全 文 ：中南大学学报(医学版)
JCentSouthUniv(MedSci)　 2008, 33(5)
　　Dateofreception　 2007-06-08
Biography　OUSheng(1981-), male, master sdegree, mainlyengagedintheresearchofnanomedicine.
Correspondingauthor　ZHANGYang-de, E-mail:nhec@ 2118.com
Foundationitems　ThisworkwassupportedbyHigh-techResearchandDevelopmentProgram(863Program)ofP.R.China(2002AA216011)
·ARTICLES· ·论　著·
Preparationandantibacterialactivityofcompound
chitosan-compoundYizhihao-nanoparticles
OUSheng1 , ZHANGYang-de2
(1.NationalKeyLaboratoryofNanobiologicalTechnology, MinistryofHealth, Changsha410008;
2.NationalHepatobiliary＆EntericSurgeryResearchCenter, CentralSouthUniversity, Changsha 410008, China)
Abstract:　Objective　Topreparechitosan(CS)-compoundYizhihao-nanoparticles(NP)
andtoinvestigateitsantibacterialactivity.Methods　CSNPswereformedbytheincorporation
ofCSandNa3 PO4 .CS-compoundYizhihaoNPswerepreparedbyion-cross-linking.Theparti-
clesizesandsurfacechargesofCSNPsweredeterminedbyMalvernZetasizer1000-HASanda-
tomicforcemicroscope(AFM), respectively.TheantibacterialacitivityofCS-compoundYizhi-
hao-NPswasstudiedinvitroandcomparedwiththatofcompoundYizhihaopowder.Results　
MalvernZetasizer1000-HASandAFMdemonstratedthatthediameterofCS-compoundYizhihao
NPswas(1 37.00 ±14.28)nmandCSNPshad(1 6.90 ±1.32)mVpositivesurfacechar-
ges.Theminimalinhibitoryconcentrations(MIC)ofCS-compoundYizhihaoNPsonStaphylo-
coccusaureus, Pneumococus, β -hemolyticstreptococcus, andEscherichiacoliwere1∶32 , 1∶32 ,
1∶16 , and1∶2 , respectively.Theminimalbactericidalconcentrations(MBC)ofCS-compound
Yizhihao-NPsonStaphylococcusaureus, Pneumococus, β -hemolyticstreptococus, andEscherichia
coliwere1∶16 , 1∶16 , 1∶8 , and1∶2 , respectively.TheantibacterialeficacyofCS-compound
Yizhihao-NPstoStaphylococcusaureus, Pneumococcus, andβ -hemolyticstreptococcushadbeen
improvedsignificantly(P<0.05).ConclusionCS-compoundYizhihao-nanoparticleshaveob-
viousantibacterialactivitytotheStaphylococcusaureus, Pneumococcus, andβ -hemolyticstreptococ-
cus, whichlaystheexperimentalfoundationfornewpreparationoftraditionalChinesemedicinein
futureresearch.
Keywords:　chitosan;　compoundYizhihao;　nanoparticle;　preparation;　antibacte-
rialactivity
[ JCentSouthUniv(MedSci), 2008 , 33(5):0369-06]
369
中南大学学报(医学版), 2008, 33(5)　
复方一枝蒿 -壳聚糖纳米药物的制备及
其体外抗菌作用的研究
欧晟 1 , 张阳德 2
(1.中南大学卫生部纳米生物技术重点实验室 , 长沙 41 000 8,
2.中南大学卫生部肝胆肠外科研究中心 , 长沙 410 00 8 )
[摘要 ] 　目的:制备复方一枝蒿 -壳聚糖纳米药物并研究其体外抗菌作用 。 方法:通过乳化交
联法合成复方一枝蒿 -壳聚糖纳米药物 。 分别用激光粒度分析仪 (MalvernZetasizer100 0-HAS)和原
子力显微镜(AFM)检测该种纳米药物的粒径分布以及表面电位 。 通过体外抗菌实验检测此种纳
米药物的体外抗菌能力 , 并与传统复方一支蒿颗粒的抗菌能力作比较 。 结果:该种纳米药物粒径
分布均一 , 为(13 7.00 ±1 4.2 8)nm;表面携带正电荷 , Zeta电位值为(1 6.90 ±1.32)mV。 其对金
黄色葡萄球菌 、肺炎球菌 、乙型溶血性链球菌和大肠杆菌的最小抑菌浓度分别为 1∶3 2, 1∶32 , 1∶16
和 1∶2, 最小杀菌浓度分别为 1∶16 , 1∶16 , 1∶8和 1∶2。与传统复方一支蒿颗粒相比 , 该纳米药物对
金黄色葡萄球菌 、肺炎球菌及乙型溶血性链球菌的抗菌能力均有所改进 (P<0.05)。 结论:复方
一枝蒿 -壳聚糖纳米药物对金黄色葡萄球菌 、肺炎球菌 、乙型溶血性链球菌均具有明显的体外抗菌
