全 文 ：ProtectiveefectofethylacetateextractofPongamiapinnatarootson
ethanol-inducedgastricmucosalinjuriesinrats
LIUKe-Yun1, 2 , ZHUYi1＊ , DONGZhi3 , LIJing3 , HUANGLing3 , CHENGuo-Biao1
(1.DepartmentofPharmacology, InstituteforDrugControlofHainanProvince, Haikou　570216, China;
2.MedicalSchool, HubeiInstituteforNationalities, Enshi　445000, China;3.Departmentof
Pharmacology, ChongqingUniversityofMedicalSciences, Chongqing　400016, China)
Abstract:AIM　Toinvestigatethetherapeuticefect
ofethylacetateextractfromPongamiapinnataroots
(PREA)onethanol-inducedgastriclesions.METH-
ODS　Theexperimentalgastricmucosalinjurieswere
preparedbyigethanoltorats, andtheprotectiveefect
ofPREAwasevaluatedbycalculatinglesionindex,
observingpathologicalchanges, andmeasuringthe
contentsofnitricoxide(NO)andmalondialdehyde
(MDA), andtheactivityofsuperoxidedismutase
(SOD)fromgastricmucosaltissue.Inaddition, gas-
tricsecretaryandgastricwaladherentmucuswere
studiedwiththepylorus-ligationratmodel.RESULTS
Comparedwiththemodelcontrolgroup, PREA(50,
150and450 mg·kg-1 , ig)dose-dependentlypreven-
tedthegastricmucosaldamagesinducedbyethanol,
itsinhibitionrateswere28.7%, 57.7% and78.7%,
respectively.Thepathomorphologylesionsofmucosal
tissuewereobviouslyameliorated.PREAobviouslyan-
tagonizedtheethanol-inducedelevationofMDAcon-
tent, andreductionofNOlevelandSODactivityof
gastricmucosa.PREAsignificantlyreducedgastric
juicevolume, freeacidity, totalacidityandtotalacid
output, butdidn′tafectthepepsinactivity.Moreover,
PREAobviouslyincreasedadherentmucusquantityof
stomachwal, aswelasfreemucusquantitydissolved
ingastricjuiceofpylorus-ligationrat.CONCLUSION
PREAhasprotectiveefectonethanol-inducedgastric
mucosalinjuries, whichsuggeststhatPREAmaybe
usedforprotectionortreatmentofhumanethanol-
　
　　Receiveddate:2007-01-08　Accepteddate:2007-08-10
　　Foundationitem:TheprojectsupportedbyNaturalSci-
enceFoundationofHainanProvince(30530)
　　Biographies:LIUKe-Yun(1974-), male, nativeofEn-
shi, HubeiProvince, masterofpharmacology, mainresearch
fieldistraditionalChinesemedicinepharmacologyandexploita-
tion;ZHUYi(1962-), male, nativeofZunyi, GuizhouProv-
ince, PhD, professor, mainresearchfieldistraditionalChinese
medicinepharmacologyandexploitation.
　　＊Correspondingauthor.E-mail:hnzhuyi@126.com
inducedgastriclesions.
Keywords:Pongamiapinnataroots;ethylacetate;
ethanol;gastricmucosa
CLCnumber:R285
Documentcode:A
ArticleID:1000-3002(2007)06-0476-06
Ethanol-inducedgastriclesionisacommon
diseaseinclinicsthataffectsbillionsofpeople
healthintheglobe.Butthereisnotadrugthat
canpreventortreatitthoroughlybecauseofits
complexpathogenesisandunknownetiology.
