全 文 ：214 ChinJNatMed May2008 Vol.6 No.3 2008年 5月 第 6卷 第 3期
Anti-HIV-1 ActivitiesofHemslecinsAandB
TIANRen-Rong1, 5 , CHENJian-Chao2 , 4 , ZHANGGao-Hong1 , QIUMing-Hua2 ,
WANGYun-Hua1, 4 , DULi3, 4 , SHENXu3 , LIUNai-Fa5 , ZHENGYong-Tang1, ＊
1LaboratoryofMolecularImmunopharmacology, KeyLaboratoryofAnimalModelsandHumanDiseaseMechanisms, KunmingInstitute
ofZoology, ChineseAcademyofSciences, Kunming650223;
2StateKeyLaboratoryofPhytochemistryandPlantResourcesinWestChina, KunmingInstituteofBotany, ChineseAcademyofSci-
ences, Kunming650204;
3StateKeyLaboratoryofDrugResearch, ShanghaiInstituteofMateriaMedica, ChineseAcademyofSciences, Shanghai201203;
4GraduateSchooloftheChineseAcademyofSciences, Beijing100039
5SchoolofLifeScience, LanzhouUniversity, Lanzhou730000 , China
Availableonline20 May2008
【ABSTRACT】 AIM:Tostudyanti-HIV-1 activitiesoftwotriterpenoids, hemslecinsAandBfromaChinesemedicinalplant
Hemsleyajinfushanensis.METHODS:ThecelularinhibitoryassaysonsyncytiaformationinducedbyHIV-1, p24 antigenproduc-tioninHIV-1 acutelyinfectedC8166 cells, cel-to-cellfusionbetweenHIV-1chronicalyinfectedH9celsandnormalC8166cells,andp24 productionfromHIV-1 chronicalyinfectedH9cellswereassayed.TheeffectsonNCp7 zincejectionandinhibitoryactivi-
tiesagainstHIV-1 reversetranscriptase(RT)andprotease(PR)werealsodetected.RESULTS:ThehemslecinsAandBwithin-
hibitoryefectson(1)syncytiaformationinducedbyHIV-1 withEC50 valuesof3.09and2.53μg mL-1 respectively, (2)p24an-tigenproductioninHIV-1 acutelyinfectedC8166celswithEC50 of3.97 and18.90 μg mL-1 respectively, and(3)fusionbetweenHIV-1chronicalyinfectedH9celsandC8166celswithEC50 of1.76 μg mL-1 and11.95μg mL-1 , respectively.ButhemslecinsAandBfailedtoshowinhibitoryactivityagainstHIV-1 RT, PR, andNCp7 zincejection.HemslecinAnothemslecinBshowedslightinteractionwithHIV-1integrase.Inco-cultureassay, hemslecinsAandBshowedmoreeffectinpretreatedC8166cellsgroup
thannopretreatedgroup.CONCLUSION:ThehemslecinsAandBarebioactivecompoundsagainstHIV-1andtheiractionmech-anismmightbecorrelatedwithvirusentry.
【KEYWORDS】 Hemsleyajinfushanensis;Hemslecin;Tritererpenoids;Anti-HIVagents;Naturalproducts【CLCNumber】 R285.5 【Documentcode】 A 【ArticleID】 1672-3651(2008)03-0214-05
【Receivedon】 2007-04-13
【FoundationItem】 ThisprojectwassupportedbyKeyScientificand
TechnologicalProjectsofChina(2004BA719A14)andYunnan
(2004NG12), theKnowledgeInnovationProgramofCAS(KSCX1-YW-
R-24)
【＊Correspondingauthor】 ZhengYong-Tang, Ph.D, Professor:Tel
＆Fax:86-871-5195684;E-mail:zhengyt@mail.kiz.ac.cn
1 Introduction
Becauseoftheincreasingproblemsofdrugre-sistanceandsideefectswiththeuseofcurentlyap-proved anti-HIV-1 drugs, such asreversetran-scriptase(RT), protease(PR), integrase(IN)andfusioninhibitors, thereisanurgentneedforthede-velopmentofnewanti-HIV-1 agentscontinualy.Inrecentyears, avarietyofcompoundsfromnaturewerereportedtohaveanti-HIV-1activityandasleadcom-poundsformoreefectivedrugs[ 1, 2] .Terpenewasonesuchcompoundswithanti-HIV-1 action.Someof
themhaveshownsignificantanti-HIV-1 activities,
whereasotherswereHSVandHCVinhibitors[ 3-5] .Asanti-HIV-1 agents, manyderivatesofterpenoidhavebeensynthesized.Theiranti-HIV-1 activitiesweremeasuredandthemechanismoftheiractionhasbeenelucidated.First, theycaninteractwithviralglycopro-teinandinhibitcelfusioninducedbyHIV-1.Se-cond, theycaninhibitactivityofRTandIN.Third,someofthemdisruptassemblyandbuddingoftheHIV-1 throughinhibitingGagprocessing.Exceptforthemechanismdescribedabove, sometriterpenoidshavebeenreportedtohaveimmunomodilatoryactivi-ty.Theycanshiftthebalancesbetweentype1 and
