
b b b
b
RTL ITL
80
60
40
Sec
20
0
c
Normal Sham control STZ control VW Ext 200
VW Ext 100 VA-40 VA-20 VA (40)+Picro(2)
Fig.2. Effectofdrugtreatmentsonbehavioralparametersonelevatedplusmaze: results:mean±SD;(n=6);analyzedbytwowayANOVAfollowedbyBonferroni’s testwhereasap<0.05vsNC,bp<0.05vsICVSTZ,andcp<0.05vsVA(40).
significant,ascomparedtoVA-40andinsignificanttoICV-STZ con-trol(Fig.2).
Effectofdrugtreatmentsonlipidperoxidationlevel
InductionwithICVSTZ(3mg/kg)producedsignificant(p<0.05) increaseinbraintissuelipidperoxidationlevel,ascomparedto nor-maluntreatedrats.However,V.wallichiiextract100and200mg/kg, VA20and40mg/kgproducedsignificantreversalbydecreasing thelevelofbrainlipidperoxidation,ascomparedtoSTZcontrol (p<0.05).Treatmentwithpicrotoxin(2mg/kg)showedsignificant increaseinlipidperoxidationreactionscharacterizing neurodegen-eration,ascomparedtoVA40(Fig.3).
Effectofdrugtreatmentsontissueglutathionelevel:
InductionwithICVSTZcausedsignificant(p<0.05)decreasein tissueGSHlevel,ascomparedtonormaluntreatedrats.However,V. wallichiiextractindoses100and200mg/kg,VA(20and40mg/kg) producedsignificant(p<0.05)reversalintissueGSHlevel,as com-paredtoICVSTZcontrol.Theeffectproducedbypicrotoxinwas significant,ascomparedtoVA40andshoweddecreaselevelof reducedglutathione(Fig.4).
Discussion
Thefindingsfrompresentinvestigationrevealedthe GABAer-giceffectofvalericacidfromV.wallichiiinameliorationofICV-STZ inducedneurodegenerativedementiainrats.Extractionofdried
rhizome of V. wallichii wasdone by percolation methodusing
dichloromethane.Valericacid,achiefconstituentpresentinV. wal-lichii,identifiedbyFT-IRandyieldwasfoundtobe0.8%w/vin presentstudy.
ICV-STZadministrationis widelyusedmodelfor speculating
theefficacyofneuroprotectiveagentsandtoexplore
pharmaco-logicalinterventionsunderlyingneurodegenerationandmemory
deficitsintheformofdementiaasbotharecharacterizedby
pro-gressive deterioration of memory, cerebral glucose and energy
metabolism, and production of oxidative stress (Prakash et al., 2015).STZ(3mg/kg)wasgivenviaintracerebroventricularroute toinduceneuronaldamagewithoutalteringperipheralblood glu-coselevel.ICV-STZisreportedtocauseanincreaseintauprotein
and amyloid-inbrain, therebygeneratinginflammatory
reac-tionsandfreeradicalsandultimatelyresultscelldeath,apoptosis
anddiminutionoflearningandmemoryinexperimentalanimals
(Weerateerangkulletal.,2008).Theanimalsofboththesexeswere usedinpresentstudywithanideathatthediseaseisprevalentin both.Thefactthatestrogenisknowntoimprovememoryistaken carebyequallydistributingthemaleandfemaleratsinallgroups, includingthatofcontrol.
