全 文 ： 英语园地
Receiveddate:2007-06-06; Reviseddate:2007-12-30
Briefintroductionofauthor:ZHOUYing, 1971-, Female, Ph.D.Associ-
ateProfesorofGuizhouUniversity, Supervisorforpostgraduates.Theresearch
workisfocusingonthenaturalproducts.
CommunicationauthorsynopsisHUANGYi-fu, 1961-, male, borninChang-
sha, Huanna, PH.D.AssociateProfessorofUniversityofKentucky(U.S.
A.).Theresearchworkisfocusingonthebiopharmaceuticalsandnatural
products.
IdentificationoftheAntibacterialActivityfromLeaf, VineandRootof
LoHanKuo(Momordicagrosvenori)
ZHOUYing1 , HUANGChi-fu2＊
(1.DepartmentofPharmaceuticalEngineering, CollegeofLifeScience, GuizhouUniversity, Guiy-
ang, 550025, China;2.ColegeofDentistry, UniversityofKentucky, Lexington, Kentucky40503,
U.S.A.)
Abstract:ObjectiveThisresearchintendedtostudyandidentifytheantibacterialactivityofLoHanKuoplant
(Momordicagrosvenori)byextractingandpurifyingbioactivesubstancesfromvariouspartsofLoHanKuoplant.
MethodsVariousextractswerepreparedfromLoHanKuofruit, leaf, vine, androot, andtheantibacterialac-
tivityoftheseextractsweretestedusingacolorimetricdetectionmethodandagarplating.ResultsExtractsofLo
HanKuoalpartsexhibitedstrongantibacterialactivityagainsttheoralbacteria, S.mutans.Tofurtherisolate
thebioactivefractions, theextractswerepurifiedwithAmberlitechromatographyandthepurifiedfractionswere
testedforantibacterialactivity.Thepositivefractionsshowingtheantibacterialactivitywerefurthertestedby
bloodagarplatingmethod.ConclusionTheexperimentaldatademonstratedthatextractsofLoHanKuoleaves,
vines, fruitandrootsexhibitedstrongantibacterialactivities.ThisstudysuggeststhatvariouspartsofLoHanKuo
havethepotentialtobeusedforpharmaceuticalpreparationsanddietarysupplementstotreatvarioushumanail-
ments, suchasinfection.
Keywords:Momordicagrosvenori, Colorimetricdetectionmethod, Amberlitechromatography, Antibacterialac-
tivity
Clcnumber:R285.5 Documentcode:A ArticleID:1008-0805(2008)07-1797-03
1 Introduction
ThroughoutthehistoryofChinapeoplehaveusedawidevariety
ofplantsandherbstohelpprevent/treatchronicandacutediseases,
improveimmunitytoinfection.Oneplantthathasbeencommonly
usedistheLoHanKuofruit(Momordicagrosvenori).LoHanKuo
fruitsaresmal-sizedfruitswithverysweettaste.LoHanKuofruits
arenativetoGuangxiGuilinregion, alsogrowninHunan, Guizhou,
Jiangxiandmanyotherprovinces.TheLoHanKuofruithasbeenu-
tilizedforcenturiesastraditionalmedicinetotreatsorethroatand
coughing.LoHanKuofruitsaretypicallyharvestedinlatefaland
driedimmediately.ThedriedLoHanGuofruitsareusedaswholeor
grindedaspowder, typicalyusedinbeverage, suchasteaandsoup.
ManyatemptstoidentifytheactivecompoundsfromLoHan
Kuofruitsweremadeoverintheearlyfifties.Thoseeffortsresulted
intheidentificationofintensivesweeteningcomponents, knownas
mogrosides(mogrosideItoVI).Thesecompoundsaretriterpenoids,
withglucosemoietyatachedtoachemicalstructurecalledmogrol
group[ 1].ThebiologicalactivitiesofLoHanGuofruitshavebeen
studied[ 2 ～ 5].PreviousresearchdemonstratedLoHanKuofruitex-
tracthadantibacterialactivity.Althoughsomepublicationsreport
somebioactivitiesofLoHanKuofruits, litleresearchhasbeendone
onthenon-fruitpartsofLoHanKuo.Traditionally, LoHanGuo
fruitsareharvestedinlatefalandthedriedvine/leafarecutoff.