type2 ThelpercelsandmaintainCD4+ Tcelnum-ber.SomeothershavebeenreportedtoinduceHIV-1outoflatencyandregulateextensiveseriesofgenesunderthecontrolofproteinkinaseC(PKC).Manyofthemtargetedmorethanoneprocess.Oneofthem,diterpenoidlactonefromAndrographispaniculota, has
ChineseJournalofNaturalMedicines6(2008)214-218
doi:10.3724/SP.J.1009.2008.00214
ChineseJournalofNaturalMedicines
TIANRen-Rong, etal:Anti-HIV-1 ActivitiesofHemslecinsAandB
2008年 5月 第 6卷 第 3期 ChinJNatMed May2008 Vol.6 No.3 215
undergoneaphaseIclinicaltrialandindicatedexcel-
lentefect[ 3] .TheplantsofthegenusHemsleyahavebeenusedasherbalmedicinesinChinainthetreatmentofbacilary dysentery, bronchitis and tuberculosis
etc[ 6, 7].25-O-acetyl-23, 24-dihydrocucurbitacinFanddihydrocucurbitacinF, namedhemslecinsAandBre-spectively(Fig.1), isolatedfrommanyplantsofthisgenushavebeenusedtocureinflammatorydiseasesinclinical, suchasenteritis, diarheaandbronchitis,
etc[ 8] .Invitroanti-HIV-1 activityofhemslecinsAandB, purifiedfromaChinesemedicinalplantHems-leyajinfushanensis, wasstudiedinthepresentstudy.
2 MaterialsandMethods
2.1 SpeciescolectionThetubersofH.jinfushanensiswerecolectedfromJinfushanMountain, ChongqingCity, China, inyear2000.TheplantmaterialwasidentifiedbyProf.ZhanWen-Jin, andaspecimenhasbeendepositedintheHerbariumoftheKunmingInstituteofBotany,ChineseAcademyofSciences.
2.2 ExtractionandisolationofhemslecinsThehemslecinswereextractedandisolatedasre-
portedpreviously[ 9] .Briefly, air-driedandpowderedtubers(2.0 kg)ofHemsleyajinfushanensiswereex-tractedwith95%ethanolunderreflux(3×8L), andfiltered.Afterconcentrationofthecombinedfiltrateundervacuum, 411 gresiduewasgoten.Theresidue(322g)wasabsorbedon800gofsilicagel(200-300mesh)andchromatographedon2.5 kgsilicagelcol-umnelutedwithagradientsystemofCHCl3 , CHCl3-Me2CO(15∶1, 95∶1)andCHCl3-MeOH(9∶1, 7∶3)togivefivefractions(Frs.1-5).HemslecinA(39.1 g)wasrecrystalizedfromFr.2 inacetone.HemslecinB(49 mg)wasisolatedandpurifiedfromfraction3(2.83g)byrepeatedsilicagelcolumnchromatogra-phyelutedwithCHCl3 /MeOH(20∶1, 15∶1, 12∶1).ThestructureofhemslecinsAandBareshowninFig-ure1.ThepurityofhemslecinsAandBdeterminedbyHPLCanalysiswereover99% anddisolvedinDM-SO.ThemolecularweightofhemslecinsAandBwere
562and520 respectively.
2.3 ReagentsAnti-FcIgGwaspurchasedfromSigma.Horse-radishperoxidase(HRP)labeledgoatanti-humanIgGwaspurchasedfromSino-AmericaBio.Co.(China).P5F1, amouseanti-p24, monoclonalantibody(McAb)andrabbitpolyclonalagainstHIV-1p24wereprepared
byourlaboratory[ 10].RecombinantHIV-1 proteasewasexpressedandpurifiedinourlaboratory[ 11] .
2.4 CelsandvirusesCellinesusedinthisstudy(C8166, H9 andH9/HIV-1IIIB)weremaintainedinRPMI-1640 sup-plementedwith10% heat-inactivatednewborncalfserum(Gibco).Thecelsusedinalexperiments
Fig.1 StructuralformulaofhemslecinsAandB
wereinlog-phase.HIV-1IIIB wasobtainedfromtheculturesupernatantofH9/HIV-1IIIBcels.Virus50%tissuecultureinfectiousdoses(TCID50)weredeter-minedbyasensitive7-dayendpointtitrationassayusingC8166 celandcalculatedbytheReedandMuenchmethod.Virusstockswerestoredinsmalaliquotsat-70 °C.