The performance of animals during behavioral assessments
for spacial memoryusing morris water mazeand for memory
cognition using elevated plus maze are well documented to
estimateextent of neuronal injury (Sachdevaet al.,2014).The performanceinnormalandshamcontrolgroupsweresignificantly increasedastheanimalsshowedprogressivedecreaseinescape
latencies during assessments from 17th to 19th day. ICV-STZ
treatmenttoratsshowedsignificantincreaseinescapelatencyon morriswatermazeandretentiontransferlatencyonelevatedplus
mazecharacterizingimpairmentinlearningandmemorydueto
neurodegenerationwhichareinconfirmationtoearlierliterature (Tiwarietal.,2009).Thissignifiestheincreasedneuronaldamage, reactiveoxygenspeciesandtherebyoxidativestress,amyloid-beta
deposition and execution of inflammatory cascades resulting
15
10
Lipid pero
xidation (nM/mg tissue) 5
0
Normal Sham control
STZ control
VW Ext (200)
VW Ext (100)
VA (40) VA (20) c
b
b b
b a
VA (40)+ picro (2)
Fig.3. Effectofdrugtreatmentsonlipidperoxidationlevel:results:mean±SD;(n=6);analyzedbyonewayANOVAfollowedbyBonferroni’stestwhereasap<0.05vsNC, bp<0.05vsICVSTZ,andcp<0.05vsVA(40).
40
30
GSH (uM/mg tissue)
20
0 10
Normal Sham control
STZ control
VW Ext (200)
VW Ext (100)
VA (40) VA (20) b
c b
b b
a
VA (40)+ Picro (2)
Fig.4. Effectofdrugtreatmentsontissueglutathionelevel:results:mean±SD;(n=6);analyzedbyonewayANOVAfollowedbyBonferroni’stestwhereasap<0.05vsNC, bp<0.05vsICVSTZ,andcp<0.05vsVA(40).
latency, attenuated oxidative and inflammatory reactions and
thereby ameliorated ICV-STZ induced neurodegeneration. This
effectofplantextractmaybeduetothepresenceofVA.These find-ingwereconfirmedbytheresultsobtainedingroupstreatedwith picrotoxin2mg/kg,aGABA-AantagonistfollowedbyVA-40mg/kg
administration as it showed increased RTL and escape latency
whichinferredthatblockageofGABAreceptorsbypicrotoxinand
makesVAunabletocorrectICV-STZinducedneurodegeneration
inratsandtherewasnoimprovementinmemory.
TheoxidativestresscausedbyICVSTZadministrationincreases lipidperoxidationanddecreasesGSHlevel.Lipidperoxidation,a measureoffreeradicalgenerationwasincreasedonday21inbrain ofratstreated withICV-STZ.Furtherproductionoffree radicals causedsimultaneousdecreaseinglutathionelevels.Glutathione isanessentialtripeptide,anantioxidantfoundinallanimalcells. Itreactswiththefreeradicals,neutralizethemand protectthe cellsfromsingletoxygen,hydroxylradicalandsuperoxideradical damage.TreatmentwithV.wallichiiextract(100and200mg/kg) andVA (20 and 40mg/kg)significantly reduced lipid peroxida-tionreactionsandrestoredantioxidantdefensesystemofcell.The pathophysiologicalprocessesofoxidativestressinchronic condi-tionsarelinkedwithalsoattributedwiththeupstreamregulation
ofGABAinneuronalcells whichwasblockedbyadministration
ofpicrotoxinandhenceshowedincreasedlipidperoxidation reac-tionsandloweredglutathioneinrats.ThepropertyofV.Wallichi
forscavengingfreeradicalsisalsoreported(SudhanshuRaoetal., 2012).
GABAis a majorinhibitory neurotransmitter in mammalian
cerebralcortex.Temporalcortexisanareagreatlyaffectedby neu-ropathichallmarksofneurodegeneration.Itdecreases13–17%of benzodiazepinebinding,whichsuggeststhatthereisadecrease ofGABAARs inAD(Limonetal.,2012).VAelevatesGABAlevels throughtheinactivationof␣-ketoglutaratedehydrogenase(Luder etal.,1990).ItdecreasesdegradationofGABAmediatedbyGABA transaminaseandthusinhibitsGABAcatabolism.Itenhancesthe responseofGABA-Areceptor(Montietal.,2009)andthusactsas GABA-Aagonist(Frumkes andNelson, 1995).Thefindingsfrom presentinvestigationmayconcludethatV.wallichiiandits con-stituentvalericacidhassignificantneuroprotective potentialto amelioratememorycognitionandretentionpossiblythroughGABA receptormodulationinrats.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare
that the procedures followed were in accordance with the
Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthe publicationof patientdata.
