全 文 ：水黄皮根乙醇提取物的抗炎镇痛作用及其急性毒性的实验研究(英文)
刘可云 1 , 朱 毅 2 , 董 志1 , 陈国彪 2 , 赵毓梅 2 , 李 备 2 , 刘 春 2 , 李 靖 1
(1.重庆医科大学药理学教研室 ,重庆 400016;2.海南省药品检验所 ,海南 海口 570216)
Receiveddate:2006-06-10
Foundation:SupportedbyNaturalScienceFoundationofHainanProvince(No.30530)
Introductiontoauthor:LIUKe-yun(1974 ～ ), male, masterofpharmacology, HubeiInstituteforNationalities.e-mail:liukeyunqiqi@yahoo.
corn.on
Correspondenceto:profZHUYi, Doctorofpharmacology, thetutorofMaster, theInstituteforDrugControlofHainanProvince, e-mail:hnzhuyi
@ 126.com.profDONGZhi, Doctorofpharmacology, thetutorofDoctor, ChongqingUniversityofMedicalSciences, e-mail:zhidong@ 126.com.
关键词:水黄皮根;抗炎;镇痛;急性毒性
摘要:目的:研究水黄皮根乙醇提取物的抗炎镇痛作用及其急性毒性。方法:抗炎实验采用二甲苯致小鼠耳肿胀法及大
鼠棉球肉芽肿法;镇痛实验采用小鼠热板和醋酸扭体实验;采用 Bliss法测定了其半数致死量(LD50)。结果:PRE可明显
抑制二甲苯所致小鼠耳廓的肿胀度 ,抑制大鼠棉球肉芽肿的增长;可明显对抗醋酸所致小鼠的扭体次数增加 ,提高热板
所致小鼠的痛阈值;其 LD50为 6.371 8 g/kg, LD50的 95 平均可信限为 5.408 4 ～ 7.723 2 g/kg。结论:水黄皮根乙醇提取
物具有明显抗炎镇痛作用 ,并且其急性毒性很小。
中图分类号:R285.6 文献标识码:A 文章编号:1001-1528(2007)02-0179-05
Experimentalstudyonanti-inflammationandanalgesiaeffect
andacutetoxicityofextractfromPongamiapinnataroots
LIUKe-yun1 , ZHUYi2 , DONGZhi1 , CHENGGuo-biao2 , ZHAOYu-mei2 , LIBei2 , LIUChun2 , LI
Jin1
(1.DepartmentofPharmacology, ChongqingUniversityofMedicalSciences, Chongqing400016 , China;2.DepartmentofPharmacology, theInstitutefor
DrugControlofHainanProvince, Haikou570216 , China)
KEYWORDS:Pongamiapinnataroot;anti-inflammation;analgesia;acutetoxicity
ABSTRACT:AIM:TostudytheefectofextractfromPongamiapinnatarootsonanti-inflammationandanalgesia
andacutetoxicity.METHODS:ThemodelsofmiceearedemainducedbyxyleneandCotonpeletgranulomain
ratstoobservetheanti-inflammationefectofPREviaoraladministration.TheefectofPREonanalgesiawastest-
edbymeasuringthelatentperiodlickinghindfootwiththehotplatemethodandcountingbodytwistinginducedby
aceticacidinmice.TheacutetoxicityofPREwasmeasuredbythemethodofBliss.RESULTS:PREcouldsig-
nificantlyinhibittheearedemacausedbyxyleneinmice, granulomahyperplasiacausedbycotoninrats.Itcould
significantlyprolongthepainthresholdonhot-plateinmice, reducethewrithingtimesinmice.TheLD50 ofPRE
was6.371 8 g/kg, its95 confidentlimitwas5.408 4-7.723 2 g/kg.CONCLUSION:PREhasobviousefect
onanti-inflammationandanalgesiaandtheloweracutetoxicity.