2.5 CytotoxicityasayThecytotoxicitywasmeasuredbyMTTmethodasdescribedpreviously[ 12].Theconcentrationcausingthereductionofviablecelsby50% (CC50)wasde-termined.
2.6 SyncytiumreductionasayPrimaryanti-HIV-1 screenwasmeasuredthroughsyncytiumreductionassayaspreviouslydescribed[ 13] .Themultiplicityofinfection(MOI)was0.05.AZTwasusedaspositivedrugcontrol.Thenumberofsyncytiawascounted3 dayspost-infectionand50%efectiveconcentration(EC50)wascalculated.
2.7 InhibitionofHIV-1 replicationinacutelyinfec-tedC8166 andMT2InhibitionofHIV-1 replicationrepresentedbythereductionofp24 antigenproductionwasdeter-
minedbyp24 antigencaptureELISA[ 14] .Thesuper-natantcolectedatday3 forC8166 oratday6 forMT2 wasusedforp24 antigencaptureELISA.Theplatewasreadat490 nm/630 nmonBio-TekELx
800ELISAreaderwithin30min.Theinhibition(%)onp24antigenexpressionandEC50 werecalculated.
2.8 InhibitionofHIV-1replicationinchronicalyin-fectedH9cels
ChronicalyinfectedH9 /HIV-1IIIB (4 ×104 )celswereculturedwithvariousconcentrationoftest-edcompounds.Zidovudine(AZT)andIndinavir(IDV)wereaddedasdrugcontrol.After3daysincu-bation, thecelswerewashedtoremovecel-freeviru-sescompletelybeforeaddingcompoundsagain, andtheculturewascontinuedforthreedayswithfreshmediacontainingcorespondingconcentrationcom-pounds.Thesupernatantwascolectedandcelvia-bilitywasmeasuredbyMTTmethodasdescribedabove, andthep24 antigenlevelwasmeasuredbyp24 antigencaptureELISA.
2.9 Co-cultureassayTheco-cultureassaywasperformedasdescribed
TIANRen-Rong, etal:Anti-HIV-1 ActivitiesofHemslecinsAandB
216 ChinJNatMed May2008 Vol.6 No.3 2008年 5月 第 6卷 第 3期
inliteraturewithsomediferences[ 14].Inthisexperi-
ment, C8166 cels(6×104cels/wel)andH9/HIV-
1IIIB cels(1 ×104 cels/wel)wererespectivelypre-treatedwithvariousconcentrationsoftestedcom-poundsfor4 h, thencorrespondingco-culturedcels
H9/HIV-1IIIB(1×104 cels/wel)orC8166 (6×104cels/wel)wereadded.Andthetwocelslinewereaddedsimultaneouslywithcompounds.DSwaspositivedrugcontrol.Thesyncytiaformationwereexaminedat
8 hourspost-infection.ThepercentageofinhibitionandEC50 werecalculated.
2.10 EfectsonHIV-1 targetsinvitroTheinteractionbetweencompoundandHIV-1integrasewasdeterminedbySPRusingaBIAcore
3000TMbiosensorsystem(BiacoreInc., Piscataway,
NJ)asdescribedpreviously[ 15] .Thekineticratecon-stantsfordissociation(kd)wasobtainedbyfitingthereal-timedatausingBIAevaluationsoftware.InhibitionassaysofrecombinationHIV-1 PRactivity, RTactivityandNCp7zincejectionwereper-
formedasdescribedinliterature[ 15, 16] .Theefectsof
compoundsattheconcentrationof200 μg mL-1 wastestedandthepercentageofinhibitionwascalculatedthroughcomparingtocontrol(withoutcompound).
3 Results
3.1 Anti-HIV-1 activitiesCytotoxicitiesweremeasuredinparalelwiththedeterminationofantiviralactivities.TheCC50softhehemslecinsAandBagainstC8166 were86.69 μg
mL-1 and77.68 μg mL-1 , respectively, andCC50sonMT2 werebothabove200 μg mL-1 (Fig2A;Table
1).Toexaminetheanti-HIV-1activitiesofhemslecinsAandB, thesyncytiaformationandp24 antigenpro-ductionweremeasured.Resultsshowedthathems-lecinsAandBnotonlyinhibitedsyncytiaformationinC8166celsinduceddirectlybyHIV-1ⅢB, butalsoin-hibitedp24 antigenproductioninHIV-1 acuteinfec-tionC8166celsandMT2cels, withEC50svaluesran-gingfrom2.23 to18.90μg/ml(Fig2B;Table1).
