全 文 ： 英语园地
Receiveddate:2007-06-03; Reviseddate:2007-10-20
Briefintroductionofauthor:ZHOUYING, 1971-, Female, Ph.D.Asso-
ciateProfessorofGuizhouUniversity, Supervisorforpostgraduates.There-
searchworkisfocusingonthenaturalproducts.
IdentificationoftheAntibacterialActivityfromLoHanKuoFruit
HOUYing1 , GUOBai-shu2 , ZHENGYan2 , HUANGChi-fu2＊
(1.CollegeofLifeScience, GuizhouUniversity, Guiyang, 550025, China;2.ColegeofDentistry,
UniversityofKentucky, Lexington, Kentucky40503, U.S.A.)
Abstract:ObjectiveThisresearchintendedtostudyandidentifytheantibacterialactivityofLoHanKuofruit.
MethodsThefruitextractwaspreparedfromdryLoHanKuofruitandthenscreenedforantibacterialactivityu-
singacolorimetricdetectionmethod.Tofurtherisolatethebioactivefractions, thefruitextractwasseparatedinto
smalerfractionsviahigh-performanceliquidchromatography(HPLC)andthefractionsweretestedagainfor
antibacterialactivity.Thepositivefractionsshowingantibacterialactivitywerefurthertestedbybloodagarplating
method.ResultsLoHanKuofruitextractexhibitedstrongantibacterialactivityagainsttheoralbacteria, S.mu-
tans.AfterseperatedbyHPLC, twomajorHPLCfractionsofLoHanKuofruitextract, fraction18 ～ 19and34 ～
35 , demonstratedsignificantinhibitionfororalbacteriaS.mutans, whileotherfractionsexhibitedsome, butnot
completeinhibition.Furthermore, theantibacterialactivityseemstobenotrelatedwithmogrosidesbecausethe
puremogrosideVexhibitednoantibacterialactivity.ConclusionThisisthefirststudydemonstratingthatLoHan
Kuofruitextractexhibitedstrongantibacterialactivitybutnotduetothepresenceofmogrosides.
Keywords:Momordicagrosvenori; HPLCfractionation; Antibacterialactivity; Mogrosides
Clcnumber:R285.5 Documentcode:A ArticleID:1008-0805(2008)06-1544-03
1 Introduction
Dentalcariesisthepredominantcauseoftoothlossinchildren
andyoungadults.Cariesisabacterialinfectioncausedbyspecific
bacteria, albeit, thisisnotlimitedtoamonospecificpathogen[ 1 , 2].
Thepresenceofmutansstreptococciandlactobacilihasbeenasso-
ciatedwithcariesdevelopment.Althoughtreatingoralbacteriawith
conventionalantibioticsisanoption, plant-basedantibiotics
productscouldbeimportantalternativestothetraditionalantibiotics
tocombattherecentdevelopmentofantibiotics-resistantbacterial
strainsduetotheoveruseofover-the-counterantibiotics[ 3～ 5].
PlantmaterialsandplantpartshavebeenusedasmedicineinChina
totreatdiseasesforcenturies.Plantsaretherichsourcefordrugdis-
coveryduetothesecondarymetabolitesproduced.Plant-basedan-
tibioticsproductscanserveasanimportantsourceforfuturethera-
peutictreatmentofinfectiousdiseases.
LoHanKuo(Momordicagrosvenori)hasbeenusedinChinafor
centuriesassweeteningagent[ 6].LoHanKuofruitisalsousedto
treatsorethroatandcoughingformanyyears.However, verylitle
criticalscientificresearchhasbeenconductedtoevaluatethepoten-
tialtherapeuticfunctionsofLoHanKuocomponents.Somestudies
reportedthatthesweeteningagentmogrosidesaretheprincipalcom-
ponentsfortheantibacterialactivityofLoHanKuofruit, especially
mogrosideV [ 7 ～9].Butthosestudieseitherlackofgoodbioassaysor
pureLoHanKuofruitextractsamples.Thosestudieswereincom-
pletebecauseonlyfruitextractorpartialypurifiedmogrosideswere
used.
Thisstudycriticalyevaluatedtheanti-bacterialactivityofLo
HanKuousingHPLCfractionsandpuremogrosideVcompound.
HPLCfractionationwasperformedtoisolatefruitextractintofrac-
tions, andthebioactivitiesofthesefractionsweretestedwithfunc-
tionalbioassays.OurexperimentdatademonstratedthattheLHKex-
tractnotonlyhadtheintensivesweeteningcapacity, butmightalso
havepotentialhealthbenefits, includinginhibitingthegrowthof
Streptococcusmutansthatwouldbepredictedtoreflectananti-caries
activity.