1 Introduction
Pongamiapinnata(Linn)Pierre(Leguminosae,
Papilionaceac;synonym, PongamiaglabraVent), isa
mediumsizedglabroustree, foundthroughoutIndia
andGuanxi, HainanProvinceofChina.Theseedoilis
usedastraditionalChinesemedicinetotreatvarious
ailments, suchassarcoptidosis, rheumatism, running
soreetc[ 1] .Inflammatorydiseasesincludingdiferent
typesofrheumaticdiseasesareverycommonthrough-
outtheworld.Althoughrheumatismisoneoftheoldest
179
2007年 2月
第 29卷 第 2期
中 成 药
ChineseTraditionalPatentMedicine
February2007
Vol.29 No.2
knowndiseasesofmankindafectingthemajorityof
population, nosubstantialprogresshasbeenmadein
achievingapermanentcure.Thegreatestdisadvantage
inpresentlyavailablepotentsyntheticdrugsliesin
theirtoxicityandreappearanceofsymptomsafterdis-
continuation.Therefore, thescreeninganddevelop-
mentofdrugsfortheiranti-inflammatoryandanalgesia
activityisstilinprogressandthereismuchhopefor
findinganti-inflammatoryandanalgesiadrugsfromin-
digenousmedicinalplants.Theanalgesiaandanti-in-
flammationactivityandacutetoxicityonPongamiapin-
natarootsstilremainunexplored.The70 ethanolic
extractofP.pinnataroots(PRE)wasevaluatedina-
cute, chronicmodelsofinflammationandanalgesiaac-
tivity.Inaddition, theacutetoxicityofmicewasalso
studiedtoevaluateitstoxicity.
2 MaterialsandMethods
2.1 Experimentalanimals
Experimentswereperformedusingkunminalbino
mice(18.2±2.1)gandSDrats(180 ±20)g, pro-
curedfromtheLaboratoryAnimalResourceSectionof
GuangdongProvince(China), thecertificatenumber
isSCXK(Yue)2003-0002.Altheanimalsweremain-
tainedunderanairconditionedenvironment(room
temperature(22±2)°C.humidity(55±15) with
12 hlight, 12 hdarkcycle.Micewerefreeaccessto
waterandfoodandhousedincolonycages(fiveani-
malspercage).Altheanimalswereacclimatizedto
thelaboratoryenvironmentfor5 dbeforetheexperi-
ment.Tenanimalspergroupcomprisingoffivemales
andfivefemales, wereusedineachexperiment, un-
lesotherwisespecified.Theanimalswerefastedover-
nightjustpriortotheexperimentbutalowedfreeac-
cesstodrinkingwater.
2.2 Plantmaterialandpreparationofethanolicex-
tract
PREweretheextractofPongamiapinnataroots
thatwerecolectedinHaikou(HainanProvince, Chi-
na)inMarch20 , 2006 andidentifiedbyGuobiao
Chen, atraditionalChinesedrugspecialistintheInsti-
tuteforDrugControlofAinanProvince, China.A
voucherwasdepositedattheInstituteforDrugControl
ofHainanProvince, China.ThepowderofPongamia
pinnataroots(2.410 kg)wasrecirculatedthreetimes
with70 ethanolat60°C, 2heachtime, theethanol
mixtureswerefilteredandconcentratedunderreduced
pressureat60°Cbytheinstrumentofturningevapora-
tion.Theextractwas0.259 kg(10.75 ofthestart-
ingmaterial).PREandstandardreferencedrugwas
dissolvedinamedium[ 5 gCMC-Na, (GuoyaoChem-
icalCompany, batchnumberisF2005-718)dissolved
in1 000.0 mLdistiledwater] tomakeupthesuspen-
sion.Aldrugormediumwereadministeredoralyto
miceindosevolumeof20mL/kgbodyweight, andto
ratwere15mL/kg.Thesuspensionwaspreparedever-
ytimejustpriortoadministration.
2.3 Acutetoxicitystudy(oral)
AccordingtothemethodofBliss[ 2] , basedonthe
resultsobtainedfrompreliminarytoxicitystudy, thele-
thaldose(100 )is10.0 g/kgandthesurvivaldose
(100 )is2.0g/kg.ThePREwereadministratedo-
ralyingradeddosesof10.0, 7.5, 5.63 , 4.22, 3.16
and2.37 g/kg(theratiois1:0.75)tosixdiferent
groupsofmice.Algroupswereadministratedoraly
onlyonetime, thencloselyobserveredforanyabnor-
malortoxicmanifestationandformortalityupto14 d.
AccordingtothemethodofBliss, usingBlisssoftware
forcalculationtocalculatethehalflethaldose(LD50).
