全 文 ：AnticonvulsiveactionofPineliaPedatisectaSchotextractprepared
byethanol-modifiedsupercriticalCO2 extraction
CHENJing-Jing, YANGRong, WANGMing-Zheng＊ , CHENGYin-Xia, HEXin-Gu,
MAYong-Gang, YANGLi-Hua, HEQiao-Yan
(DepartmentofPharmacology, ShanxiMedicalUniversity, Taiyuan 030001, China)
Abstract:AIM Toinvestigatetheanticonvulsiveac-
tionofsupercriticalCO2 ethanolextractfromPineliaPedatisectaSchot(SEE-CO2PP).METHODS Theratconvulsivemodelwasinducedbypenicilinlocal-
izedinjectedinratcortex.TheefectsofSEE-CO2PP
onthelatencyofseizureandchangesofconvulsivebe-
haviorswereinvestigated.Thelatencyofepileptiform
discharge, andfrequencyandamplitudeofhighest
waveincortexandhippocampuswererecordedby
usingRM6240Cmultichannelphysiologicalsignalcol-
lectionandanalysisrecorder.Atthesametime, the
contentsofglutamicacid(Glu), asparticacid(Asp),
glycine(Gly)andγ-aminobutyricacid(GABA)in
hippocampusweredeterminedwithhighperformance
liquidchromatography.RESULTS SEE-CO2PP15
and30 g·kg-1 , ig, prolongedthelatentperiodofsei-
zureandweakenedtheextent.SEE-CO2PPalsopro-longedthelatentperiodofepileptiformdischarge, re-
ducedthefrequencyanddecreasedamplitudeofthe
highestwaveinbothcortexandhippocampus.Moreo-
ver, SEE-CO2PPincreasedthecontentofGABAinhippocampus, butthelevelsofGly, AspandGluhad
noobviouschanges.CONCLUSION SEE-CO2PPinhibitstheepileptiformdischargeandconvulsivebe-
haviorsofconvulsivemodelrats, whichsuggestsSEE-
CO2PPhasanticonvulsiveaction.Keywords:PineliaPedatisectaSchot;penicilin;
convulsions
CLCnumber:R285.5
Documentcode:A
ArticleID:1000-3002(2007)06-0449-06
Receiveddate:2006-12-06 Accepteddate:2007-11-08
Foundationitem:TheprojectsupportedbyNaturalSci-
enceFoundationofShanxiProvince(20041109)
Biographies:CHENJing-Jing(1982 -), female, post-
graduate;WANGMing-Zheng(1952 - ), male, professor,
mainresearchfieldisneuropharmacology.
＊Correspondingauthor. E-mail:wang5693693@163.com
Tel:(0351)4962091
PinelliaPedatisectaSchot(PPS)isoneof
thecommonlyantiepilepticdrugsinthepre-
scriptionoftraditionalChinesemateriamedica.
StudiesshowedthattheextractofPPSincold
water(30℃)couldsignificantlyantagonize
convulsioninmiceinducedbystrychnine, and
lowerdownmortalityofconvulsantmice, but
hadnoeffectonmaximalelectroshockmod-
el[ 1] .YetitisstilunknownwhetherPPScould
antagonizethepenicilin(PNC)-inducedsei-
zure.Thisstudyinvestigatedtheinfluenceof
supercriticalCO2 ethanolextractfrom PPS
(SEE-CO2PP)onthePNC-inducedseizureandepileptiformdischarge.Meanwhilethecontents
ofneurotransmitersrelatedtoepilepsyinhippo-
campusweredeterminedbyhighperformance
liquidchromatography(HPLC)inordertoex-
ploretheanticonvulsivemechanism ofSEE-
CO2PP.
1 MATERIALSANDMETHODS
1.1 Animals
FortyWistarratsofbothsexesweighing
180-220 gandaged10-12 weeks(Animal
Center, ShanxiMedicalUniversity, China)
wereused.Allanimalswereindividuallykept
inanaturallight-darkcycleandalhadfreeac-
cesstofoodandwateruntilabout12 hbefore
theexperiment.Duringtheexperiment, thela-
boratorywaskeptquiet.