Therefore, thescreeninganddevelopmentof
drugsfromChinesemedicinalplantsforanti-
gastriculcerinducedbyethanolisstilinpro-
gress.Pongamiapinnata(Linn.)Pierre(P.
pinnata)isamediumsizedglabroustreepopu-
larlyknownaspoongaoilpongamiaseededin
China.P.pinnatagrowsintheseasideandthe
bankofriversandstreamsofaloverHainan
ProvinceofChina, andplantedasavenuetrees
ingardens.DiferentpartsofP.pinnatahave
beenusedinmedicineforbronchitis, whooping
cough, rheumaticjointsandinflammatorydisea-
ses.TheflavonoidsofP.pinnatawereoneof
themainchemicalingredients[ 1] .Itwasrepor-
tedthat7 flavonoidswereisolatedfromethanol
extractsbytheethylacetateextractmethod,
and70% ethanolextractshadanti-gastriculcer
efectsinpylorus-ligatedmodels[ 2] .Inourlab
theefectivefractionsfromP.pinnatarootson
experimentalgastriculcerhadbeenscreened
andfoundtheethylacetateextractfromP.pin-
nataroots(PREA)wastheeffectivefraction.
·476·
中国药理学与毒理学杂志
ChinJPharmacolToxicol　　
2007年 12月;
2007　　Dec;　
21(6):476-481
21(6):476-481
SointhisresearchtheefectofPREAonetha-
nol-inducedgastricmucosallesionsinratswas
evaluated.
1　MATERIALSANDMETHODS
1.1　Animalsandreagents
MaleandfemaleSprague-Dawley(SD)
rats, 180 -220 g, werepurchasedfromthe
CenterofExperimentalAnimalofGuandong
Province.ThecertificatenumberisSCXK
(Yue) 2003-0002. Theywerehousedin
polyprolenecagesandmaintainednormallabo-
ratoryconditionsat21-23 ℃ ona12 hlight-
darkcycleandfedastandardrodentchowand
water.
Alcianblue8GS(F20051221)waspur-
chasedfromShanghaiChemicalReagentCom-
pany, ChinaNationalMedicineGroup.Detec-
tionkitsfornitricoxide(NO), malondialde-
hyde(MDA), superoxidedismutase(SOD)
andpepsinwerefromNanjingJianchengBioen-
gineeringInstitute.Sodiumcarboxymethylcel-
lulose(CMC-Na)wasfromGuoyaoChemical
Co., batchnumberF2005-718.Cimetidine
(Cim)wasfrom HainanDrugManufactory
Ltd., batchnumber050701.
1.2　Drugpreparation
P.pinnatarootswerecolectedinHaikou
(HainanProvince, China)inMarch20, 2006
andidentifiedbyCHENGGuo-Biao, atradi-
tionalChinesemateriamedica(TCMM)spe-
cialistinInstituteforDrugControlofHainan
Province, China.Thevoucherwasdepositedat
InstituteforDrugControlofHainanProvince,
China.ThepowderofP.pinnataroots(2.41
kg)wasrecirculated3 timeswith70% ethanol
(1∶8, V/V)at60℃, 2heachtime.Theetha-
nolmixtureswerefilteredandconcentratedun-
derreducedpressureat60℃ withtheinstru-
mentofturningevaporation.Thepowderedex-
tractofethanolextractwas188.0 g, andthe
yieldwas7.8%.Thepowderedextractwasex-
tractedbyusingsequentialpetroleumetherand
ethylacetatefor3times, andthenthepowdered
extractofethylacetate(49.4 g), PREA, was
obtainedafterrecoveringethylacetate.The
yieldofPREAwas2.0%.Themaincompo-
nentsofPREAwereflavonoids, theircontent
was60.9%, determinedbyTCMMspecialistin
InstituteforDrugControlofHainanProvince.
PREAandCimweredissolvedinCMC-Na(5g·
L-1)tomakethesuspensionsjustpriortodrug
administration.