2.4 Anti-inflammatoryactivty
2.4.1 StudyofPREonacuteinflammation[ 2]
2.4.1.1 EfectofPREonxylene-inducedearedema
Inthistest, fiftymaleandfemalemiceweredi-
videdintofivegroupsequalyaccordingtorandom
numbertable.Basedontheresultsofacutetoxicity,
thedosesforfutherpharmacologicalstudieswerefixed
tobe0.21 , 0.63, 1.89g/kginmice(p.o.).Theoth-
ergroupadministed(p.o.)0.002 g/kgDexametha-
sone(DXM)wasusedaspositivecontrolandthesame
volumeofmediumasnormalcontrol, onceperday, for
5 dcontinuously.Sixtyminutesaftertheseventhad-
ministration, 0.05mLofxylenewasappliedtothesur-
faceoftherightear.Xyleneapplicationmicewere
kiled2 hlaterandbothearswereremoved.Circular
sectionsweretakenbyacorkborerwithadiameterof
8 mm, andweighted.Theincreaseinweight(Ear
sweling)causedbytheiritantwasmeasuredbysub-
tractingtheweightoftheuntreatedleftearsectionfrom
thatofthetreatedfightearsections.
180
2007年 2月
第 29卷 第 2期
中 成 药
ChineseTraditionalPatentMedicine
February2007
Vol.29 No.2
2.4.2 StudyofPREonchronicinflammation
2.4.2.1 Cotonpeletgranulomainrats.
Autoclavedcotonpeletsweighing(30 ±1)mg
eachwereimplantedsubcutaneouslythroughsmalin-
cisionmadealongtheflankregionoftheratsanesthe-
tizedwithether.Thediferentgroups(eight, each
group)ofratswereadministeredthePRE(0.14,
0.42 and1.26 g/kg, p.o.)andDexamethasone
(0.002 g/kg, p.o.)oncedailyfortenconsecutive
daysfromthedayofcotonpeletinsertion.Thecontrol
groupreceivedmediumalone.Algroupsontheelev-
enthday, altheratsweresacrificedandthecoton
peletscoveredbythegranulomatoustisuewereex-
cisedanddriedinhotairovenat60 °Ctilaconstant
weightwasachieved.Granulomaweightwasobtained
bysubtractingtheweightofcotonpeleton0 d(be-
forestartofexperiment)fromtheweightofthecoton
peletoneleventhday(attheendofexperiment).
2.5 analgesiaactivity[ 2] .
2.5.1 Hot-platetest
Thetemperatureofmetalsurfacewasmaintained
at(55.0 ±0.5)°C.Latencytoadiscomfortreaction
(lickinghindpaws)wastakenaspainthresholdinfe-
malemice.Thecut-oftimewas60 s.Thefiftyvalid
femalemicewereselected(thepainthresholdwerede-
terminedin5to30seconds)andweredividedintofive
groups.Thediferentgroupsofmicewereadministered
thePRE(0.21, 0.63and1.89g/kg, p.o.)andAs-
pirin(0.2 g/kg, p.o.)oncedailyforthreeconsecu-
tived.Inthethirdday, thepainthresholdwasdeter-
minedbeforeadministrationand30, 60, 90 and120
min, respectively.
2.5.2 Writhingtest
Micewerekeptsinglyinaclearplasticobserva-
tionalcage(35 cm×25 cm×15 cm).andwerepre-
treatedwithPREorAspirinbyadministration(P.O.)
60 minpriortointraperitoneal(i.p.)injectionof
0.6 aceticacidinavolumeof0.1 mL/10.0 gper
mice.Afterthei.p.injectionofaceticacid, the
numberofwrithesexhibitedfor15 minwerecounted.
0.2 g/kgAspirinwasusedaspositivecontrol.The
writhesandprolongationofthelatencytimeswerecom-
paredwiththenormalcontrol.
2.6 Statisticalanalysis
Altheexperimentaldatawereexpresedbyx±s.
TheauthoradoptedSPSS10.0 statisticalsoftwareto
analyzethediferencesbetweenmedicalgroupandex-
cipientcontrolgroupbyStudentsttest.AvalueofP
<0.05orleswasconsideredstatisticalysignificant.