Right to privacy and informed consent. The authors have
obtainedthewritteninformedconsentofthepatientsorsubjects mentionedinthearticle.Thecorrespondingauthorisinpossession ofthisdocument.
Author’scontribution
SVandRGdesignedthestudy.SVandVGhaveperformedthe
experiments.RGandSVinterpretedtheresearchfindings.SVand KLDworkedforisolationofValericacidandcharacterization.RG andSVdraftedthemanuscript.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgement
TheauthorsarethankfultoShooliniUniversity,Solan,for pro-vidingresearchfacilities.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.02.008.
References
Barnes,J.,2002.HerbalMedicines.PharmaceuticalPress,London.
Chauhan,N.S.,2006.ImportantMedicinalandAromaticPlantsofHimachalPradesh, 2nded.IndusPublishingCompany,NewDelhi.
Chen,S.,Zheng,J.C.,2012.Translationalneurodegeneration,aplatformtoshare knowledgeandexperienceintranslationalstudyofneurodegenerativediseases. Transl.Neurodegener.1(1),http://dx.doi.org/10.1186/2047-9158-1-1. Cole,S.L.,Vassar,R.,2007.TheAlzheimer’sdisease-secretaseenzyme,BACE1.Mol.
Neurodegener.22(2),http://dx.doi.org/10.1186/1750-1326-2-22. Ellman,G.L.,1959.Tissuesulphydrylgroups.Arch.Biochem.Biophys.82,70–77. Fratiglioni,L.,Winblad,B.,Strauss,E.V.,2007.PreventionofAlzheimer’sdiseaseand
dementia.MajorfindingsfromtheKungsholmenProject.Physiol.Behav.92, 98–104.
Frumkes,T.E.,Nelson,R.,1995.FunctionalroleofGABAincatretina:I.Effectsof GABAAagonists.Vis.Neurosci.12,641–650.
Gendron,T.F.,Petrucelli,L.,2009.Theroleoftauinneurodegeneration.Mol. Neu-rodegener.13(4),http://dx.doi.org/10.1186/1750-1326-4-13.
Gruenwald,J.,2004.PDRforHerbalMedicines.MedicalEconomics,Montavale,pp. 852–856.
Hirtz,D.,Thurman,D.J.,Gwinn-Hardy,K.,Mohamed,M.,Chaudhuri,A.R.,Zalutsky, R.,2007.Howcommonarethe“common”neurologicdisorders?Neurology68, 326–337.
Houghton,P.J.,1988.Reviewofpharmacologyofconstituentsandmedicinaluses.J. Ethnopharmacol.22,121–142.
Katzman,R.,2008.TheprevalenceandmalignancyofAlzheimerdisease:amajor killer.AlzheimersDement.4,378–380.
Kokate,C.K.,Purohit,A.P.,Gokhale,S.B.,2007.Pharmacognosy.NiraliPrakashan, NewDelhi.
Kuller,L.H.,Lopez,O.L.,2008.AlzheimersDement.Isittimeforachangeinfocus? Dementia4,S77–S84.
Leathwood,P.D.,Chauffard,F.,1985.Aqueousextractofvalerianreduceslatencyto fallasleepinman.PlantaMed.51,235–262.
Limon,A.,Reyes-Ruiz,J.M.,Miledi,R.,2012.LossoffunctionalGABAAreceptorsin theAlzheimerdiseasedbrain.PNAS109,10071–10076.
Loeb,C.,Patrone,A.,Besio,G.,Balestrino,M.,Mainardi,P.,1990.Theexcitatory aminoacidantagonistamino-phosphono-valericacid(APV)providesprotection againstpenicillin-inducedepilepticactivityintherat.EpilepsyRes.6,249–251. Luder,S.A.,Parks,K.J.,Frerman,F.,Parker,D.W.,1990.Inactivationofbeefbrain ␣-ketoglutaratedehydrogenasecomplexbyvalproicacidandvalproicacid metabolites.J.Clin.Invest.86,1574–1581.