1.3　Evaluationofgastricmucosadamage
Theratswererandomlydividedinto 6
groups, includingnormal, model, PREA(50,
150 and450 mg·kg-1)andCim(140 mg·
kg-1)groups.PREAorCimwasgiven(ig)to
ratsonceadayfor5dexcepttheratsofnormal
andmodelgroups, whichweregiven(ig)
0.5% CMC-Na(15 mL· kg-1 )insteadof
PREAorCim.Alratsexceptthatinnormal
groupweregiven(ig)0.8 mLabsoluteethanol
(99.5%)[ 4] 2 hafterthelastadministrationof
drugs.Onehourlateraltheanimalsweresac-
rificed, stomachswereremovedandopeneda-
longthegreatercurvaturetoobservethelesions
macroscopicaly.Scoringstandardwasasfol-
lows:intactgastricmucosawasdefinedas0;
point-shapedbleeding, point-shaped erosion
andstrip-shapedlesionofnomorethan1 mm
werealdefinedas1;strip-shapedlesionwith
widthmorethan1mmwasdoublyscored.Total
scoresofeveryratwereconsideredaslesionin-
dex.Inhibitionratiooflesionformation(%)=
(A-B)/A×100% (A, Bwerethelesionin-
dexofmodelgroupanddrug-treatedgroup, re-
spectively).
1.4　Histologicalanalysis
Strip-shapedtissuespecimensfromgastric
antrumwerequicklycutofundericebathafter
theevaluationofgastricmucosallesion.The
specimenswerefixedin40 g· L-1 neutrally
buferedformaldehyde, embeddedinparaffin.
Sectionsof5 μmthickwerecut, placedonad-
hesive-coatedslides, andstainedwithhematox-
ylinandeosin(HE)forhistopathologicalanal-
ysis(cooperatedwithDepartmentofHistology
andEmbryology).
·477·中国药理学与毒理学杂志　　2007年 4月;21(2)
1.5　MeasurementsofNOandMDAcon-
tentsandSODactivity
Afterthestrip-shapedtissuespecimenshad
beencutoffromratstomach, gastricmucous
membraneswerequicklystrippedoffromthe
restofstomachonice.Gastricmucosahomoge-
natesof10% werepreparedwithice-coldsa-
line.Homogenateswerecentrifugedat2000×g
for10minat4℃ andthesupernatantwassaved
formeasurementofNOandMDAcontentsand
SODactivityaccordingtotheinstructionsof
correspondingkits.Proteincontentwasdeter-
minedusingtotalproteinquantificationkitwith
theCoomassie-bluemethod.
1.6　Determinationofgastricsecretionand
gastricwaladherentmucusquantity
Groupingandadministrationoftheexperi-
mentalratswerethesameasthatofethanol-in-
ducedmodels, butnonormalgroup.Fromd3
ofthedrugadministrationtheratswerefasted,
and2 hafterthefifthdrugadministrationthe
pylorusofeachratwasligatedunderetheranes-
thesia[ 5] .Theratswerekeptbeingfastedand
5hlatertheratsweresacrificed.Thestomachs
wereremovedandthevolumeofgastricjuice
wasdeterminedaftercentrifugationat2000 ×g
for10 min.Aciditywasassessedbytitration
against0.01 mol·L-1 NaOH.Theactivityof
gastricpepsinwasassessedbyaminoaciddeox-
idizationmethod.Adherentmucusquantityof
stomachwalandfreemucusquantitydissolving
ingastricjuiceweremeasuredbyAlcianblue
method[ 6] .
1.7　Statisticalanalysis
Theresultswereexpressedas x±s.Statis-
ticalanalysiswasdonewithSASsystem(8.0).
DatawereanalyzedusingF-test, andq-testto
comparetheresultsbetweeneverytwogroups.
P<0.05wastakenassignificant.