3 Results
3.1 Acutetoxicitystudy(oral)
ThetimesofmovementreducedafterfeedPREfor
someofmice.Beforedeath, themicehadtoxicosis
symptomofalowrespiratoryrate, gaspingbreathand
flappingofnosewing, andsoon.Theresultsofal
groupswererecordedintable1.Accordingtothemeth-
odofBliss, thelinearregressionequationwasY=
0.048 044 +6.157 1 Log(D).TheLD50 was
6.371 8 g/kg, its(Feilercorected)95 confident
limitwas5.408 4 -7.723 2 g/kg, andLD5 was
3.444 4 g/kg, LD95 was11.787 g/kg(table1).
Table1 Theresultsofdifferentdosesof
PREoralygiventomice
dose
/g/kg
log10
dose
/x
n Deathnumber
percentage
mortality
/
Probability
unit
/Y
Regression
Probability
unit/Y
10 1 10 9 90.00 6.281 7 6.205 2
7.5 0.875 06 10 6 60.00 5.252 9 5.435 9
5.63 0.750 51 10 4 40.00 4.747 1 4.66 9
4.22 0.625 31 10 2 20.00 4.158 5 3.898 2
3.16 0.499 69 10 0 0.00 — 3.124 7
2.37 0.374 75 10 0 0.00 — 2.355 4
3.2 EfectofPREonxylene-inducedearedema
Themeanincreaseinearedemawasabout13.47
±4.2 mginthevehicle-treatedcontrolmice.PRE
(0.21, 0.63 and1.89 g/kg, p.o.)significantly(P
<0.05, P<0.01)reducedthemeanearedema
weightat1 hafterbeingappliedthexylene.PRE
(0.21, 0.63and1.89g/kg, p.o.)exhibitedanti-in-
flammatoryactivityinadose-dependentmannerwith
thepercentinhibitionoferaedemaof33.41, 47.51
and56.79, respectively, ascomparedwiththecontrol
group.However, thestandardpositivedrug, DXM
(0.002 g/kg, p.o.)showedhighlysignificant(P<
0.01)anti-inflammatoryactivitywiththepercentinhi-
bitionof46.69(table2).
181
2007年 2月
第 29卷 第 2期
中 成 药
ChineseTraditionalPatentMedicine
February2007
Vol.29 No.2
Table2 EffectofPREonxylen-inducedear
edemainmice(x±s, n=10)
Group Dose/g/kg
Xylene-induced
earedema/mg Inhibition/
Control - 13.47±4.2 -
DXM 0.002 7.18±1.13＊＊ 46.69
PRE 0.21 8.97±1.37＊ 33.41
0.63 7.07±0.80＊＊ 47.51
1.89 5.82±2.29＊＊ 56.79
＊P<0.05;＊＊P<0.01vs.controlgroup.
3.3EfectofPREoncotonpeletgranulomainrats
PRE(0.21, 0.63 and1.89 g/kg, p.o.)
significantly(P<0.05, P<0.01)reducedthegranu-
lomaformationwithpercentageinhibitionof4.73,
17.03 and21.22 ascomparedwithDXM(0.002 g/
kg), whichshowedsignificant(P<0.01)inhibition
ongranulomaformationwiththepercentinhibitionof
25.72(table3).
Table3 EffectofPREoncottonpelletgranuloma
inrats(x±s, n=10)
Group Dose/g/kg
Weightofcoton
peletgranuloma/mg Inhibition/
Control - 109.04±3.95 -
DXM 0.002 80.99±1.37＊＊ 25.72
PRE 0.14 103.88±3.56＊ 4.73
0.42 90.46±1.87＊＊ 17.03
1.26 85.90±1.18＊＊ 21.22
＊P<0.05;＊＊P<0.01vs.controlgroup.
3.4 EfectofPREonpainthresholdofmiceinthe
hot-platetest(table4)
PRE(0.21, 0.63 and1.89g/kg, p.o.)signifi-
cantly(P<0.05, P<0.01)increasedthepain
thresholdat30min, 60 min, 90minand120minafter
administrationascomparedwithAspirin(0.2 g/kg),
whichshowedsignificant(P<0.05, P<0.01)in-
creasedthepainthreshold(table3).