Therootsarecoveredwithsoilandprotectedfromseverewintercold
condition.Aslitlestudieshavebeendoneonthenon-fruitpart,
theLoHanKuoleafandvinearenotutilizedatal.Verylitlebioac-
tivityareknownaboutthenon-fruitpartsofLoHanKuo.
Thisstudyaimstopreparetheextractsandstudytheantimicro-
bialactivityofLoHanGuoleaf, vine, androotextracts.Ourexperi-
mentaldataindicatedthatextractsofLoHanKuoleaf, vine, androot
havepotentantimicrobialactivities, especialyagainstoralorgan-
isms, suchasStreptococcusmutansandA.actinomycetemcomitans.
Thepotentanti-microbialactivitiesarefoundinthefruit, leaf,
vine, androotofLoHanKuoplant.Thisisthefirstexperimental
studythatdemonstratedthepotentantibacterialactivitiesexistingin
thenon-fruitpartsofLoHanKuo.Thepotentanti-microbial
chemicalsarenotthesweeteningagents, suchasthemogrosidesof
thefruitasthebioactivitywidelyexistsintheleaf, vine, androotof
LoHanKuo.Theusageofnon-fruitpartofLoHanKuotoproduce
antimicrobialchemicalswilhavemajoreconomicimpactastheleaf/
vineisverycheaporitcanbeobtainedataverylowcost.
2 MaterialsandMethods
2.1 InstrumentsAmberliteXADpurificationcolumn, Phenomenex
Gemini5μlC18 , System:Waters510 pumps[ 2] , Waters484 Tuna-
bleAbsorbanceDetector;Microplatereader:MRXplatereader(Dy-
natechLaboratories).Evaporationequipment:SavantSC-110
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SpeedVac, SavantRT-100RefrigeratedCondensationwithaSavant
VP100 oilpump;waterpurificationequipment:Milli-Q;Amberlite
XADresin, Methanol, ResazurinandEthanolwerepurchasedfrom
Sigma, USA.BloodagarplateswereobtainedfromRemelCompany,
USA.
2.2 BacterialstrainandgrowthconditionTwooralbacterialspecies
wereusedinthisstudy.A.actinomycetemcomitans(ATCC714)and
Streptococcusmutans(ATCC 25175), werepurchasedfromATCC.
TSBYEmediaandAnaerobeBrothwerepurchasedfromOxoidLtd.
Growthconditionswereat37℃ inanaerobiccondition.
2.3 LoHanKuoextractionFruit, leaf, vine, androotofLoHan
Kuowerecollectedfromfieldanddried.Plantextractswereprepared
fromLoHanKuopartsbycrushing10 gramsofdryLoHanKuoma-
terialsusingamortarandpestle, andfolowedbyadding20 mlof
50% EtOH.ExtractsofvariousLoHanKuopartswereallowedto
shakefor24 hoursinaflask.Afterfilteredthroughfilterpaper, 1ml
offilteredextractwasthenlyophilized8 hoursintodrypowder.The
driedextractsofleaf, vine, androotwereobtainedthroughthelyoph-
ilizationprocessandwerere-suspendedin100μlof50% EtOH.
2.4 Chromatographypurificationprocedure 100 gramsofLoHan
Kuoleaf, vine, androotwereseparatelygrindedandboiledin500
mlofwaterfor30 min.Thewaterextractionwasrepeatedforthree
times.Theextractsolutionwasfilteredandthesupernatantwas
saved.1000mlofthesupernatantwerepassedthrougha100mlnon-
polarAmberliteXADcolumnwhichwaspre-equilibratedwithwa-
ter.Then, thecolumnwaswashedwith1000mlwatertoremovenon-
bindingmaterials.Thecolumnwaselutedwith10%to100% ethanol
solution.The200 mlof10 fractionswerecollectedandanalyzedfor
antibacterialactivity.
