全 文 ：天然产物研究与开发 NatProdResDev2009, 21:208-213
文章编号:1001-6880(2009)02-0208-06
　
　
　ReceivedOctober1, 2007;AcceptedDecember16, 2007
＊CorespondingauthorTel:86-731-8836834;E-mail:zhangyan210@
126.com
萼翅藤枝叶挥发油及其抗菌活性的研究
张　艳＊ ,杨栋梁 ,刘佳佳
中南大学化学化工学院医药生物化工所 , 长沙 410083
摘　要:萼翅藤枝叶挥发油由 GC/MS检测。 树叶挥发油的 52种成分中 , 氧化石竹烯(13.79%)、棕榈酸
(11.91%)和 β-石竹烯(10.45%)是主要成分。同时 ,树枝挥发油中的 10种成分占总量的 99.99%,其中主要的
化学成分为棕榈酸(59.18%), 亚油酸(12.70%)和邻苯二甲酸丁辛酯(8.21%)。 用滤纸扩散法 , 分别测定了
枝 、叶挥发油对 8种微生物的抑制效果。枝 、叶挥发油均具有很强的抗菌效果 , 并且抗细菌活性优于抗真菌活
性。叶挥发油比枝挥发油具有更广谱的抑菌效果 ,且对所试的大多数菌株都具有更高的活性。
关键词:萼翅藤;挥发油;GC/MS;抑菌活性;细菌;真菌
中图分类号:Q946.85;R284.1 文献标识码:A
VolatileOilsinLeavesandBarksofCalycopterisfloribunda
ZHANGYan＊ , YANGDong-liang, LIUJia-jia
InstituteofBiochemicalEngineering, ChemistryandChemicalEngineeringCollege,
CentralSouthUniversity, Changsha410083 , China
Abstract:ThevolatileoiloftheleavesandbarksofCalycopterisfloribundawasexaminedbygaschromatography/mass
spectrometry(GC/MS).Fifty-twoconstituentswereidentifiedinleavesoil;caryophyleneoxide(13.79%), n-hexade-
canoicacid(11.91%)andβ-caryophylene(10.45%)werefoundtobethemajorcomponents.Tenconstituentsiden-
tifiedrepresentabout99.98% ofthetotalbarksoil;amongthese, n-hexadecanoicacid(59.18%), linolicacid
(12.70%)andbutyloctylphthalate(8.21%)werethemajorconstituents.Theantibacterialandantifungalactivities
ofbothoilsweretestedagainsteightmicroorganismsbydiskdifusionmethodinvitro.Theoilsexhibitedstrongantimi-
crobialactivity, andwereprovedtobemoreactiveagainstthetestedbacteriathanthefungi.Theleavesoilexhibited
broaderantibacterialspectrumandhigherinhibitionactivityagainstmostlymicrobethanthebarksoil.
Keywords:Calycopterisfloribunda;volatileoil;GC/MS;antimicrobialactivity;bacteria;fungi
Introduction
C.floribundaisanendemicevergreenlianaspeciesto
Asiantropicmountains.ItisthesinglememberofCa-
lycopterisLam.includedinCombretaceae.C.floribunda
mainlydistributeatBurma, India, SingaporeandChi-
na.InChina, itonlyoccursinecosystemstypicalofthe
highlandsandIndianOceanwarmcurentofwestYun-
nanProvince, atanaltituderangingfrom300to650m
andischaracterizedbyacidiclateriticsoilandShorea
assamicaDyervegetation.Moreover, thisplantisoneof
theendangeredplantsinChina;itsdistributingatwest-
ernYunnancanpowerfulyexplainthetropicnorthern
fringequalityofplantsinthisarea, soithassignificant
valueattheChineseflorastudy[ 1] .