Table4 EffectofPREonpainthresholdofmiceinthehot-platetest(x±s, n=10)
Group Dose/g/kg
Painthreshold(s)
Anterior Posterior
30/min 60 /min 90/min 120/min
Control - 15.1±5.43 15.2±5.16 14.6±5.54 15.9±4.16 14.8±2.89
Aspirin 0.20 15.6±6.32 20.5±6.44＊ 25.9±7.96＊＊ 33.1±10.14＊＊ 28.4±8.75＊＊
PRE 0.21 16.7±6.21 17.6±4.32 20.6±5.80＊ 20.1±5.84＊ 18.7±4.56
0.63 16.6±25.6 25.6±6.15＊＊ 33.3±16.38＊＊ 29.1±12.54＊＊ 26.3±6.68＊＊
1.89 15.4±5.18 27.4±6.51＊＊ 37.6±11.71＊＊ 36.9±12.23＊＊ 33.3±11.21＊＊
＊P<0.05;＊＊P<0.01vscontrolgroup.
3.5 TheanalgesicefectofPREonpainthresholdon
aceticacid-inducedwrithingtestinmice
PRE(0.21, 0.63 and1.89 g/kg, p.o.)signifi-
cantly(P<0.05 orP<0.01)increasedthepain
thresholdin15minafterintraperitoneal(i.p.)injec-
tionof0.6 aceticacid.PRE(0.21, 0.63and1.89
g/kg, p.o.)exhibitedanalgesicactivityinadose-de-
pendentmannerwiththepercentinhibitionof14.2,
57.4 and71.6, respectively, ascomparedwiththe
controlgroup.However, thestandardpositivedrug,
Aspirin(0.2 g/kg, p.o.)showedhighlysignificant
(P<0.01)analgesicactivitywiththepercentinhibi-
tionof73.38 (table5).
4 Discussion
DiferentpartsofPongamiapinnatahavebeenrec-
ommendedasaremedyforvariousailmentsinIndia.
Theseedandseedoilofthisplanthavebeenusedfor
Table5 TheanalgesicefectofPREonpainthreshold
onaceticacid-inducedwrithingtestinmice(x±s, n=10)
Group Dose/g/kg Writhesnumbers Inhibition/
Control - 16.9±2.55 -
Aspirin 0.20 4.5±1.20＊＊ 73.38
PRE 0.21 14.5±1.20＊ 14.2
0.63 7.2±1.33＊＊ 57.40
1.89 4.8±1.47＊＊ 71.60
＊P<0.05;＊＊P<0.01vs.controlgroup.
treatingvariousinflammatoryandinfectiousdiseases
suchasleucoderma, leprosy, lumbago, muscularand
articularrheumatism[ 3] .Otherwise, theseedoilhas
antibacterialactivity, Theactivityismainlyduetothe
inhibitionofcel-membranesynthesisinthebacteri-
at[ 4] .Theleavesofthisplanthasobviousanti-inflam-
matoryactivitywithoutulcerogenicactivitysuggesting
itspotentialasananti-inflammatoryagentforuseinthe
treatmentofvariousinflammatorydiseases[ 5] .The
leavesofthisplanthasalsoantinociceptiveandantipy-
182
2007年 2月
第 29卷 第 2期
中 成 药
ChineseTraditionalPatentMedicine
February2007
Vol.29 No.2
reticactivities[ 6] .EfectofmethanolicextractofP.
Pinnataroots(PPRM)isstudiedagainstvariousexperi-
mentalgastriculcermodelsandofensiveanddefensive
gastricmucosalfactorsinrats.Itshowtendencytode-
creaseaceticacid-inducedulcerafter10 daystreat-
ment.UlcerprotectiveefectofPPRMisduetoaug-
mentationofmucosaldefensivefactorslikemucinse-
cretion, lifespanofmucosalcels, mucosalcelglyco-
proteins, celproliferationandpreventionoflipidper
oxidationratherthanontheofensiveacid-pepsinse-
cretion[ 7] .Butanti-inflammationandanalgesiaefect
andacutetoxicityofextractfromPongamiapinnata
rootsstil remain unexplored in domestic. The
PongamiapinnataiswidelydistributedinGuangxi,
Taiwan, HainanProvinceetc.So, wehavebeenstud-
yingitspharmacologicalactionandtoxicity, inorderto
developpreferablytheafluentmedicineresource.