2　RESULTS
2.1　EffectofPREAongastriclesionsin-
ducedbyethanol
Inmodelgroupthelargebandlikehemor-
rhagicerosionsintheglandularstomachwas
evident(Fig1, Tab1).PREA(50, 150 and
450 mg·kg-1)dose-dependentlypreventedthe
Fig1.　EfectofethylacetateextractfromPongamiapinnataroots(PREA)ongastriclesionsinducedby
ethanolinrats.Drugsweregiven(ig)onceadayfor5d.Twohoursafterthelastadministration, 99.5% ethanol0.8mLwasig
toalratsexceptnormalgroup.Onehourlatertheratsweresacrificedandgastriclesionswereobservedmacroscopically.A:normal;
B:model;C:PREA50 mg·kg-1;D:PREA150 mg·kg-1;E:PREA450 mg·kg-1;F:cimetidine(Cim)140mg·kg-1.
·478· ChinJPharmacolToxicol　2007　Dec;21(6)
Tab1.　EffectofPREAongastriclesionsinduced
byethanolinrats
Group Dose/mg·kg-1 Lesionindex Inhibitionrate/%
Normal 　0±0
Model 119±19
PREA 50 85±15＊＊ 28.7
150 50±19＊＊ 57.7
450 25±14＊＊ 78.7
Cim 140 37±11＊＊ 69.2
SeeFig1forthetreatments.Totalscoresofeveryratwerecon-
sideredaslesionindex.Thescoringstandardandinhibitionrate
calculationseeMATERIALSANDMETHODS1.3. x±s, n=
8.＊＊P<0.01, comparedwithmodelgroup.
gastricmucosaldamagescomparedwithmodel
group.TheefectofPREA 450 mg·kg-1 was
likelystrongerthanthatofCim140 mg·kg-1.
2.2　EffectofPREAonhistopathological
changes
Thestructureofgastricmucosafromnor-
malgroupwasdistinct:intactanduninterupted
epithelia, gastricfundalglandliningupinorder
andnohyperemia.Inthemodelgroup, itfound
thefocalanddifusemucosalepitheliadamage,
edema, splitofepitheliallaminapropriaofmu-
cosatissueandirregularglandarrangement.
Gastricmucosalcapilarieswereseverelydilated
andcongested, andalotofredbloodcelswere
exudatedout.However, pathomorphologicalle-
sionsofmucosatissueinthegroupspretreated
byPREA(50, 150 and450 mg·kg-1)and
Cim(140 mg·kg-1)wereobviouslyameliora-
tedatdifferentdegrees.Especialytherewere
onlydilatedcapilaries, slightedemaanda
smalquantityofnecrosistissuesinPREA450
mg·kg-1 group.
2.3　EfectofPREAonNOandMDAcon-
tentsandSODactivityofgastricmucosa
NOcontentandSODactivityofgastricmu-
cosahomogenatesinmodelgroupsignificantly
decreasedcomparedwiththatinnormalgroup.
PREA50, 150 and450 mg·kg-1 dose-depend-
entlyinhibitedethanol-induceddecreaseofNO
levelandSODactivity.MDAcontentingastric
mucosaofmodelgroupobviouslyincreased
comparedwiththatofnormalgroup.PREA50,
150 and450 mg·kg-1 significantlyprevented
theincreaseofMDAcontentcomparedwiththat
ofmodelgroup(Tab2).
2.4　EfectofPREAongastricsecretion
AsshowninTab3, PREA(150 and450
mg· kg-1 )significantlyreducedthegastric
juice, freeacidity, totalacidityandtotalacid
output.However, alldosesofPREAshowedno
efectonpepsinactivity.