作用 , 为我国传统中药新剂型的开发奠定了实验学基础 。
[关键词 ] 　 壳聚糖;　复方一枝蒿;　纳米粒;　制备;　抗菌作用
[中图分类号 ] 　R94 4　[ 文献标识码 ] 　A　[文章编号 ] 　1 672-7 34 7(20 08)05-0 369-06
　　 Chitosan(CS)isthedeacetylatedderivativeof
chitinandlargelyavailableintheexoskeletonsof
shelfishorinsects, whichisoneoftheabundant, re-
newable, nontoxic and biodegradable carbohydrate
polymers.Itissingularalkalinepolysaccharidein
nature[ 1-2] .Recently, CShasatractedincreasingat-
tentionasabiomaterialandpharmaceuticalexcipient
fordrugdeliverybecauseofitsfavorablebiological
properties[ 3-8] .Besidesitslowtoxicityandbiodegra-
dation, CShassomepropertiesofmucoadhesiveas
welasanimportantdrugpenetrationenhancement
capacityacrossmucosalbariers[ 9-12] .Thediferent
drugcariersofCSnanoparticles(NPs)havebeen
exploredbyseveralresearchers[ 13-15] .Itisclearthat
CSNPshavesuchmanydistinctivepropertiesas
targeting, delayed releasing, and increasing drug
absorption.
CompoundYizhihaoisakindofChineseherbal
medicinewhichisoftenusedtocureupperrespirato-
rytractinfection.Inordertoimproveitstherapeutic
eficacy, CSNPswereformedduringtheprocessof
incorporatingCSwithNa3 PO4 andusedasthedrug
carrierwrappedcompoundYizhihaointhisstudy.
Wedeterminedtheparticlesizesandsurfacecharges
oftheCSNPsbyMalvernZetasizer100 0-HASand
atomicforcemicroscope(AFM), respectively, and
examinedtheantibacterialacitivityofCS-compound
YizhihaoNPsinvitro.
1　MATERIALSANDMETHODS
1.1 　 Materials　 　 CS (deacetylationdegree≥
90% , viscositydegree<100 cps)waspurchasedfrom
ShanghaiBo aoBiotechnologyLimitedCompany(Chi-
na).CompoundYizhihaowasmadebyPharmaceutical
Factory ofXinjiang MedicalUniversity (China).
Na3PO4 wassuppliedbyJiangsuLianyungangChemical
AgentFactory(China).D-Glucosewasprovidedby
GIBCOBRL(USA).Ultrapurewater(MiliQPlus,
MiliporeIbericaS.A., Spain)wasusedthroughout.
ThemaininstrumentswereZetasizer1000-HSA
(MalvernInstruments, Southborough, MA), AsylumRe-
searchMFP-3DAFMSystems(Japan), NanoscopeIV
BioscopeTM (DigitalInstruments, China), Refrigeration
Centrifuge(Universal16R, German), ElectronicAna-
370
Preparationandantibacterialactivityofcompoundchitosan-compoundYizhihao-nanoparticles　OUSheng, etal.
lyticalBalance(SHIMADZU AUW120D, German),
MagneticStirringApparatus(JinfangFuhuaApparatus
LimitedCompany, China).
1.2　Methods
1.2.1　PreparationofCS-Na3 PO4 NPs　　CSNP
waspreparedbypolymeriddispersionmethod.3 mLof
CSsolution(1.44 g/Linaceticacidsolution, in
whichthemasratioofchitosantoaceticacidwas1∶
1.5)wasslowlymixedtogetherwith1.2 mLofan
aqueousNa3 PO4 solution(0.84 g/L)atroomtemper-
atureundermagneticstiring, andthereactionwasleft
overnighttoasurethecompletionofpolymerization.
NPswereisolatedbycentrifugationat9 000 r/minin
for40 minandthesupernatantwasremoved;finaly,
thesuspensionwasfilteredbypoly(ethylenetereph-
thalate)nuclearmembranefilter(diameterofporesis
0.22 μm)andCSNPswasformed.