SeveraltherapeuticpropertiesareassignedtoCom-
bretaceaespecies, suchasinvigoratingthespleen, dia-
betescharacterizedbypolyphagia, anti-ascariasis, anti-
enterobiasisandsoon.C.floribundahasfounditsus-
ageintheAsiantraditionalmedicineincludingAyurve-
da, folkandUnanimedicine[ 2] .Thephytochemical
screeningrevealedthepresenceofflavonoids, alka-
loids, tanninsandsaponins[ 3] .Thestem-leafinfusion
isusedindysentery, feveraswelasemesisandasa
tonictohealstomachulcers.Thefruitisusedinthe
treatmentofjaundiceandtorelievepruritus[ 4, 5] , while
thetendercopper-colouredleaveshavelaxativeandan-
thelminthicproperties[ 6] .Theacetoneextractofthe
leavesistoxictoroundworms(Ascarislumbricoides)
invitroinamediumcontainingbileandsodiumcar-
bonateanalogoustointestinalenvironment.Fivecyto-
toxicbiflavonoids:calycopterin, calycopterone, isocaly-
copterone, 4-demethylcalycopteroneand4, 5-dihydroxy-
3, 3, 6, 7-tetramethoxyflavonewereisolatedfromethan-
olicextractandethylacetateextractofC.floribunda
leaves[ 7, 8] .Themainflavonoid“Calycopterin” (4, 5-
dihydroxy-3 , 6, 7, 8-tetramethoxyflavone)[ 9] hasant-
helmintic, antiviral(invitroinhibitionofpoliovirus)
[ 7] andanticanceractivity(anti-proliferativeandanti-
aromatase) [ 10] .Cytotoxicactivitywasobservedin
brineshrimp[ 10] andhumancancercellines[ 11] .
Besidesthereportofthepresenceofphenolicbifla-
vonoidcompoundslikecalycopterone, calyflorenone
andneocalycopteroneinethanolextract[ 7, 8] , thereis
nearlynopublishedinformationavailableonthephyto-
chemistryofC.floribunda.Toprovideinformationfor
furtherutilizationofthisplant, GC/MS(gaschroma-
tography/massspectrometry)andpeak-area-normali-
zationmethodwereappliedtoanalysesthechemicalin-
gredientsinitsvolatileoilsqualitativelyandquantita-
tively;additionaly, theantibacterialandantifungalac-
tivitiesofoilswerestudiedbydiskdifusionmethodin
vitroaswel.
MaterialsandMethods
Sampleextraction
TheleavesandbarksofC.floribundawerecolected
fromYingjiangCounty, YunnanProvince, thePeople s
RepublicofChinainJune2006, andthesamplewas
authenticatedbyProfessorLiuJiajiaatCentralSouth
University.Thevolatileoilswereextractedrespectively
from50 gofpreviouslyair-driedandpowdered(20
meshparticlesize)leavesandbarks.Theextraction
wascariedoutbysteamdistilationinClevengerappa-
ratusfor5 h.Theoilsobtainedweretakenwith1 mL
hexaneandthesolutionswerestoredinafreezerfor
GC/MSanalysisandbiologicaltest.
GC/MSanalysis
Analysesoftheoilsinvolvedinjectionof1 μLofthe
hexanesolutioninagaschromatographHP5890ser.I
PluscoupledwithamassspectrometerHP 5989B
Chem.Stationoperatingintheelectronimpactmodeat
70 eV, electronmultiplierenergy1600 V andion
sourcetemperature200 ℃.Forgaschromatographic
analyses, aHP-5MScapilarycolumnoffusedsilica
(30 m×0.30)andheliumascariergasataflowrate
of1.0 mL/minwereused.Temperaturesofthecolumn
wereprogrammedstartingat100℃ for1minandrai-
singto280 ℃ at8 ℃/min.Identificationofthecom-
poundswasbasedoncomparisonofthecorresponding
massspectrawithdatafromthelibrariesWiley-275and
Wiley/NBS.Relativeamountsofconstituentsinthe
volatileoilswereassumedtobeproportionaltotheare-
asunderthecorespondingchromatogrampeaks.
Antimicrobialassay
Bacterialandfungalstrains
Inordertoinvestigatetheantimicrobialeficacyofthe
volatileoils, thepathogenicbacteria:Staphylococusau-
reus, Bacilussubtilis(gram-positive), Escherichiacoli,
Pseudomonasaeruginosa(gram-negative)aswelas
pathogenicfungus:Candida albicans, Trichophyton
rubrum, Aspergilusniger, Peniciliumcitrinumwereun-
dertaken.Althemicrobialstrainswereprovidedby
MicrobialTypeCultureColection, InstituteofMicrobi-
alTechnology, CentralSouthUniversity, China.
Antimicrobialinvestigations
BacteriawereculturedinMueler-HintonAgarculture
mediumandincubatedinbiochemicalincubatorat37
℃, whereas, fungiwereculturedinPotatoDextrose
Agarculturemediumandincubatedindiurnalgrowth
incubatorat28 ℃.
