全 文 ：·Originalarticle·
Anticataractpotentialofphylanthusniruriingalactoseinducedcataractogenesisofrat
SureshKumarGupta1 , VivekanandhanKalaiselvan2 , AshishSharma1 , SushmaSrivastava1 ,
ShyamSunderAgrawal1
1DepartmentofOcularPharmacology, DelhiInstituteof
PharmaceuticalSciencesandResearch, Sector-II, Pushp
Vihar, M.BRoad, NewDelhi-17, India
2KovaiMedicalCenterandHospital, ColegeofPharmacy
Coimbatore-35, India
Correspondenceto:VivekanandhanKalaiselvan.KovaiMedical
CenterandHospital, ColegeofPharmacyCoimbatore-35,
India.vivekarts@redifmail.com
Received:2009-03-27　　Accepted:2009-05-30
Abstract
· AIM:Toevaluateanticataracteffectofphyllanthus
niruri(PN)bothinvitroandinvivogalactoseinduced
cataract.
· METHODS:AqueousextractofPN was evaluated
againstgalactose-inducedcataractbothinvitroandin
vivo.Galactosaemiccataractwasinduced in ratsby
feeding300g/Lgalactosediet.PNwasadministeredoraly
atthree-doselevels 75, 150 and 300 mg/kg ofbody
weight.Ratlensesweresubjectedtoosmoticstressin
vitro by incorporating galactose (30mmol/L) in the
culturemedium.TheeffectofPN(720 and880μg/mL)on
theglutathione(GSH)andpolyolslevelswasstudied.
· RESULTS:PN significantly delayed the onsetand
progressionofcataractinvivo.Inadditiontothedelayin
reachingvariousstagesofdevelopmentofcataract, stage
IVdidnotdevelopwithlowerdosestilthecompletionof
experimentalperiod.LensestreatedwithPN 880μg/mL
concentrationshowedhigherlevelsofGSHanddecreased
levelsofpolyolsinvitro.Invivo, 75mg/kgsignificantly
delayed the onset and progression of cataract as
comparedtocontrol.
· CONCLUSION:PN delayedtheprocessofcataracto-
genesisintheexperimentalmodels.However, further
studyisrequiredtoextrapolatetheuseinhumanbeings
forthepreventionofcataract.
· KEYWORDS:galactosecataract;glutathione;polyols;
phylanthusniruri
DOI:10.3969/j.issn.1672-5123.2009.06.001
GuptaSK, KalaiselvanV, SharmaA, SrivastavaS, AgrawalSS.
Anticataractpotentialofphyllanthusniruriingalactoseinduced
cataractogenesisofrat.IntJOphthalmol(GuojiYankeZazhi)2009;
9(6):1011-1015
INTRODUCTION
Cataract, theopacificationofthelensoftheeye, istheleadingcauseofblindness, itaccountsforapproximately
50% ofalblindnessworldwide.WorldHealthOrganization
launchedVision 2020, toeliminatecataractaspriority
diseases[ 1] .InIndia, cataractisresponsibleforalmost80%
ofblindness[ 2, 3] .Apartfrom senilecataractvariousrisk
factorssuchasnutritionaldeficiency, sunlight, smoking,
environmentalfactors, lackofconsumptionofantioxidantsand
diabetesareknowntoincreasetheriskofcataract[ 4-7] .
Diabeteshasbeenconsideredtobeoneofthemajorrisk
factorsofcataract.Many invitroand invivoexperimental
studiessupporttheviewthatdiabetesisoneofthecausesof
cataract[ 8 , 9] .Duringhyperglycemiaextracelularglucose
difusesintothelensuncontroledbythehormoneinsulin, the
lensisoneofthebodypartsmostafectedindiabetes
melitus.Theproteinsofthelensareextremelylong-lived,
andthereisvirtualynoproteinturnover, whichcanleadto
postranslational modification[ 2] . Ingestion of excesive
galactosehasbeenshowntoinducetheformationofcataracts
inseveralspeciesofexperimentalanimals.Thecataractogenic
efectisprimarilyrelatedtothesynthesisandaccumulationof
excessivesorbitol(polyols)inthelensfibersandconsequent
osmotic stress[ 10, 11] . Sorbitolis synthesized by aldose
reductaseutilizingNADPH anddoesnoteasilycros cel
membranes;itcanaccumulateincelsandcausedamageby
disturbingosmotichomeostasis.Anotherpathophysiological
mechanismofcataractformationincludesdeficientglutathione
levelscontributingtoafaultyantioxidantdefensesystem
withinthelensoftheeye[ 12] .Normalythelenscontains
significantlevelsofreducedglutathione(GSH), whichkeeps
theproteinsintheirreducedform.However, thereare
significantlylowerlevelsofGSH incataractouslenses.