1.2.2　PreparationofCS-compoundYizhihaoNPs
　　CS-compoundYizhihaoNPswerepreparedbythe
method ofion-cros-linking. 10 mL ofcompound
Yizhihaosolution(100 g/Lindistiledwater)was
slowlyaddedinto20 mLofCSNPssolutionandmixed
bymagneticstirringapparatusatroomtemperaturefor5
h, thenwascentrifugedat6 000 r/mininfor30 min.
Thesupernatantwasremovedandtheprecipitationwas
washedbyddH2O beforebeingresuspendedby150
mmol/LNaClsolution;finaly, themixturewaslyophi-
lizedwithsteadystateandCS-compoundYizhihaoNPs
wereformed.
1.2.3　CharacterizationofCSNPandCS-com-
poundYizhihaoNP　　ThesizeandZetapotentials
ofCSNPspreparedunderthementionedabovecondition
wasanalyzedusingZetasizer3 000 analyzer(Malvern
InstrumentsLtd., Southborough, MA, USA).Alsam-
pleswerediluted3 -foldin0.1 mol/Lsodium chlo-
ride.
ThesizeandsurfacemorphologyofCS-compound
YizhihaoNPswereanalyzedbyAJ-IIAFMandZetasi-
zer3 000 analyzer.Sampleswerediluted50-foldand
3-foldin0.1 mol/Lsodiumchloriderespectively.
1.2.4　ExaminationofantibacterialactivityofCS-
compoundYizhihaoNPs[ 16-17] 　　MHbrothandMH
agarplatewith2-folddilutionsofCS-compoundYizhi-
haoNPssolutionandcompoundYizhihaopowderwere
usedtoinvesttheirantibacterialactivityinvitroand
comparethemwitheachother.Atfirst, 1 mLofdrug
solutionwasdilutedbyMH brothorMH serum broth
fromthe1sttubetothe10thtube.The11thtubewas
bacterialcontrolandthe12thtubewasNHbrothwith-
outbacterialcontrol.Thedilutionsfromthe1sttubeto
the10thtubewere1 ∶2 , 1∶4 ～ 1∶224.Althese
tubeswerealignedinto4 rows.1st～ 11thtubesin
eachrowwereaddedinto0.1 mLofdiferenttesting
bacterialfluids(Staphylococcusaureus, Escherichiacoli,
β -hemolyticstreptococci, Streptococcuspneumoniae)re-
spectively.The12thtubewasonlyaddedintoMH
broth.Andthen, eachtubewasculturedat37 ℃ for
24 h.
The1st～ 6thtubesweredilutedto10-2 , 10-3 ,
10
-4 , 10-5 , 10-6 , 10-7 , respectively, eachofwhich
wereaddedinto10 mLnutrientagar, miscingbene, and
thenthesolutionofeachconcentrationwaspouredinto3
platesbeforebeingculturedat37℃ for24 h.
1.3　Statisticalanalysis　　SPSS12.0 softwarefor
Windows(SPSSInc, IL, USA)wasusedforanalysis.
Continuousvariableswereexpressedasx±s.Statistical
analyseswereperformedwithnon-parametric.Avalues
ofP<0.05 wasconsideredstatisticalysignificant.
2　Results
2.1　CS-compoundYizhihaoNPsformation　　
　　TheparticlesizeandzetapotentialofCSNPswere
measuredbyZetasizer3 000 analyzer(Fig.1).The
sizeofCSNPswas(96.13 ±1 1.21)nm, whileZeta
potentialanalysisontheNPrevealedapositivesurface
chargeof(16.90 ±1.32)mV.
AFM observationrevealedthatCS-compoundYizhi-
haoNPswereweluniform andthesizeswerebetween
80 nmand20 0 nmindiameter(Fig.2).Thesizeof
CS-compoundYizhihaoNPswas(137.00 ±14.28)
nm.
2.2　AntibacterialactivityofCS-compoundYizhi-
hao-NPs　 　 Theresultsofthisexperimentwere
showninTab.1 andTab.2.Theminimalinhibitory
concentration(MIC)andminimalbactericidalconcen-
tration(MBC) ofCS-compound YizhihaoNPsand
CompoundYizhihaopowderwereshowninTab.3.