Stockculturesweremaintainedat4 ℃ onthenutrient
agarslant.Activeculturesforexperimentswerepre-
paredbytransferingoneloopfulofcelsfromstock
culturestosolidnutrientagarmediumandincubated
withoutagitationfor24 hrespectively.Themicrobial
strainsweresubculturedforthreetimes, andtransfer
oneloopfulofthethirdsubculturedcelsfromsolidme-
diumtoculturesflaskofliquidmedium, thenshake
cultivatedfor24h.Theculturesweredilutedtoachieve
opticaldensitiescorrespondingto106 -107 colonyform-
ingunits(CFU/mL)bysterilewater.
Inordertodeterminetheantimicrobialactivityofthe
volatileoils, diskdifusionmethodwasfolowed.Dilu-
209Vol.21 　　　　　　ZHANGYan, etal:VolatileOilsinLeavesandBarksofCalycopterisfloribunda　
tionsoftheinoculawereculturedonsolidagarmedium
byspreading0.1 mLinoculumsuspensionuniformly;
verifytheabsenceofcontaminationandchecktheva-
lidityoftheinoculum.Sterilefilterpaperdiscs(6 mm
indiameter)weresoakedwiththerequireddosesof
undilutedvolatileoil(1, 2, 4and6μL)andplacedon
thesurfaceofinoculatedplates.Theplateswereincu-
batedat37 ℃for24h(forbacteria)andat28℃ for
48 h(forfungi)respectively.Thediametersofthein-
hibitionzoneswerethenmeasuredinmilimeters.The
diametersoftheinhibitionzonesweredesignated(-)
notdetected, fordiameterslessthan6 mm;litlesensi-
tive(+), fordiameters8-10 mm;sensitive(++),
fordiameters10-15 mm;verysensitive(+++), for
diametersmorethan15 mm.Altheexperimentswere
performedintriplicate.
ResultsandDiscussion
Chemicalanalysis
TheoilsobtainedfromleavesandbarksofC.floribun-
dawerequantitativelyandqualitativelyanalyzedby
GC/MS, thetotalioncurrentchromatogramofthevola
tileoilsfromC.floribundaleavesandbarksshowedin-
Fig.1 andFig.2.Additionaly, themainconstituentsof
thestudiedvolatileoilsandrespectiverelationpercents
aregiveninTable1andTable2.
Table1　AnalysisofvolatileoilfromtheleavesofC.floribunda
No. Compound Content(%) No. Compound Content(%)
1 2-Pentylfuran 0.86 27 α-Caryophylene 1.87
2 trans-2-(2-Pentenyl)furan 0.92 28 Cyperene 0.65
3 2, 4-Heptadienal 0.43 29 β-Ionone 2.18
4 o-Cymene 0.51 30 β-Cubebene 1.15
5 Hyacinthin 0.56 31 β-Eudesmene 0.96
6 cis-Linalooloxide 0.92 32 τ-Elemene 0.46
7 β-Linalool 0.68 33 τ-Himachalene 0.57
8 Nonanal 1.38 34 Spathulenol 2.63
9 α-Thujone 0.33 35 Caryophylleneoxide 13.79
10 2, 6-Nonadienal 0.55 36 Cedrenol 0.70
11 2-Nonenal 0.54 37 Widdrol 0.73
12 Decanal 0.87 38 Epiglobulol 0.87
13 Safranal 0.42 39 Junipercamphor 3.17
14 β-Cyclocitral 0.56 40 Myristicacid 1.40
15 Nonanoicacid 0.71 41 bis-(p-Chlorophenyl)ether 2.57
16 α-Ionene 1.01 42 Methylhinokiate 1.22
17 trans-Cinnamaldehyde 0.85 43 Hexahydrofarnesylacetone 5.22
210 NatProdResDev　　　　　　　　　　　　　　　　　　　　 　Vol.21
18 1, 1, 5, 6-Tetramethylindane 0.81 44 Dibutylphthalate 0.37
19 trans-Isoeugenol 0.35 45 Farnesylacetone 1.12
20 1, 1, 6-Trimethyl-1, 2-dihydronaphthalene 0.78 46 Isophytol 0.98
21 1, 5, 8-Trimethyl-1, 2-dihydronaphthalene 0.60 47 n-Hexadecanoicacid 11.91
22 1, 1, 6-Trimethyl-1, 2-dihydronaphthalene 0.57 48 Butyloctylphthalate 0.90
23 3-(2, 6, 6-Trimethyl-1-cyclohexen-1-yl)-2-propenal 0.77 49 Phytol 6.50
24 β-Caryophylene 10.45 50 Linolicacid 0.72
25 4-(2, 4, 4-Trimethyl-cyclohexa-1, 5-dienyl)-but-3-en-2-one 1.69 51 Oleicacid 3.78
26 α, β-Dihydropseudoionone 0.76 52 Linolenicacid 1.13
Table2　AnalysisofvolatileoilfromthebarksofC.floribunda
No. Compound Content(%) No. Compound Content(%)
1 2-Pentylfuran 1.20 6 n-Hexadecanoicacid 59.18
2 D-Limonene 5.23 7 Butyloctylphthalate 8.21
3 4-Carvomenthenol 0.92 8 Phytol 2.06
4 Myristicacid 1.22 9 Linolicacid 12.70
5 Dibutylphthalate 2.45 10 Oleicacid 6.81
Theleavesproducedgreen-yelowoilinahighyield
(1.11%;w/wondryweightbasis).52 components
thatcorespondedto96.43% ofthechromatographied
oilwereclearlyidentified.Theoilwascomposedof
71.72% ofsesquiterpeneswhiletherestweremainly
monoterpenes.Thedominantcomponentsintheoilfrom
leaveswerecaryophyleneoxide(13.79%), n-hexade-
canoicacid(11.91%), β-caryophylene(10.45%).