Therefore, preventionofpolyolaccumulationandmaintaining
GSHleveltopreventcataractanddiabeteshasreceived
considerableatention.
Agreatnumberofmedicinalplantsarereportedtoposessanti
diabeticandoferprotectioninvariouspathologicalconditions
suchascardiovasculardiseases, neurodegenerationetc[ 13-15] .
Alargenumberofplants/speciesarenowwelrecognizedto
posesshypoglycemicpotential[ 16-18] .Manyofthesehypog-
lycemicagentshavenotbeeninvestigatedfortheirbeneficial
efectson secondarycomplicationsofdiabetessuch as
cataract.Itwouldbeofgreatimportancetoevaluateboth
pharmacologicalyandbiochemicaly, whichmightbehelpful
inthebetermanagementofsecondarycomplicationsof
diabetes.
Phylanthusniruri(Euphorbiaceae)commonlyknownas
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bhui-amla, hasbeenusedinAyurvedicmedicineforover
2 000 yearsandhasawidenumberoftraditionaluses
includingdiabetes[ 19-21] .Thecurentstudywasundertaken
withtheaim ofinvestigatingtheanticataractpotentialof
aqueousextractofphylanthusniruri(PN) wholeplant
againstgalactosecataractinratsaswelasagainstgalactose
inducedmorphologicalandbiochemicalchanges invitro.
TogethertheseresultsimplythatPNmaybeexploredasan
anticataractogenicagentfordiabeticcataract.
METHODS
Materials　LyophilizedaqueousextractofPNwasobtained
fromPromedExportsPvtLtd.(NewDelhi, India).Dulbecco
modifiedEaglesmedium(DMEM)wasprocuredfrom Hi
MediaLaboratories(Mumbai, India).Galactosewaspur-
chasedfromSDfine-ChemLimited(Mumbai).Streptomycin
andpenicilinwereobtainedfromHindustanAntibioticsLtd.
(Pune, India).
Animals　Theanimalsinthecurrentstudyweretreatedin
accordancewiththeinstitutionalguidelinesandAsociation
forResearchinVisionandOphthalmologystatementforthe
useofanimalsinresearch.Forinvivostudy, Wistarratsof
eithersex, weighing60-80gweredividedintocontroland
treatedgroupsand for in vitrostudy, thelenseswere
enucleatedfrom thewistarrats(60-80g)ofeithersex
belongingtothenormalgroup(withoutanytreatment)for
cardiovascularandantifertilitystudiesbeingconductedinthe
institutewereused.
Lensorganculture　Thelenseswerecarefulyenucleated
fromeyeswithaposteriorapproach.Eachisolatedlenswas
placedinaFalconplasticcultureplate(24-wel)containing
2mLofDMEM supplementedwith 200mL/Lfetalbovine
serum, 100g/mLofstreptomycin, and100IU/mLpenicilin.
Thelenseswereincubatedat37℃ under900g/Lmoisture,
950mL/Lair, and50mL/LCO2gasatmospherefor2 hours.Thedamagedlensesthatdevelopedartificialopacitieswere
discardedandonlytransparentlensesweretakenforthe
subsequentinvitroexperiment.
Galactose-inducedOsmoticStress InVitro　Transparent
culturedlenseswererandomlydividedintonormal, galactose
onlyandtwotreatmentgroupseachcomprisingsixlenses.