1.2 Drugs, reagentsandinstruments
PPSwasfromMedicalMaterialCompanyof
ShanxiProvince, anditsmaincomponentsare
saponins, aminoacids, alkaloids, etc[ 2] .Itwas
processedwithethanol-modifiedsupercritical
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2007年 12月;
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CO2 extractionmethodbyDepartmentofPhyto-
chemistry, ShanxiMedicalUniversity, byusing
HA221-50-06 supercriticalityextractioninstru-
ment(Hua′anSupercriticalityExtractionLimit-
edCompany, Nantong, JiangsuProvince, Chi-
na).PPS(5kg)wascrushedandthenextracted
withfluidCO2 for3 hataflowrateof200 mL·
min-1 undertheconditionsof33 MPaand
70℃.70% ethanolwasaddedintotheextrac-
tionmixtureataflowrateof10 mL·min-1
throughouttheprocess.SEE-CO2PPwasidenti-fiedthatthecontentoftotalalkaloidswas
12.64% byspectrophotometricmethod.The
extractwasdissolvedwithsalodoilandstored
inrefrigeratorforuse.ThedosesofSEE-CO2PPwereexpressedasthecorrespondingquantityof
crudematerial, whichcalculatedaccordingto
thequantityratioofSEE-CO2PP tocrudematerial.
PNCwasfrom NorthernChinaMedical
LimitedCompany.TritonX-100 wassupplied
byShanghaiBiologyEngineeringLimitedCom-
pany.Acetonitrile, 2-aminoethylisothiouronium
bromide(AET), pyridoxal-5-phosphate(PLP)
andhomoserinewereobtainedfromSigmaCom-
pany.JiangwanⅠ-Cbrainstereotacticapparatus
wasfrom ChuanshaHuamuAgriculturalMa-
chineFactory, Shanghai, China;RM6240C
multichannelphysiologicalsignalcolectionand
analysisrecorderwasfromChengduInstrument
Factory, Chengdu, China;Beckman344HPLC
system from BeckmanInstrumentCompany,
USA;LDZ4-0.8 automaticequilibriumcentri-
fugefromBeijingClinicalCentrifugeFactory,
China;andTGL-168highspeeddeskcentrifuge
wasformAntingScientificInstrumentFactory,
Shanghai, China.
1.3 Preparationofpenicilinkindlingrat
model
Ratswererandomlydividedinto5 groups:
normalcontrolgroup, ignormalsaline(NS);
modelgroup, igNS;solventcontrolgroup, ig
salodoil;SEE-CO2PP15and30g·kg-1 groups,igSEE-CO2PP, eightratspergroup, onceaday.Ond2 afterthefinaladministrationofSEE-
CO2PP, altheratswereanesthetizedwithure-
thane(1 mg·kg-1 , ip), andplaced2 stainless
steelelectrodesrespectivelyinthefrontalcortex
(sensorimotorcortex:posterior, 3.0 mm;left,
2.0 mm;height, 0.5 mmfrombregma)and
hippocampus(posterior, 3.8mm, left, 2.0mm,
height, 2.6mm)accordingtothebrainatlasof
Konig, and connected the electrode with
RM6240Cmultichannelphysiologicalsignalcol-
lectionandanalysisrecorder.Whenelectroen-
cephalogram(EEG)displayedsharp, spike, or
sharp(orspike)-slowwavecomplexafter400U
PNCinjectedinsubdural(sensorimotorcortex)
withmicrosyringe, itwasshowedthatthemodel
waspreparedsuccessfuly.
1.4 Electroencephalogramrecordingmethod
ElectrodewasconnectedwithRM6240C
multichannelphysiologicalsignalcolectionand
analysisrecorderafterinsertedintofrontallobe.
Thecerebellarelectronicsignalwasinputedand
itsspectralanalysiswasmadewithRM6240C
software.TheEEGrecordingparametersin-
cludedcollectionfrequency1 kHz, scanning
speed800 msperdivision, sensitivity1 mV,
time0.2s, filterwavefrequency10Hzandlow
passfiltering15 Hz.ThenormalEEGwasob-
servedfor10 minandrecordedfor1 -2 min
(excludingtheinfluenceofoperatedtrauma).
AfterPNCwasgiven, EEGwascontinuously
observedfor5min, thenrecordedfor1minev-
ery5minuntilto1 h.Meanwhile, theratbe-
haviorswereobserved.