Thebarksproducedyelowishoilinayieldof0.60%
(w/wondryweightbasis).11 constituentswereiden-
tifiedrepresentingabout99.98% ofthetotaloil.A-
mongthese, 91.41%belongtotheclassofsesquiterpe-
nes.Themajorconstituentsweren-hexadecanoicacid
(59.18%), linolicacid(12.70%)andbutyloctyl
phthalate(8.21%).
Thus, itseemsthatvolatileoilsofleavesandbarksare
somewhatsimilarincomposition.Oneisthatn-hexade-
canoicacidistheconstituentwhichdetectedmuchin
bothvolatileoilsanalyzed, especialyinbarksoil.Mo-
reover, theoccurrenceofsesquiterpenesasthepredom-
inantvolatileoilsconstituentsisanotherchemicalchar-
acteristicofC.floribunda.While, theleavesvolatileoil
difersfromthatofthebarksbyitshighcontentof
caryophylenes, whicharescarceinbarksoil.Sofar,
duetotheparticularityofC.floribunda, itscertaintax-
oninCombretaceaeisinvestigatedbysomeresearch-
ers.Infact, Goutsiouetalshowedthatchemicalprofiles
ineachpopulationareundergeneticcontrol[ 12] , which
strengthensHarborneandTurner spointthatvolatile
terpenoidsmaybeusefulinstudiesofplantpopulation
structure[ 13] .Theabovecommentsofsimilaritiesand
diferencesbetweenvolatileoilsofleavesandbarks
needtobetakenwithcautionaswelastaxonomicpo-
tentialities, becauseofthepossibilityofinfraspecific
variationoftennotedindistributionsofvolatileterpe-
noids, thisinfavorofustofurtherinvestigatetheche-
motaxonomicoccurrenceofC.floribunda.
Table3　AntimicrobialactivitiesoftheleavesandbarksvolatileoilsofC.floribunda
Microbialstrains Leavesoil(μL) Barksoil(μL)
1 2 4 6 1 2 4 6
Staphylococcusaureus + ++ +++ +++ - + + ++
Bacilussubtilis - + ++ ++ - + ++ ++
211Vol.21 　　　　　　ZHANGYan, etal:VolatileOilsinLeavesandBarksofCalycopterisfloribunda　
Escherichiacoli + + + ++ - - + +
Pseudomonasaeruginosa - - + + - - + ++
Candidaalbicans ++ ++ +++ +++ - + + ++
Trichophytonrubrum + + ++ ++ - - - -
Aspergilusniger - - + + - - - -
Penicilliumcitrinum - - - - - - + ++
　　Note:Thediametersoftheinhibitionzonesweredesignated(-)notdetected, fordiameterslessthan6mm;littlesensitive(+), fordiameters8-10
mm;sensitive(++), fordiameters10-15mm;verysensitive(+++), fordiametersmorethan15mm.
Antimicrobialactivities
ThevolatileoilsoftheleavesandbarksofC.floribunda
werefurtherstudiedregardingsomeoftheirbiological
activities.Bothoilsweretestedforbactericidalandfun-
gicidalefectsandtheresultsareshowninTable3.In
general, theoilsexhibitedastrongantimicrobialactivi-
ty, andprovedtobemoreactiveagainstthetestedbac-
teriathanthefungi.Theleavesoilexhibitedabroader
antibacterialspectrumthanthatofthebarksoil, and
hadhigherinhibitionactivityagainstmostlymicrobe.