371
中南大学学报(医学版), 2008, 33(5)　
Tab.1　CulturalresultofbacteriainNHbroth
Organism 1∶2 1∶4 1∶8 1∶16 1∶32 1∶64 bacterialcontrol non-bacterialcontrol
Staphylococcusaureus - - - - ± + + -
Pneumococus - - - - ± + + -
Streptococus - - ± + + + + -
Escherichiacoli + + + + + + + -
　　-:Clear;±:Litlemuddy;+:Muddy
Tab.2　CulturalresultofbacteriainNHagar
Dilution AveragenumberofbacteriaStaphylococcusaureus Pneumococcus β-hemolyticstreptococus E.coli
1∶8 - - - -
1∶16 - - 2.2×107 2.3×107
1∶32 9.0×107 1.3×107 2.8×108 2.9×107
1∶64 1.2×108 2.6×107 3.6×108 3.5×107
1∶128 1.6×108 1.9×107 4.5×109 4.6×107
1∶256 2.6×108 4.8×107 1.5×109 1.8×107
1∶512 2.5×108 2.5×108 2.5×109 2.8×107
Bacterialcontrol 1.5×109 3.2×109 3.5×109 3.5×107
Tab.3　MICandMBCofCS-compoundYizhihao-nanoparticles
Organism CompoundYizhihaopowderMIC MBC
CS-compoundYizhihaoNPs
MIC MBC
Staphylococcusaureus 1∶16 1∶8 1∶32＊ 1∶16＊
Pneumococus 1∶16 1∶8 1∶32＊ 1∶16＊
β-hemolyticstreptococcus 1∶8 1∶4 1∶16＊ 1∶8＊
Escherichiacoli ≤1∶2 ≤1∶2 ≤1∶2 ≤1∶2
　　ComparedwithcompoundYizhihaopowder, ＊P<0.05
372
Preparationandantibacterialactivityofcompoundchitosan-compoundYizhihao-nanoparticles　OUSheng, etal.
3　Discussion
Themethodofemulationcross-linkingwasusedto
prepareCS-compoundYizhihaoNPsinthisstudy.The
formationofCSNPsoccursspontaneouslywiththein-
corporationofNa3 PO4 andCS [ 14-18] .Inthepresent
study, thenegativelychargedNa3 PO4 solutionanddis-
sociativeaminoofCSwereusedtoform NPsbythe
ionicinteraction.
From theseresults, wefoundthatCS-compound
YizhihaoNPscaninhibitthegrowthofStaphylococcus
aureus, β -hemolyticstreptococi, Streptococcuspneumoni-
aeefectively;butitdid nothaveanytherapeutic
efectstoEscherichiacoli.Ithasbeenalsodemonstra-
tedthattheMIC andMBC valuesofCS-compound
YizhihaoNPsweresignificantlydecreasedcomparedwith
compoundYizhihaopowder(P=0.03 , P=0.02 , re-
spectively), whichindicatedthattheantibacterialactivi-
tyofNPshasbeenimprovedefectively.Themecha-
nismbywhichNPexerteditsefectwasthoughttobea
physicaloutcome, withtheNP stericalyhinderinga
closeireversibleatachment, itsgoodprolongedrelease
efectenhancingtheeficacyofdrugsloadedbyitand
particlesizesinthesubmicronrangefacilitatingthe
penetrationoftheNPsthroughthecelularmembrane.
Staphylococcusaureus, β -hemolyticstreptococci, Strepto-
coccuspneumoniaearealthepathogenicbacteria, which
cancauseupperrespiratorytractinfection.Asthisre-
sult, ifCS-compoundYizhihaoNPshavenobiotoxicity,
wecanuseittocureupperrespiratorytractinfection,
suchaspharyngitis, laryngitis, tracheitisandsoon.In
ordertodosomefurtherresearchandtopromotethe
developmentofapplication ofChinesemedicine, we
shouldinvestthebiotoxicityofthiskindofNPsinthe
future.
In thisstudy, itisevidentthatCS-compound
YizhihaoNPscouldbepreparedbythemethodofemu-
lationcross-linkingandtheprocessisveryeasyand
safe.CSNPscouldbeconsideredasapromisingnew
drugdeliverysystemwhichisabletoprovidehighand
long-lastingmucosalandhumoralimmuneresponses.
Ourfurtheraimistoinvestigatetheexactmechanismof
CS-compoundYizhihaoNPwhenitexertstheantibacte-
rialproperties.
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(EditedbyPENGMin-ning)
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