NormallenseswereincubatedinDMEM alone, whereas
controlgrouplenseswereincubatedinDMEMsupplemented
with30mmol/Lofgalactose.Mediuminthetreatedgroups
wasadditionalysupplementedwithtwodiferentconcentra-
tionsofPN(720and880g/mL)alongwithgalactose.Althe
lensesindiferentgroupsweremaintainedfor24 hoursatthe
above-mentionedexperimentalconditionsofincubation.Post
incubation, thelenseswereexaminedforthepresenceofany
opacity, andphotodocumentationwasdone.Thereafter,
lenseswerewashed, weighed, andprocesedfortheesti-
mationofbiochemicalparameters.Eachlenswashomogenized
in1mLof0.1mol/L-phosphatebufer(pH 7).Thehomo-
genatewasdividedintotwoequalparts.Onepartwasusedfor
theestimationofGSHandtheotherforpolyols.
EstimationofGSH　TheGSHcontentwasestimatedbythe
methodofMoron etal[ 22] .Thehomogenatewascentrifugedat
5 000rpmfor15 minutesat4℃.Tothesupernatant, 0.5mL
of100g/Ltrichloroaceticacidwasaddedandrecentrifuged.
Theprotein-freesupernatantthusobtainedwasreactedwith
4mLof0.3mol/LofNa2HPO4(pH8.0)and0.5mLof0.4g/L(w/v)5, 5-dithiobis-2-nitrobenzoicacid.Theabsorbanceof
theresultingyelowcolorwasreadspectrophotometricalyat
412nm.Aparalelstandardwasalsomaintained.
EstimationofPolyols　Polyolestimationwasdonebythe
methoddescribedbyWestandRapoport[ 23] .Thehomogenate
wasreactedwith0.6mol/Lperchloricacid.Precipitatewas
removedbycentrifugationandthesupernatantwasneutralized
with2mol/LNaOH.Againtheprecipitatewasremovedby
centrifugationandclearsupernatantwasreactedwith, freshly
prepared0.125mol/Lstannouschlorideand2g/Lchromotropic
acid.Theabsorbanceofpurplecoloredcomplexwasmeasured
spectrophotometricalyat570nm.
Paralelstandardwassubjectedtotheabove-mentionedsteps
forthecalculationofpolyolinthesamples.
GalactoseCataract InVivo　 Wistarratsofeithersex,
weighing60-80gwasdividedintocontrolandtreatedgroups
(ineachgroupn=8).Feeding300g/Lgalactosediettoal
groupsadlibituminducedcataract.
Sevendayspriortostartofgalactosediet75, 150and300mg/kg
bodyweightdoseofPNindistiledwater(asavehicle)were
givenoralyonceadaytothetreatedgroupandcontinuedtil
theendoftheexperiment.Incontrolgrouponlydistiled
waterandthegalactosedietweregiven.Eyeswereexamined
throughaslitlampafterdilatingtheratpupilwith10g/L
tropicamide.Thestagesofcataractweregradedaccordingto
Sippelsclassification[ 24] .
StatisticalAnalysis　Aldatawereexpressedasmean±SD.
Thegroupswerecomparedbyone-wayANOVAusingpost-hoc
Dunnetstest, withaP<0.05 consideredassignificant.
RESULTS
FoodIntakeAndBodyWeights　Therewasnosignificant
efectoffeedingofgalactoseorgalactoseplusPNextract(75,
150 and300mg/kgbodyweight)onfoodintakeandbody
weightoftheanimalsduringtheentirecourseofthestudy.
LensMorphology　 AlthelensesinDMEM alonewere
transparent.However, lensesafter24 hoursofincubationin
thepresenceofgalactosedevelopeddenseopacity.Incor-
porationofPN(720 and880g/mL)intheculturemedium
preventedthedevelopmentofopacitytodiferentextent.
Sixty-fourpercentoflensremainedtransparentwiththe
supplementationofPNextractattheconcentrationof720g/mL
andrestofthelensesdevelopedfaintopacity.Atthedoseof
880μg/mL, PNwasmoreefective.Only16 percentoflenses
showedfaintopacitywhile84% weretransparent.
EfectofPN onGSH andPolyols　Toinvestigatethe
posiblemechanismsofdiferentialefectsofphylanthus
niruriatdiferentdosesongalactose-inducedcataract, levels
ofGSHandpolyolswereestimatedwhicharerelatedtothe
oxidativestressandpolyolpathway.