2 MaterialsandMethods
2.1 BacterialstrainandgrowthconditionOralbacteriawasusedin
thisstudy.Streptococcusmutans(ATCC25175)waspurchasedfrom
ATCC.TSBYEmediaandAnaerobeBrothwerepurchasedfromOx-
oidLtd.Growthconditionswereat37℃ inanaerobiccondition.
2.2 InstrumentsHPLCcolumn:PhenomenexGemini5μlC18 , Sys-tem:Waters510 pumps[ 2] , Waters484TunableAbsorbanceDetec-
tor;Microplatereader:MRXplatereader(DynatechLaboratories).
Evaporationequipment:SavantSC-110 SpeedVac, SavantRT-
100 RefrigeratedCondensationwithaSavantVP100 oilpump;water
purificationequipment:Mili-Q;
2.3 ChemicalsMethanol, resazurinandethanolwerepurchased
fromSigma, USA.BloodagarplateswereobtainedfromRemelCom-
pany, USA.
2.4 LoHanKuoextractDryLoHanKuofruitwaspurchasedfrom
TheLuohanguoCompanyofChineseGuangxiYongfu.LoHanKuo
fruitextractwaspreparedfromLoHanKuofruitbycrushing10grams
ofdryLoHanKuofruitusingamortarandpestleandfolowedby
adding20 mlof50% EtOH.LoHanKuoextractwasallowedto
shakefor24 hoursinaflaskbeingfilteredthroughfilterpaper.One
mloffilteredextractwasthenlyophilized8 hoursintodrypowder.
Thedriedextractobtainedthroughthelyophilizationprocesswasre-
suspendedin100μlof50% EtOH.
2.5 CelldensitymeasurementofS.mutanscultureTheefectof
LHKextractonthegrowthofS.mutanswasexaminedbymeasuring
thecelldensityatOD600nmoftheculturesinTSBYEmedia, andwascomparedtogrowthinanequivalentconcentrationofsucroseinTS-
BYEmedia.Briefly, overnightbacterialculturewasdiluted10 times
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infreshculturemedia.VariousconcentrationsofLHKextract(10,
5, 0 mg/ml)weresuspendedintheTSBYEmedia.500μlofanS.
mutansovernightculturewasinoculatedintoculturetubescontaining
5 mloftheLHKsolutionsandincubatedat37℃ inananaerobic
chamberfor24hr.SucroseinTSBYEmediaandTSBYEmediaalone
wereusedaspositiveandnegativecontrolsrespectively.At24 hr,
theOD600nmoftheculturesolutionsweremeasured.
2.6 HPLCpurificationprocedure 300 mloftheLHKextract
(165mg/ml, dissolvedinwater)wasfilteredwithULTRAFREE-
MC0.45μmFilterUnitandwasinjectedusingWaters717plusAu-
tosamplertotheWaters510 HPLCsystem(containsWaters510
HPLCPump, Waters484TunableAbsorbanceDetector, WatersSys-
temInterfaceModuleandMilennum 32 software).AnRP18 7 μm,
19 mm×150 mmWatersSymmetryColumnwasused.Flowrate:1.
0 ml/min.Withastepgradientusingmixturesofmethanol/water,
fiftyfractionswerecolectedwith(4 mlperfraction)Spectra/Chrom
CF-1 FractionColector.Alfractionsweredriedunderroomtem-
peratureusingevaporatingequipment(containsSavantSC-110
SpeedVac, SavantRT-100 RefrigeratedCondensationandSavant
VP100oilpump).
2.7 BioassayscreeningofLoHanKuofractionsEachfraction(1
ml)fromtheHPLCwaslyophilizedandtestedagainstS.mutansu-
singthehighthroughputscreening(HTS)method(10).Lyophilized
extractsweredissolvedin50μlof50% EtOHandtestedforactivity
throughtheHTSprocedurebyadding1 μlofeachextracttoselected
welsofa96 -welplatecontaining100μlofTSBYEmediumand
10% ofbacteriafromtheovernightculture.Theplatewasthenincu-
batedinananaerobicchamberbetween16 and18hours.Afterincu-
bation, 3μlofthecolorimetricindicatorresazurinwasaddedtoeach
wel.Thelivebacteriacouldmetabolizetheblueresazurinintopink
color, whilethedeadbacteriacouldnot.Theplatewasthenalowed
toincubatefortheresazurintobemetabolizedbythebacteriaprovi-
dingthechangeincolor.WelscontainingtheS.mutansrequired1
hrofincubation.AreadingusingaDynex96 welplatereaderis
takenforeachbacteriaat600nm.Typicallywellswithviablecelwill
rangeinpinkcolorgivinganOD595 readingof0.200to0.600.Welswith nonviablebacteriawillbebluegivinganOD595 readingabove2.000.