2.5 BioassayscreeningofLoHanKuofractions 1 mlofeachfrac-
tionpurifiedfromAmberliteXADchromatographywaslyophilizedand
testedagainstS.mutansusingtheHTS(high-throughputscreen-
ing)proceduredescribedbelow [ 6].Lyophilizedextractsweredis-
solvedin50 μlof50% EtOHandtestedforbioactivityasfollowing
procedurebyadding1μlofeachextracttoselectedwellsofa96 -
welplatecontaining100μlofTSBYEmediumand10% ofbacteria
fromtheovernightculture.Theplatewasthenincubatedinananae-
robicchamberbetween16 and18 hours.Afterincubation, 6 μlof
thecolorimetricindicatorresazurinwasaddedtoeachwell.Thelive
bacteriacouldmetabolizetheblueresazurinintopinkcolor, whilethe
deadbacteriacouldnot.Theplatewasthenallowedtoincubatefor
theresazurintobemetabolizedbythebacteriaprovidingthechange
incolor.WelscontainingtheS.mutansrequired1hrofincubation.
AreadingusingaDynex96 wellplatereaderwastakenforeachbac-
teriaat600nm.Typicallywelswithviablecelwilrangeinpinkcol-
orgivinganODreadingof0.200 to0.600.Welswithnonviable
bacteriawilbebluegivinganODreadingabove2.000.
2.6 BloodagarplatingFractionswithbluecolorsinthe96 -well
platethatshowedbiologicalactivityviatheHTSwerediluted10000x
andplatedinduplicateonbloodagarplates(Remel).Controls
werealsoplatedalongwithwelsthatdidnotshowactivityaroundthe
activeones.Theplateswereincubatedat37℃ inananaerobiccon-
ditionfor48hours.Thecolonies(CFU)werecountedoneachagar
plateaftertheincubation.
3 Results
TheeffectofvariousLHKextractsonthegrowthofS.mutans
wasexamined.TheextractsofLoHanKuoleaf, vine, stemandroot
demonstratedsignificantinhibitionofS.mutansgrowth.Fig.1shows
antibacterialactivityofLoHanKuoleafextract.Ascomparedtoneg-
ativecontrol, thestrongestbioactivityfractionsarefrom 60% to
100%ethanol.Fig.2 showsthatLoHanKuofruitextracthassimi-
larantibacterialactivityprofile.Ascomparedtonegativecontrol, the
strongestbioactivityfractionsarefrom 70% to100% ethanol.Anti-
bacterialactivityofLoHanKuorootextractalsoarefrom 70% to
100% ethanol(Fig.3).Fig.4 showsantibacterialactivityofLo
HanKuovineextract, thestrongestbioactivityfractionsareinthe
rangefrom60% to100% ethanol.
Tofurtherinvestigatethechemicalpropertyofthebioactivecom-
ponentsofLoHanKuo, thebioactivefraction90% and100%ofthe
leafextractwerecolected.Afterremovingtheethanol, fractionswere
fractionatedbyetherthreetimes, anetherandwaterfractionswere
obtained.Theetherfractionhadthesignificantantibacterialactivity,
whilethewaterfractiondidnothaveanyantibacterialactivity(Fig.
5).Theantibacterialcomponentsappearedtobelowpolarcom-
pounds.
Amberlitecolumnfractionsareindicatedasthepercentageofethanolconcentra-
tion.Controlisshownontheleftwherenoextractwasused, onlyS.mutans
areinoculatedonthebloodagarplates.Thenumberofcoloniesformedonthe
platesareshownasCFU.
Fig.1 EfectsofLHKleafextractonS.mutansgrowth.
Amberlitecolumnfractionsareindicatedasthepercentageofethanolconcentra-
tion.Controlisshownontheleftwherenoextractwasused, onlyS.mutans
areinoculatedonthebloodagarplates.Thenumberofcoloniesformedonthe
platesareshownasCFU.
Fig.2 EffectsofLHKfruitextractonS.mutansgrowth.
Amberlitecolumnfractionsareindicatedasthepercentageofethanolconcentra-
tion.Controlisshownontheleftwherenoextractwasused, onlyS.mutans
areinoculatedonthebloodagarplates.Thenumberofcoloniesformedonthe
platesareshownasCFU.