GalactoseproducedasignificantdiferenceinGSHinthe
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galactose-onlygrouplensesincomparisonwithnormallenses.
TreatmentwithPN (720 and 880μg/mL) significantly
restoredtheGSHconcentrationFigure1.
EfectofPNwasstudiedonthepolyollevelsinthelenses
incubatedinmediumwithgalactose.Ourresultsshowedagradual
Figure4　EffectofPhyllanthusniruri(PN)onprogression
patternofgalactosecataractinvivo.Treated1:PN 75mg/kg;
Treated2:PN150mg/kg;Treated3:PN300mg/kg
increaseinpolyollevelincontrolgroup.However, PNat720
and880μg/mLconcentrationwasfoundtoinhibitpolyol
accumulationinthetreatedgroup.TreatmentwithPN -
880μg/mL in the medium significantly inhibited the
accumulationofpolyolincomparisontothatofthenormal
lensesandwithnosignificantstatisticaldiferenceinbetween
thetreatmentgroups(Figure2).
EfectofPNonGalactoseCataractinRat　Therateof
progressionofcataractinthecontrolandthetreatmentgroup
wascompared, anoveralgradepointaveragereferedas
opacityindex(OI)wascalculatedforeachgroupondiferent
days, basedonslitlampexamination.TocalculateOI,
normaleyesweregivennopoint, stageI, II, IIandIVwere
given1, 2, 3 and4 pointsrespectively.
Thediferentstagesofcataractinbothcontrolandtreated
groupsonvariousdaysareshowninFigure3.Theresultsof
thepresentstudyshowedthat100% eyesinthecontrolgroup
hadopacitybythe30thdayoftheexperiment.Oncomparing
theratesofprogressionofcataractinthetreatmentgroups,
onsetwasfoundtobesignificantlydelayedascomparedtothe
controlgroup.Itwasobservedontheendof30thdaynota
singleeyewasfoundinStageIVcataractinthedoseof75mg/kg
bodyweight.Thesamegroupshowed6.25% eyeswerein
normal;62.50% eyeswereinStageI, 18.75% eyeswerein
StageIIand12.50% eyeswereinStageII(Figure4).
DISCUSSION
Althoughcataractisthemostprevalentdisorderleadingto
visualimpairment, pharmacologicalinterventiontoinhibitor
todelaythelensopacificationisyetattheexperimentstage.
Severalfactorsareinvolvedintheinductionofthisdisease
process, butexactmechanismofcataractformationisstilnot
veryclear.Studiesareongoingtoexplorethemechanismof
cataractogenesisusingvariousmodelsofcataract.Among
variousexperimentalmodels, thegalactosemodeliscommonly
used, asitproducesagreaterincreaseinitsreducedform,
galactitol, thandoesglucoseandthefactthatgalactitoldoes
notfurthermetabolizeasdoessorbitol, thereducedformof
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glucose[ 25] .Galactosemodelisreasonabletoassumethatthe
factorsinitiatingthegalactosecataractinyoungratsarevery
similartothoseinvolvedinthehumangalactosecataract[ 26] .
Thelensopacitiesinratsthatarefedgalactose, likethosein
humangalactosemicsubjects, slowlydisappearwhenratsare
placedondietsfreeofgalactose.Threeposiblemechanisms
thatmaybeinvolvedincataractformationasaresultof
hyperglycemiaorhypergalactosemiaarethepolyolpathway,
oxidation, andnon-enzymaticglycation[ 27] .
Phylanthusniruriisanherbfoundincentralandsouthern
India, al partsoftheplantareused medicinaly. Its
beneficialefectsforliverespecialyinthetreatmentof
jaundicehavebeenprovedinclinicalstudies[ 28] .Itposseses
excelenthypoglycemic, antioxidantanddiureticproperties[ 20, 29] .
Therefore, wehaveinvestigatedphylanthusniruriforitsanti-
cataractpotentialagainstgalactose-inducedcataractinrats.
TheamountofGSHinthelensdecreasesalmostineverytype
ofcataract.TheroleofGSHinthemaintenanceoflensclarity
isofconsiderableinterest;whichservesthemajorantioxidant
functioninthelensandkeepstheproteinsintheirreduced
form[ 30, 31] .WefoundthatthelevelsofGSHincreasedinthe
lensesoftheratstreatedwithphylanthusniruri, whichmay
directly/ indirectly inhibitthe consumption of GSH.