Tab2.　EffectofPREAonnitricoxide(NO)andmalondialdehyde(MDA)contentsandsuperoxidedis-
mutase(SOD)activityofgastricmucosadamagedbyethanolinrats
Group Dose/mg·kg-1 NO/μmol·g-1 protein
MDA
/mmol·g-1 protein
SODactivity　
/mmol·min-1·g-1 protein　
Normal 3.5±1.2 2.9±0.6 43.8±8.8
Model 2.5±0.7＊ 6.1±1.7＊＊ 29.1±10.3＊＊
PREA 50 4.0±1.3# 3.7±1.0## 40.8±9.4#
150 5.2±1.4## 2.8±0.5## 52.1±12.8##
450 5.6±1.6## 2.4±0.3## 63.0±13.5##
Cim 140 4.8±1.2## 3.0±0.8## 46.4±12.1##
SeeFig1forthetreatments. x±s, n=8 .＊P<0.05, ＊＊P<0.01, comparedwithnormalgroup;#P<0.05, ##P<0.01, compared
withmodelgroup.
·479·中国药理学与毒理学杂志　　2007年 12月;21(6)
Tab3.　EffectofPREAongastricsecretioninpylorus-ligationrats
Group Dose/mg·kg-1
Gastricjuice
/mL
Freeacidity　
/mmol·L-1　
Totalacidity　
/mmol·L-1　
Totalacidoutput
/mmol·h-1
Pepsinactivity　
/mg·min-1· L-1　
Model 6.2±0.9 84±9 112±15 0.14±0.03 46±10
RREA 50 5.9±1.0＊ 81±13 107±13 0.12±0.03 45±9
150 5.2±0.7＊ 56±12＊＊ 81±16＊＊ 0.08±0.01＊＊ 43±12
450 4.6±1.2＊＊ 37±12＊＊ 67±15＊＊ 0.06±0.03＊＊ 45±10
Cim 140 3.7±1.0＊＊ 41±9＊＊ 68±14＊＊ 0.05±0.02＊＊ 33±8＊
ThetreatmentswerethesameasFig1 exceptthepylorus-ligationratmodelused. x±s, n=8.＊P<0.05, ＊＊P<0.01, compared
withmodelgroup.
2.5　EffectofPREAongastricmucusse-
cretioninpylorus-ligationrats
Tab4 showedthatPREA(150 and450
mg·kg-1)significantlyincreasedadherentmu-
cusquantityofstomachwal.PREA450 mg·
kg-1 alsoincreasedfreemucusquantitydissol-
vingingastricjuice.
Tab4.　EfectofPREAongastricmucussecre-
tioninpylorus-ligationrats
Group Dose/mg·kg-1
Adherentmucusof
stomachwall/mg
Freemucusdissolving
ingastricjuice/mg
Model 0.73±0.27 0.54±0.11
RREA 50 0.77±0.25 0.57±0.09
150 1.04±0.27＊ 0.59±0.10
450 1.21±0.19＊＊ 0.64±0.06＊
Cim 140 0.77±0.19 0.56±0.05
SeeTab3 forthetreatments. x±s, n=8.＊P<0.05, ＊＊P<
0.01, comparedwithmodelgroup.
3　DISCUSSION
Ethanol-inducedgastricmucosaldamage
inratsisoftenusedasanexperimentalmodel
whenscreeningdrugsforanti-ulceractivitybe-
causeitrepresentsthemostcommoncauseof
gastriculcerinman.Studiesinthepasthave
shownthatthe70% ethanolextractsfromP.
pinnatarootspreventedgastricmucosalesion
withpylorusligatedmodels[ 3] .Prabhaetal[ 7]
reportedthatmethanolicextractofP.pinnata
rootsshowedsignificantprotectionagainstgas-
tricmucosaldamageinducedbyaspirin, but
notagainstethanol-inducedgastriculceration.
InthisstudyitwasdemonstratedthatPREA
significantlydecreasedethanol-inducedgastric
mucosalesionindexandobviouslyrelieved
pathologicalchanges.Theseresultsdidnota-
greewiththeresultsofPrabhaetal.Itwaspos-
siblethatthemethodofbuildingmodelandthe
drugdosageweredifferent.