Majdi,M.,Chen,H.S.V.,2009.2NMDA-gatedionchannelresearchanditstherapeutic potentialsinneurodegenerativediseases:areview.J.Recep.LigandChannelRes. 2,59–73.
Marder,M.H.,Viola,C.,Wasowski,S.,Fernandez,J.H.,2003.6-Methylapigeninand hesperidin:newvalerianaflavonoidswithactivityontheCNS.Pharmacol.Biol. Behav.75,537–545.
Monti,B.,Polazzi,E.,Contestabile,A.,2009.Biochemical,molecularandepigenetic mechanismsofvalproicacidneuroprotection.Curr.Mol.Pharm.2,95–109. Nadkarni,K.M.,1976.IndianMedica,3thed.PopularPrakashan,Bombay. Ohkawa,H.,Ohishi,N.,Yagi,K.,1979.Assayforlipidperoxidesinanimaltissuesby
thiobarbituricacidreaction.Anal.Biochem.95,351–358.
Panijel,M.,1985.Treatmentofmoderatelysevereanxietystates.Therapiewoche35, 4659–4668.
Prakash,A.,Kalra,J.K.,Kumar,A.,2015.NeuroprotectiveeffectofN-acetylcysteine againststreptozotocin-inducedmemorydysfunctionandoxidativedamagein rats.J.BasicClin.Physiol.Pharmacol.26,13–23.
Pokorski,R.J.,2002.Differentiatingage-relatedmemorylossfromearlydementia.J. Insur.Med.34,100–113.
Rissman,R.A.,Mobley,W.C.,2011.Implicationsfortreatment:GABAAreceptorsin aging,DownsyndromeandAlzheimer’sdisease.J.Neurochem.117,613–622. Ron,B.H.,Willis,C.V.,Bone,K.,Morgan,M.,2000.Herbalproducts:active
con-stituents,modeofactionandqualitycontrol.Nutr.Res.Rev.13,47–77. Sachdeva,A.K.,Kuhad,A.,Chopra,K.,2014.Naringinamelioratesmemorydeficits
inexperimentalparadigmofAlzheimer’sdiseasebyattenuatingmitochondrial dysfunction.Pharmacol.Biochem.Behav.127,101–110.
Sah,S.P.,Mathela,C.S.,Chopra,K.,2010.Valerianawallichii:aphyto-pharmacological review.J.Pharm.Res.3,2337–2339.
Sattigeri,B.M.,Balsara,J.J.,Jadhav,J.H.,2012.Effectofpicrotoxinandcyproheptadine pretreatmentonsodiumvalproateinducedwetdogshakebehaviorinrats.Int. J.Biol.Med.Res.3,1606–1608.
Schneider,L.S.,Dagerman,K.S.,Insel,P.,2005.Riskofdeathwithatypical antipsy-choticdrugtreatmentfordementia.JAMA294,1934–1943.
Subhan,F.,Karim,N.,Gilani,A.H.,Sewell,R.D.E.,2009.TerpenoidcontentofValeriana
wallichiiextractsandantidepressant-likeresponseprofiles.Phytother.Res.24,
686–691.
Tiwari, V., Kuhad, A., Bishnoi, M., Chopra, K., 2009. Chronic treatment with tocotrienol, an isoform of vitamin E, prevents intracerebroventricular streptozotocin-inducedcognitiveimpairmentandoxidative-nitrosativestress inrats.Pharmacol.Biochem.Behav.93,183–189.
SudhanshuRao,N.,Mittal,S.,Meghani,E.,2012.Evaluationofantioxidant
proper-tiesofValerianawallichiitoscavengefreeradicals.AsianJ.Pharm.Clin.Res.5,
238–240.
Vohora,S.B.,Dandiya,P.C.,1992.Herbalanalgesicdrugs.Fitoterapia63,195–207. Weerateerangkull,P.,Praputpittaya,C.,Banjerdpongchai,R.,2008.Effectsofascorbic
acidonSTZinducedoxidativestressandmemoryimpairmentinrats.J.Phys. Sci.20,54–60.