Fig.3 EfectsofLHKvineextractonS.mutansgrowth.
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Amberlitecolumnfractionsareindicatedasthepercentageofethanolconcentra-
tion.Controlisshownontheleftwherenoextractwasused, onlyS.mutans
areinoculatedonthebloodagarplates.Thenumberofcoloniesformedonthe
platesareshownasCFU.
Fig.4 EffectsofLHKrootextractonS.mutansgrowth.
Amberlitecolumnfractions90%and100%wereextractwithether.Theether
andwaterfractionsweretestedagainforantibacterialactivity.Controlisshown
ontheleftwherenoextractwasused, onlyS.mutansareinoculatedonthe
bloodagarplates.Thenumberofcoloniesformedontheplatesareshown
asCFU.
Fig.5 EffectsofLHKleafextractonS.mutansgrowth.
4 Discussion
ExtractsofLoHanKuoleaf, vine, rootandfruitexhibitedanti-
bacterialactivityagainstS.mutans.ThevariouspartsofLoHanKuo
sharethesimilarantibacterialprofilesrangingfrom 60% ethanolto
100% ethanolelution.Thesimilarprofilessuggestthatthoseextracts
possiblycontainsimilarantibacterialcompoundsinleaf, vine, root
andfruit.ThisstudydemonstratedthatalpartsofLoHanKuoshare
similarantibacterialactivity.Ongoingresearchworkisfocusediniso-
latingandidentifyingthosebioactivecompoundsfromLoHanKuo
plant.
Dentalcariesisthepredominantcauseoftoothlossinchildren
andyoungadults[ 7].Cariesisabacterialinfectioncausedbyspecif-
icbacteria, albeit, thisisnotlimitedtoamonospecificpathogen[ 8].
Plant-basedantibioticsproductsfromLoHanKuoplantcouldbe
importantalternativestothetraditionalantibioticstocombatthere-
centdevelopmentofantibiotics-resistantbacterialstrainsduetothe
overuseofover-the-counterantibiotics.LoHanKuoplanthaa
beenusedasmedicineinChinatotreatsorethroatandStrepbacterial
infectionforcenturies.TheantibacterialcomponentsisolatedfromLo
HanKuoplantscanserveasanimportantsourceforfuturetherapeu-
tictreatmentoforaldiseases.AlthoughextractsofLoHanKuoleaf,
vine, rootandfruitexhibitedantibacterialactivityagainstS.mutans,
theycouldbetestedagainstmanyotherinfectiousandoralbacteria.
Acknowledgements:Thisresearchwassupportedbythegrant
R41DE17265-01 fromtheNIH/NIDCR, U.S.A.
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收稿日期:2007-06-06; 修订日期:2007-11-10
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:黄亦夫(1961-),男(汉族),湖南长沙人 ,现任美国肯塔基大学副教授 ,博士学位 ,主要从事生物制药 、天然产物方面的研究工作.
罗汉果叶茎根的抑菌活性组分的研究
周 英 1 , 黄赤夫2＊
(1.贵州大学生命科学学院 550025; 2.美国肯塔基大学口腔学院 40503)
摘要:目的 通过以活性为导向的提取分离的方法 , 首次研究罗汉果全植株中的抑菌活性组分。方法 通过提取得到罗汉
果全植株各个部分—果实 、叶 、茎和根的提取物 , 然后运用一种快速颜色指示法和血琼脂法研究检测它们的抑菌活性。
结果 罗汉果的非果实部分及果实对于口腔细菌转糖链球菌 S.mutans具有很强的抑菌活性。 为了进一步研究并分离活
性组分 , 将其粗提物通过 AmberliteXAD树脂进行分离纯化 ,再通过同样的检测方法进行抗菌活性测定 ,对有活性的组分
通过血琼脂法进行验证 。结论 罗汉果叶 、茎和根(非果实部分)以及果实都具有较强的抑菌活性;罗汉果的非果实部分
具有进一步开发为抗菌药物和保健品的潜力。
关键词:罗汉果全植株; 抗菌染色检测方法; AmberliteXAD树脂; 抑菌活性
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