Usingdiskdifusionmethod, theleavesoilwasfoundto
becompleteantimicrobialagainstalthetestedmi-
crobesexceptPeniciliumcitrinum.Itwasnotablethat
completeinhibitionagainstCandidaalbicansofleaves
oilwasobtainedonlyat1 μL, andhadobviousinhibi-
tionagainstStaphylococcusaureus, Bacilussubtilis,
Escherichiacoli, Trichophytonrubrumat2 μL.Though
thebarksoilhadshowedinhibitionagainstalthetest-
edmicrobialstrains, itoccuredonlywhenthecontent
ofoilwashigh;additionaly, noinhibitionwasobtained
forthegrowthofTrichophytonrubrumandAspergilus
niger.
Concerningthebiologicalactivitiesofessentialoilof
C.floribunda, someofthesesquiterpenesfoundinleav-
esoil, suchascaryophyleneoxide, havebeenshownto
haveanti-fungal, aswelasinsecticideandantifeedant
activities[ 14] .Althoughbeingdetectedinminora-
mounts, theterpenes:o-cymene, β-linalool, safranal, β-
cyclocitral, α-ionene, cyperene, β-cubebene, β-eu-
desmene, τ-elemene, τ-himachalene, spathulenol, ce-
drenol, epiglobulolandmyristicacid, wereexclusively
foundinleavesoil.Atpresent, however, themodeof
actionofterpenicconstituentsonmicroorganismsisnot
fulyunderstood.Butbasedontheliteraturereports,
spathulenolhasbeenshowntoactascytotoxic, cymene
andsafranalarewelknownchemicalswithpronounced
antimicrobialefects[ 15] .
Themodeofactionofterpenicconstituentsonmicroor-
ganismsisnotfulyunderstood;inviewoftheirhydro-
phobicity, itisgeneralyconsideredthattheyarein-
volvedinsuchmechanismsascytoplasmicmembrane
disturbance, coagulationofcelcontentsanddisruption
oftheprotonmotiveforce[ 16] .Additionaly, theminor
compoundsintheoilsmightalsohaveacriticalpartfor
actioninantimicrobialactivity, possiblybyproducinga
synergisticefectbetweenothercomponents.
Conclusion
Inthisstudy, thevolatileoilsoftheleavesandbarksof
C.floribundawereextractedbysteamdistilation, ana-
lyzedbyGC/MS, andmadethecomparisonbetween
theircompositionandrelativecontents.Incomparison
withbarksvolatileoil, leavesproducedvolatileoilina
higheryieldthanbarks, andmorevolatileandfragrant
substancesarefavoredasleavesconstituentsandsub-
stanceswithdiferentpropertiesareselectedinbarks
volatileoil, plentyofcompositionswithantimicrobial
efectswerecontained.Inlaterantimicrobialassay, the
antibacterialandantifungalactivitiesofleavesand
barksoilwereinvestigatedbydiskdifusionmethod.
Theleavesoilshowedastronginhibitoryefectregard-
ingthegrowthofStaphylococcusaureus, Bacilussubti-
lis, Escherichiacoli, CandidaalbicansandTrichophyton
rubrum, amoderatedefectwasobservedforPseudo-
monasaeruginosaandAspergilusniger, noefectfor
Peniciliumcitrinum.Thebarksoilshowedastrongin-
hibitoryefectregardingthegrowthofStaphylococcus
aureus, Bacilussubtilis, Pseudomonasaeruginosa, Can-
didaalbicansandPenicilium citrinum, amoderated
efectwasobservedforEscherichiacoli, noefectfor
TrichophytonrubrumandAspergilusniger.
212 NatProdResDev　　　　　　　　　　　　　　　　　　　　 　Vol.21
Itissupposedthat, thevolatileoilfromC.floribunda
leavesprobablybeanantimicrobialagent, sotheeluci-
dationofactiveconstituentinthevolatileoilmaypro-
videusefulleadtothedevelopmentofnewandefec-
tiveantimicrobialagents.Inordertofindanalternative
approachfordiscoveryofantiviralagents, furtherstud-
iesareneededformoreextensiveevaluationsofthebi-
ologicalpropertiesofthevolatilesoilsofC.floribunda.
Acknowledgements　WethanktheMicrobialType
CultureColection, InstituteofMicrobialTechnology,
CentralSouth University forproviding microbial
strains.
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