2.8 BloodagarplatingFractionswithbluecolorsinthe96 -well
platethatshowedbiologicalactivityviatheHTSwerediluted10,
000xandplatedinduplicateonbloodagarplates(Remel).Con-
trolswerealsoplatedalongwithwellsthatdidnotshowactivitya-
roundtheactiveones(e.g.Iffraction18 showedactivitywewould
platedfractions17, 18, and19.)Theplateswereincubatedat37℃
inananaerobicconditionfor48 hours.Thecolonies(CFU)were
countedoneachagarplateaftertheincubation.
3 Results
TheefectofLHKextractonthegrowthofS.mutanswasexam-
ined.LHKfruitextractdemonstratedadose-responseinhibitionof
S.mutansgrowth(Fig.1)whencomparedtothecontrolculturesof
sucroseinTSBYEmediainovernightculture.TheLHKextractre-
ducedthedensityofS.mutans.LHKconcentrationof10mg/mlsig-
nificantlyreducedtheS.mutansdensityby30%.
PreparativeHPLCofLoHanKuofruitextractwasperformedand
fiftyfractionswerecolected(Fig.2).Fig.3 displaystheprofileof
thisseparationthatwassubsequentlyusedtoexaminebiologicalfunc-
tionsoftheextractfractions.
BioassayforHPLCfractionsofLoHanKuoextractwasconduc-
tedin96-welmicroplates(350 μlwellvolume)inananaerobic
chamberat37℃.Briefly, 20 μlofS.mutansovernightculturewas
addedtoeachwellcontainingvariousconcentrationsofsucrose.The
ODofeachwellwasmeasuredatbaseline.IndividualHPLCfractions
(5 μl)wereaddedtoeachwelandincubatedovernight.Resazurin
wasthenaddedtoeachweltodetectmetabolicactivityrelatedtothe
amountofgrowththathadoccurred.Theresults, Fig.3, demon-
stratesignificantinhibitoryactivityoffractioncolectedat19 and35
min.Fig.4 showedtheantibacterialprofilesofthefiftyfractionsof
HPLC.Bloodagarplateswereusedtofurtherconfirmtheantimicro-
bialpropertiesofHPLCfractions.TheCFUonbloodagarplateswere
countedandrecorded.HPLCFraction18, 19and34, 35 hadthefe-
westCFUs.Fig.5 showsanexampleofLHKfraction#35 thatcan
significantlyinhibitthegrowthofS.mutans.Comparedtothecon-
trol, LHKfraction#35 showsthebacteriastaticactivity.Purifiedmo-
grosideVexhibitednoinhibitionagainsteitheroralbacteria, A.acti-
nomycetemcomitansandStreptococcusmutans(Fig.6).
Fig.1 DosageeffectofLHKextractonS.mutans
Fig.2 PreparativeHPLCseparationprofileofLHKextract
4 Discussion
LoHanKuoextractexhibitedantibacterialactivityagainstS.
mutans.LoHanKuofruitextractwasfurtherpurifiedwithHPLC.
Thebioactivitieswereidentified.TheantibacterialactivitiesofLo
HanKuofruitswereidentifiedintwomajorfractions.Thefractions
18 ～ 19 aremorepolarthanfractions34 ～ 35.Curentresearchwork
isfocusedinisolatingandidentifyingthebioactivecompoundsin
thosefractions.
Surprisingly, themogrosideVdidnotshowanyantibacterialac-
tivityagainstoralbacteria.Ourstudyindicatestheantibacterialactiv-
itydoesnotcomefrommogrosides.Thisexperimentaldataraisedthe
possibilitythatthestudiesonmogrosidesofLoHanKuoextractby
otherswereincompletebecausetheyusedLoHanKuoextractinstead
ofthepuremogrosides[ 7 , 8].Eventhoughthemogrosidesarethemain
componentsofLoHanKuofruits, itdoesnotmeantheantibacterial
bioactivitiescomefrommogrosidesasothercomponentscouldbere-
sponsiblefortheantibacterialactivityinLoHanKuofruit.