1.5 Observedindexes
Theseizurestageswereassignedaccording
tothemodifiedversionofRacine[ 3] .Stage0:
noreactionorbehavioralarrest;stageⅠ :rhyth-
micalticofmouthorjaw;stageⅡ:noddingor
flickingtail;stageⅢ:unilaterallimbclonus;
stageⅣ:multi-limbsclonus;stageⅤ:general-
izedrigidity-clonusseizure.AfterPNCgiven,
ratbehavingwascontinuouslyobservedfor1 h
includingepilepticextentandepilepticlatency
(thetimeelapsedfromPNCadministrationto
themomentofwhiskertwitchingforthefirst
time).StagesⅤ -Ⅵ seizurewasregardedas
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severeseizure, andstages0, Ⅰ , Ⅱ andⅢ
seizurewereasmildone.
Itwasjudgedasepileptiform discharge
whenEEGshowedsharp, spike, orsharp(or
spike)-slowwavecomplex.Thelatencyofepi-
leptiformdischarge, frequency(min-1)andam-
plitudeofthehighestwave(mV)wereob-
servedandrecorded.Theaveragefrequencyof
epileptiformdischargeperminutewascounted
withthemeanofnumbersofsharporspike
wavesofdecakisfor1min.
1.6 Determinationofasparaginicacid
(Asp), glutamicacid (Glu), glycine
(Gly)andγ-aminobutyricacid(GABA)
contentsinhippocampus
Theratsweresacrificed, andthehippo-
campuswasseparatedandstoredat-70℃ 1h
afterPNCwasinjectedinsubduralspace.PLP
(1mmol·L-1)2 mLwasaddedtothemixture
of28.1mgofAETand100μLofTritonX-100,
thenphosphatebuffer(pH7.0, 0.1mol·L-1)
wasgiventothefinalvolumeof100 mL.The
abovesolutionwasstoredat4℃.Braintissue
0.1 gwasthrewintotheaboveprecooleddis-
posalsolutionintheratio1∶50(W/V)andho-
mogenizedinthemixedwaterwithice.The
Asp, Glu, GlyandGABAcontentsweredeter-
minedwithHPLC.ChromatographicColumn:
diamonsilTMODS4.6mm×150 mmmaintained
at35℃;percolumnC18 4.6 mm×30 mm;
flow-rate:1.0 mL·min-1 , fluorometricdetec-
tion.Thestandardcurvewasdrewasfollows:a
seriesofstandardsolutionswithstandardprepa-
ration250, 125, 62.5 and25 μmol·L-1 was
arayed, then10 μLofeachsolutionwasinjec-
tedintotheHPLCsystem.Thepeakareaof
eachconcentrationwasdetermined, thestand-
ardcurvewasdrewandtheregressionequation
workedout.Asp:Y=9.54X+014, r=
0.9977;Glu:Y=5.64X+0.0744, r=
0.9985;Gly:Y=3.18X+0.107, r=0.9976;
GABA:Y=2.98X+0.117, r=0.9961.
1.7 Statisticalanalysis
Statisticalanalysiswascarriedoutwith
SPSSsoftware.Thedatawereexpressedas
x±s.Diferencesinseizurelatency, epilepti-
formdischargelatency, frequencyofepilepti-
formdischargeandamplitudeofhighestwaveof
cortexEEGwereevaluatedbyusingone-way
ANOVA.Theextentofconvulsionwasevalua-
tedusingχ2 test.
2 RESULTS
2.1 EffectsofSEE-CO2PPonconvulsionand changesofelectroencephalogram in
PNC-inducedrats
Insolventcontrolandmodelgroups, al
ratshadstageⅤ seizureafterPNCadministration
andlasteduntiltheendoftheexperiment.In
SEE-CO2PP15 g·kg-1 group, 7 ratshadmildseizureand1 ratkeptnormalbehavior;among
the7rats, only1developedstageⅤ seizureand
theseizurelastedabout40min.InSEE-CO2PP
30g·kg-1 group, 4ratshadmildseizureandno
onedevelopedsevereseizure.Theseizuredura-
tionwasapproximatelyto20min(Tab1).
Tab1. EfectsofsupercriticalCO2 ethanolex-tractfrom Pinelia Pedatisecta Schot(SEE-
CO2PP)onseizurelatencyinpenicilin(PNC)-inducedrats
Group Dose/g·kg-1 Seizurelatency/s
Normalcontrol
Model 64±11
Solventcontrol 64±11
SEE-CO2PP 15 145±24＊＊
30 162±42＊＊
SEE-CO2PP(SEE-CO2PPgroup), salodoil(solventcontrolgroup)ornormalsaline(controlandmodelgroups)wasgiven
ig, onceaday, for2 d.Ond2 afterthefinaladministration,
PNC400Uwasinjectedunderduramaterandtheseizurelaten-
cywasdetected. x±s, n=8.＊＊P<0.01, comparedwithmod-
elgroup.