ThePongamiapinnatarootsareasafetraditional
Chinesemedicine.TheLD50 is6.371 8g/kg, its95
confidentlimitis5.408 4-7.723 2 g/kg, andLD5 was
3.444 4g/kg, LD95 was11.787g/kg.Wewilstudy
thelong-termtoxicityinordertoevaluatepreferablyits
safetyformedication.Itcouldsignificantlyprolongthe
painthresholdonhot-plateinmice, reducethewrit-
hingtimesinmice.Inacuteandchronicinflammation
models, PREshowssignificantanti-inflammatoryactiv-
ity.Themechanism ofanti-inflammatoryactivityof
PLEisnotexactlyknownandneedsfurtherstudy.
Inconclusion, thepresentstudyclearlyshowsthat
70 ethanolicextractofPREpossessesgoodanti-in-
flammatoryactivityandanalgesicefect.Theadvantage
ofPREissafe.Theanti-inflammatoryandanalgesic
activitydeservefurtherstudiestoidentifythepossible
mechanismofactionaswelasestablishthetherapeutic
valueinthetreatmentofinflammatoryandpaindisea-
ses.
Reference:
[ 1] JiangsuMedicalColege.DictionaryofChineseMateriaMedica
[ M] .Shanghai, ShanghaiScientificandTechnicalPublishers,
1997.
[ 2] XuSY, BianRN, ChenX.ExperimentalMethodologyofPhar-
macology.3 rded[M].Beijing:PeopleHealthPress, 2002,
201-210, 713-735, 693-695.
[ 3] NadkamiKM.IndianMateriaMedica[ M] .PopularBookDepot,
Bombay, India, 1954(1):1001.
[ 4] BaswaM, RathCC, DashSK, etal.Antibacterialactivityof
Karanj(Pongamiapinnata)andNeem(Azadirachtaindica)seed
oil:apreliminaryreport[ J].Microbios, 2001, 105(412):183-
189.
[ 5] Srinivasank, MuruganandanK, LalJ, etal.Evaluationofanti-
inflammatoryactivityofPongamiapinnataleavesinrats[ J] .Eth-
nopharmacol.2001(78):151-157.
[ 6] Srinivasank, MuruganandanK, LalJ, etal.Antinociceptiveand
antipyreticactivitiesofPongamiapinnataleaves[ J] .Phytother-
Res.2003, 17(3):259-264.
[ 7] PrabhaT, Dora-BabuM, PriyambadaS, etal.Evaluationof
Pongamiapinnatarootextractongastriculcersandmucosalofen-
siveanddefensivefactorsinrats[J] .IndianJExpBiol.2003, 41
(4):304-310.
鳖甲煎丸对人肾小球系膜细胞增殖及细胞外基质表达的影响
范焕芳 1 , 陈志强 1 , 张雪娟1 , 张秀君 1 , 徐华洲 2 , 黄 茂 3
(1.河北医科大学中西医结合研究所 ,河北 石家庄 050031;2.河北医科大学基础医学院 ,河北 石家庄
050031;3.河北体育学院 ,河北 石家庄 050000)
收稿日期:2006-06-38
作者简介:范焕芳(1970～ ),女 ,博士研究生 ,主治医师 ,研究方向:中西医结合对肾脏病的临床及实验研究 ,电话:13931120376。
关键词:鳖甲煎丸;增殖;层黏连蛋白;Ⅳ型胶原;人肾小球系膜细胞
摘要:目的:探讨鳖甲煎丸对人肾小球系膜细胞(HMC)增殖及细胞外基质(ECM)不同成分的抑制作用。方法:应用细胞
培养技术 , 进行人 HMC培养 , 采取血清药理学方法 ,制备鳖甲煎丸药物血清 , 利用脂多糖(LPS)为刺激因子 , 应用四甲基
偶氮唑蓝(MTT)法检测各组系膜细胞的增殖情况 , 并采用酶联免疫吸附试验(ELISA)法检测 ECM中层黏连蛋白(LN)
183
2007年 2月
第 29卷 第 2期
中 成 药
ChineseTraditionalPatentMedicine
February2007
Vol.29 No.2