ThisisthefirststudythatdemonstratedmogrosideVhadnoan-
tibacterialactivity.Whethertheothermogrosides, suchasmogroside
ItoIV, haveanyantibacterialactivityarestiltobedetermined.Itis
verylikelyotherchemicalcomponentsinLoHanKuofruitarere-
sponsiblefortheantibacterialactivity.Futureresearchworkwillbe
focusedinisolatingandidentifyingthebioactivecompoundsfromLo
HanKuofruit.
Dentalcariesisthepredominantcauseoftoothlossinchildren
andyoungadults.Cariesisabacterialinfectioncausedbyspecific
bacteria, albeit, thisisnotlimitedtoamonospecificpathogen.Plant
-basedantibioticsproductsfromLoHanKuofruitcouldbeim-
portantalternativestothetraditionalantibioticstocombattherecent
developmentofantibiotics-resistantbacterialstrainsduetotheover-
useofover-the-counterantibiotics.LoHanKuofruitshavebeen
usedasmedicineinChinatotreatsorethroatandStrepbacterialin-
fectionforcenturies.TheantibacterialcomponentsisolatedfromLo
HanKuofruitscanserveasanimportantsourceforfuturetherapeutic
treatmentoforaldiseases.
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Allwellscontain:bluecolornometabolicactivityoflivebacteria,
pinkcolorpositiveforbacterialgrowthandmetabolism.Thedark
purplecolorwellisforHPLCfraction#19 andthebluecolorwelis
forHPLCfraction#35.
Fig.3 TheHTSofanti-bacterialactivityusingResazurinasameta-
bolicdetectionsystem.
Fig.4 LHKHPLCfractionsvs.S.mutans
10 μlofacontrolwellwithoutLHKfraction(Left)orbluewelwith
LHKfraction#35(Right)wasdilutedto10-4and50μlwasdistrib-
utedontoabloodagarplate.Theleftplatecontained10 timesmore
S.mutansCFUsthantherightplate.
Fig.5 TheBloodagarplatingresultsofLHK#35andthecontrol
Acknowledements:Thisresearchwassupportedbythegrant
R41DE17265-01 fromtheNIH/NIDCR, U.S.A.Wewouldlike
tothankDr.Marshal, McGillUniversity, Canadaforprovidingusthe
purifiedmogrosideVsample.
CFUsofcontrolwithoutLHKfraction(Left)orpuremogrosideV
(Middle)orfraction#35(Right).Controlandmogrosideplatescon-
tained10 timesmoreS.mutansthanthefraction#35 plate.
Fig.6 CFUinbloodagarplates
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收稿日期:2007-06-06; 修订日期:2007-10-20
作者简介:周 英(1971-),女(汉族),贵州贵阳人 ,现任贵州大学副教授 ,硕士生导师 ,博士学位 ,主要从事天然药物化学的研究工作.
＊通讯作者简介:黄赤夫(1961-),男(汉族),湖南湘潭人 ,现任美国肯塔基大学医学中心副教授 ,博士学位 ,主要从事天然药物研究工作.
罗汉果果实中抑菌活性组分的研究
周 英 1 , 郭白苏 2 , 郑 燕2 , 黄赤夫 2＊
(1.贵州大学生命科学学院 ,贵州 贵阳 550025; 2.美国肯塔基大学口腔学院 40503)
摘要:目的 确定并纯化罗汉果果实中的抑菌组分。方法 制备罗汉果干果的粗提物 ,并通过一种快速的颜色指示法检测
其抗菌活性。罗汉果果实提取物对变形链球菌(S.mutans)有很强的抑制活性。为了进一步分离活性组分及成分 ,通过
HPLC对其进行分离 , 并通过上述筛选方法对不同组分的抑菌活性进行快速筛选 , 对有活性的组分通过血琼脂法进行验
证。结果 罗汉果果实提取物对变形链球菌(S.mutans)有很强的抑制活性;将其粗提物通过 HPLC进行分离得到 50个
组分 , 罗汉果果实提取物的两个 HPLC组分 , #18-19和#34-35对于变形链球菌(S.mutans)有很强的抑制活性 ,而其它
的组分没有或只有很弱的活性。罗汉果苷 V无抑菌活性。结论 罗汉果果实提取物具有较强的抑菌活性;罗汉果果实的
抑菌活性不是由罗汉果苷 ,而是由罗汉果中另外的活性组分或成分产生的。罗汉果果实具有进一步开发为抗菌药物和
保健品的潜力。
关键词:罗汉果果实; HPLC组分; 抗菌活性; 罗汉果苷
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