Tab2 andFig1 showedtheefectsof
SEE-CO2PPoncortexandhippocampusEEGofratswithconvulsion.Fig1AandEwerenormal
EEGincortexandhippocampusrecordedbe-
foretheadministrationofPNC.Theepilepti-
formdischarge(spikeorsharpwave)appeared
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incortex(71±14)sandinhippocampus(63±
12)safterPNCadministration(Fig1BandF).
Inthecortex, themeanoffrequencyofepilepti-
formdischargewas(41 ±6)min-1 , andthe
mean amplitude ofthe highestwave was
(1.21±0.53)mV.Inthehippocampus, the
meanoffrequencyofepileptiformdischargewas
40±4)min-1 , andthemeanamplitudeofthe
highestwavewas(1.11±0.08)mV.Compared
withthemodelgroup, SEE-CO2PP15and30g·
kg-1 prolongedlatentperiodofepileptiformdis-
charge, reducedthefrequencyanddecreased
amplitudeofthehighestwaveincortex(Fig1C
andD)andhippocampus(Fig1GandH).
Tab2. EffectsofSEE-CO2PPonelectroencephalogramsincortexandhippocampusofPNC-inducedrats
Group Dose/g·kg-1
EDL/s
Cortex Hippocampus
FED/min-1
Cortex Hippocampus
HW/mV
Cortex Hippocampus
Model 71±14 64±12 41±6 40±4 1.21±0.53 1.11±0.08
Solventcontrol 71±14 64±12 42±5 41±3 1.19±0.62 1.09±0.09
SEE-CO2PP 15 136±28＊ 136±28＊ 29±7＊＊ 28±6＊＊ 0.74±0.22＊＊ 0.72±0.18＊
30 165±47＊＊ 164±46＊＊ 23±6＊＊ 23±5＊＊ 0.60±0.24＊＊ 0.59±0.28＊＊
SeeTab1 forthetreatments.EDL:epileptiformdischargelatency;FED:frequencyofepileptiformdischarge;HW:amplitudeofthe
highestwave. x±s, n=8.＊P<0.05, ＊＊P<0.01, comparedwithcorrespondingmodelgroup.
Fig1. RepresentativeresultsofexperimentinTab2.SeeTab1forthetreatments.A-D:cortex;E-H:hippocam-
pus.AandE:normalcontrol;BandF:model;CandG:SEE-CO2 PP15 g·kg-1;DandH:SEE-CO2 PP30 g·kg-1.
·452· ChinJPharmacolToxicol 2007 Dec;21(6)
Tab3. EffectsofSEE-CO2PPoncontentsofasparaginicacid(Asp), glutamicacid(Glu), glycine(Gly)andγ-aminobutyricacid(GABA)inhippocampusofPNC-inducedrats
Group Dose/g·kg-1
Asp
/μmol·g-1
Glu
/μmol·g-1
Gly
/μmol·g-1
GABA
/μmol·g-1
Normalcontrol 2.81±0.25 11.2±0.7 3.0±1.1 5.5±1.2
Model 2.97±0.49 14.3±0.8＊ 2.6±0.6 3.8±0.5＊＊
Solventcontrol 2.83±0.17 13.1±0.7 3.0±1.1 3.8±1.4
SEE-CO2PP 15 2.78±0.30 12.8±3.9 2.7±0.6 5.2±0.3#
30 2.78±0.26 12.7±1.1 2.8±0.3 5.6±0.5##
SeeTab1forthetreatments. x±s, n=8.＊P<0.05, ＊＊P<0.01, comparedwithnormalcontrolgroup;#P<0.05, ##P<0.01,
comparedwithmodelgroup.
2.2 EfectsofSEE-CO2PPoncontentsofAsp, Glu, GlyandGABAinhippocampus
ofPNC-inducedrats
Inmodelgroup, thecontentofGlusignifi-
cantlydecreased, GABAincreased, andAsp
andGlyhadnoobviouschangescomparedwith
thatofcontrolgroup.Comparedwithmodel
group, thecontentofGABA inbothdose
groupsofSEE-CO2PPsignificantlyincreased,andAsp, GluandGlyhadnoobviouschanges
(Tab3).