Alkaloids, flavonoidssaponinsandphenoliccompoundswere
foundtobepresentinphylanthusniruri[ 32] theanticataract
activityassociatedwithextractofthisplantmaybeatributed
tothepresenceoftheseconstituents.Sugarcataractformation
isasociatedwithdiabetesandgalactosemiahasbeenlinked
tothealdosereductasecatalyzedproductionofpolyols,
sorbitol, and galactitol from glucose and galactose
respectively.Accumulationofhighconcentrationsofpolyolsin
thelensleadstoexcessivehydration, gainofsodium, andlos
ofpotassiumionsduetoanincreaseinintracelularionic
strength[ 33] .Alsothereisalosofmembranepermeability
andleakageoffreeaminoacids, glutathione, myoinositol,
andothersmalmolecularweightsubstances.Theresulting
hyperosmoticstressassociatedoxidativeinsultispostulatedto
be the primarycause forthedevelopmentofdiabetic
complicationssuchascataract[ 34] .Evidencehasshownthat
therewasasignificantraiseinpolyolsingalactosemicrats.
Inthepresentinvestigationpolyollevelwassignificantly
decreasedinPNtreatedratlensesandwehavealsofound
thatthe PN extract was more efective in lower
concentration.
TheanticataractogenicefectofPNwasconfirmedfromthe
resultsofinvivostudy.Inthepresent invivostudy, oral
administrationofPN showedsignificantprotectionagainst
cataractformationintreatedrats.Theanticataractpotentialof
PNseemstoberelatedtoitsantidiabeticpropertyasevident
fromtheresultsofinvitrorganculturestudies.
In conclusion, PN showed anticataractactivity against
galactosecataractinexperimentalanimals.Theefectis
atributedtothemaintenanceofGSHaswelasinhibitingthe
accumulationofpolyolsinthelens.Thispreliminarystudyis
encouraging, butfurtherstudyisrequiredtoextrapolatethe
useofPNinhumanbeingsfortheprophylaxisorthetreatment
ofhumancataractogenesis.
Acknowledgements:WeacknowledgetheIndianCouncilof
MedicalResearchforthefinancialsupport.Theauthorsare
thankfultoPromedExportsPvtLtdforthesupplyof
standardizedextractofPN.
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珠子草对大鼠半乳糖性白内障的抑制作用
SureshKumarGupta1 , VivekanandhanKalaiselvan2 , Ashish
Sharma1 , SushmaSrivastava1 , ShyamSunderAgrawal1
(作者单位:1印度新德里 , 德里药剂科学研究所眼科药理学研
究室;2印度哥印拜陀 ,哥印拜陀医学院 , 哥印拜陀医学中心和
医院)
通讯作者:VivekanandhanKalaiselvan.vivekarts@redifmail.com
摘要
目的:本研究旨在评价珠子草在体外及体内抑制半乳糖性
白内障的作用。
方法:研究珠子草提取液在体内及体外抑制半乳糖性白内
障的作用。 300g/L半乳糖饮食诱导半乳糖性白内障大
鼠 , 75, 150及 300mg/kg3剂量水平口服珠子草。在体外
小鼠晶体培养基中掺入半乳糖(30mmol/L)建立渗透性应
激 ,研究珠子草 (720及 880μg/mL)对谷胱甘肽(GSH)及
山梨糖醇水平的影响。
结果:在体内 ,珠子草显著延缓的白内障的发生和发展。
除了延缓各期白内障的发展 ,直至试验周期结束 ,低剂量
组未发展至 IV期。在体外 , 880μg/mL珠子草培养晶状
体显示较高浓度的 GSH和山梨糖醇浓度的降低。在体
内 ,与对照组比较 , 75mg/kg珠子草显著延缓了白内障的
发生和发展。
结论:在试验模型中珠子草延缓了白内障的发展。然而 ,
将其延伸运用在人类预防白内障需要进一步的研究。
关键词:半乳糖性白内障 ,谷胱甘肽 ,山梨糖醇 ,珠子草
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