EndogenousNOhasadualactioninthe
gastrointestinaltract:protectiveefectbycon-
stitutivenitricoxidesynthase(eNOS)/NOand
proulcerogenicefectsbyiNOS/NO[ 8] .There-
fore, itwassuggestedthatdecreasingofNO
contentwascloselyrelatedtogastricmucosal
injury.Ourexperimentresultsshowedthat
PREApreviousadministrationcouldantagonize
thedecreaseofNOlevelinducedbyethanol.
Gastrointestinalmucosalcanproduceagreat
amountofoxygen-derivedfreeradicals, which
havehightoxicity, whengastricmucosaunder-
goeschemicalsubstancestimulation, ischemia,
anddisordersofenergymetabolismaswelas
neurohumorregulation[ 9] .Gastricmucosais
damagedbytheoxygen-derivedfreeradicalsby
theproductsoflipidperoxidation, suchas
MDA.SODisthefirstlineofdefenseagainst
oxygen-derived free radicals. Our results
showedthatMDAcontentsobviouslyincreased
andSODactivitysignificantlydecreasedineth-
anol-inducedgastricmucosaandPREAobvi-
ouslyantagonizedthechanges.
Ethanol-inducedgastricmucosalinjuryhas
beenalsosuggestedtobeduetoimpairmentsin
·480· ChinJPharmacolToxicol　2007　Dec;21(6)
defensivefactorssuchasgastricmucus[ 10] .
Whentestedonpylorus-ligatedrats, PREAin-
creasedgastricmucuscontent, inhibitedgastric
secretaryvolumeandreducedfreeacidityand
totalacidity, whichsuggesteditsmodulationon
gastricsecretion.However, PREAdidnothave
anysignificantefectongastricpepsinactivity.
In conclusion, PREA has protective
efectsonethanol-inducedgastricmucosalinju-
riesandmaybeusefulintheprotectionor
treatmentofgastricdamagebyethanolinman.
However, furtherstudiesareneededtoclarify
theexactmechanismofgastricprotectiveefect
ofPREA.
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水黄皮根乙酸乙酯萃取物对大鼠乙醇型胃黏膜损伤的保护作用
刘可云 1, 2 , 朱　毅 1 , 董　志 3 , 李　靖3 , 黄　凌 3 , 陈国彪 1
(1.海南省药品检验所药理学研究室 , 海南 海口　570216;2.湖北民族学院医学院 , 湖北 恩施　445000;
3.重庆医科大学药理学教研室 , 重庆　400016)
摘要:目的　研究水黄皮根乙酸乙酯萃取物(PREA)
对乙醇致胃黏膜损伤的治疗作用。方法　建立乙醇
致大鼠胃黏膜损伤模型 ,通过观察胃组织病理学改
变 、计算胃黏膜损伤指数 、检测胃黏膜组织一氧化氮
(NO)、丙二醛(MDA)含量和超氧化物歧化酶(SOD)
活性评价 PREA对乙醇型胃黏膜损伤的保护作用。
采用幽门结扎模型 ,观察 PREA对大鼠胃液分泌和胃
黏液分泌的影响。结果　与模型组比较 , PREA可剂
量依赖性地降低乙醇所致胃黏膜损伤指数 ,明显改善
胃黏膜损伤的病理变化 , 抑制乙醇引起的胃黏膜
MDA含量升高及 NO水平和 SOD活性降低 ,并显著
减少胃酸分泌 、抑制游离胃酸酸度和总酸度 ,对胃蛋
白酶活性没有明显影响 。另外 ,可显著抑制幽门结扎
模型大鼠胃腔游离黏液以及胃壁结合黏液的分泌。
结论　PREA对乙醇型胃黏膜损伤具有明显的保护
作用 ,提示 PREA可能成为预防或治疗乙醇所致胃损
伤的药物 。
关键词:水黄皮根;乙酸乙酯;乙醇;胃黏膜
　
　　基金项目:海南省自然科学基金资助项目(30530)
(本文编辑　齐春会)
·481·中国药理学与毒理学杂志　　2007年 12月;21(6)