3 DISCUSSION
Epileptiform dischargeofPNC-induced
ratsissimilartothatofabsenceseizuresinhu-
man.PNCisanantagonistoftheGABAA-
benzodiazepinereceptorcomplex, andthrough
thereceptoritcanweakenthesynapticinhibi-
toryactivitiesorreinforcethesynapticexcitato-
ryactivities.Whenexcitatorypost-synapticpo-
tential(EPSP) colligationexceedsdefinite
threshold, paroxysmaldepolarization shifts
(PDS)develops.PDSisoneofthekeyfea-
turesofepilepticelectrophysiology, andthe
spikewaveinEEGisthereflectionofPDS.
ThedevelopmentofPDShasgreatrelationwith
calciumion[ 4-6] .Thedecreaseofextracelular
Ca2+ hasveryimportantefectonsynchroniza-
tiondifusionofepileptiformdischarge.Thein-
creaseofintracelularCa2+ leadstoceldepo-
larizationwhichdevelopsabnormaldischarge,
soepileptiformdischargeappearsandspreads
quickly.Moreover, intracelularCa2+ canin-
creasethereleaseofGluanditcanintensify
theviciouscycleofseizure[ 7] .Thepresent
studyshowedthatSEE-CO2PPprolongedthela-tentperiodofthePNC-inducedseizureand
weakentheextentofseizure.Atthesame
time, SEE-CO2PPprolongedlatentperiodofepileptiformdischarge, reducedthefrequency
ofepileptiformdischargeanddecreasedampli-
tudeofthehighestwaveofcortexEEG.Sothat
SEE-CO2PPexertstheanticonvulsiveaction.
Centralneurotransmitersincludeexcitato-
ryandinhibitorytransmiters.Theincreaseof
excitatorytransmitterand/orthedecreaseofin-
hibitorytransmitercanaltertheexcitatory-in-
hibitorybalanceofneuralnetwork, whichisthe
molecularbasisofepileptiform discharge[ 8] .
GABAisregardedasthemaininhibitorytrans-
miter.Dichter[ 9] reportedthatthedecreaseof
GABAhadintimaterelationwiththeproduction
andspreadofepilepsy.GluandAspareregar-
dedasthemainexcitatorytransmiters.Thein-
creasesofGluandAspleadtopostsynaptic
membraneoverexcitedthatalterstheexcitatory-
inhibitorybalanceofneuralnetwork, andepi-
leptiform dischargeappearsintheend[ 10] .
SEE-CO2PPsignificantlyincreasedGABAlevel
inhippocampus.Itshowedthattheantiepilep-
ticmechanism ofSEE-CO2PPmayberelatedwiththeincreaseofGABA content, which
·453·中国药理学与毒理学杂志 2007年 12月;21(6)
couldactivateGABAA receptorandeventualy
increaseCl-toenterintocellsandenhancethe
inhibitoryfunctionofGABA.
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掌叶半夏超临界 CO2 乙醇萃取物的抗惊厥作用
陈靖京 , 杨 蓉 , 王明正 , 成银霞 , 何新嘏 , 马勇刚 , 杨李华 , 何巧燕
(山西医科大学药理学教研室 , 山西 太原 030001)
摘要:目的 研究掌叶半夏超临界 CO2乙醇萃取物
(SEE-CO2PP)对青霉素诱发惊厥的对抗作用。方法
采用大鼠皮质局部定位注射青霉素诱发惊厥模型 ,
研究 SEE-CO2PP对惊厥发作的潜伏期以及惊厥行
为变化的影响 ,并用 RM6240C型多道生理信号采集
处理仪记录皮质和海马痫性放电的潜伏期 、频率和
痫波最高发放波幅 ,同时应用高效液相色谱法测定
海马谷氨酸(Glu)、天冬氨酸 (Asp)、甘氨酸(Gly)
和 γ-氨基丁酸(GABA)递质的含量。结果 SEE-
CO2PP15和 30g·kg-1(ig)可延长青霉素诱发惊厥
的潜伏期 ,并减弱发作强度。 SEE-CO2PP能够延长
痫性放电的潜伏期 ,减少痫性放电的频率 ,减小皮质
和海马发放痫波的最高波幅 ,同时 , SEE-CO2PP可
以增加海马 GABA的水平 ,对 Gly, Asp和 Glu水平
无明显影响。结论 SEE-CO2PP可对抗青霉素诱
发的惊厥行为和痫样放电 ,具有抗惊厥作用 。
关键词:掌叶半夏;青霉素;惊厥
基金项目:山西省自然科学基金资助项目(20041109)
(本文编辑 齐春会)
·454· ChinJPharmacolToxicol 2007 Dec;21(6)