Table1 Thesummaryofcytotoxicitiesandanti-HIV-1 activitiesofhemslecinsAandB
Compounds Cels Virus Assays EC50(μg mL-1) CC50(μg mL-1) SI(CC50 /EC50)
HemslecinA C8166 HIV-1IIB Syncytia/MTT 3.09 86.69 25.27
p24/MTT 3.97 21.85
MT2 p24/MTT 2.23 >200 >89.65
H9 /HIV-1IIB p24/MTT 6.48 1.24 inactive
C8166 HIV-1IIB/H9 Co-culture 1.76 - -
HemslecinB C8166 HIV-1IIB Syncytia/MTT 2.53 77.68 30.70
p24/MTT 18.90 4.11
MT2 p24/MTT 14.03 >200 >14.26
H9 /HIV-1IIB p24/MTT 20.19 4.69 inactive
C8166 HIV-1IIB/H9 Co-culture 11.95 - -
EC50 istheefectiveconcentrationthatinhibits50%ofviralproduction;CC50 istheinhibitoryconcentrationthatreducescelulargrowthorviabilityofuninfectedcellsby50%;SIisselectiveindex(CC50 /EC50).Thedatashowninthetablearearepresentativeofthreeindependentexperiments.
Fig.2 Cytotoxicitiesandanti-HIV-1 activitiesofhemslecinsAandB.CytotoxicitiesonMT2 celsweremeasuredbyMTTassay(A)andinhibitionofHIV-1 p24antigenproductioninculturesupernatantwasperformedbyELISA(B).Da-
taareexpressedasmeans±standarddeviations.
3.2 Mechanismsofanti-HIV-1actionToexaminetheefectsofhemslecinsAandBonthelatestepofHIV-1 replication, thecelviabilityandp24 antigenproductionofHIV-1 chronicalyin-fectedH9 celsweremeasured.BothcompoundshadnoinhibitoryefectsonHIV-1 replicationinH9 /HIV-1IIIB(Table1).ButhemslecinsAandBinhibi-
tedthecel-to-celfusionbetweenthenormalC8166celsandtheHIV-1IIIBchronicalyinfectedH9 celinco-culture(Fig.3).TheEC50 inpretreatedC8166celswaslowerthanthatinpretreatedH9 /HIV-1IIIBorsimultaneousco-culture(Fig3).InthepretreatedC8166 group, theEC50sofhemslecinsAandBwere
1.76 and11.95μg mL-1 , respectively(Table1).At
TIANRen-Rong, etal:Anti-HIV-1 ActivitiesofHemslecinsAandB
2008年 5月 第 6卷 第 3期 ChinJNatMed May2008 Vol.6 No.3 217
thesametime, othertargetsofhemslecinsAandBonHIV-1werealsopreliminarilyinvestigatedinvitro.AstheresultsshowedthathemslecinsAandBhadnoefectsonPR, RTactivitiesandNCp7 zincejectioninvitro(Table2).ButhemslecinAslightlyboundthe
HIV-1integrase, withaKdvalueof61.45 μg mL-1(Fig4)andhemslecinBhadnoefectwithRU(Re-
sponseUnit)below 15 at25 μg mL-1 (Datenotshown).
Table2 EffectsonHIV-1 targets
Assays Percentageofinhibitionorejection(%)HemslecinsAa HemslecinsBa
HIV-1PR 12.03b 23.22b
HIV-1RT -20.39b -19.47b
zincejection -3.93c -8.09c
a, Efectswasmeasuredattheconcentrationof200μg/ml;b, Percent-
ageofinhibition;c, PercentageofNCp7zincejection
Fig.3 TheinhibitoryeffectsofhemslecinsA(A)andB(B)oncel-to-celfusionbetweennormalC8166celsandHIV-
1IIIBchronicallyinfectedH9 cells.DSwasusedaspositivedrugcontrol(C).Dataareexpressedasmeans±standarddevi-ations
Fig.4 InteractionbetweenhemslecinsAandrecombi-nantHIV-1 intergrase.Theinteractionwasanalyzedby
Biacore3000.TheconcentrationofhemslecinsAfromtoptobottomwere25, 12.5, 6.25, 3.125, 1.5625, 0.78125μg
mL-1 , respectively.
4 Discussion
Naturalproducts as the mostconsistentlysuccessfulsourceindrugdiscovery, mayofermoreopportunitiesforfindinganti-HIVdrugsorleadcom-pounds.Morethanhundredsofnaturalyderivedanti-HIVagentshavebeenisolatedandidentified, suchassulfatedsterols, terpene, alkaloid, polysaccharideand
peptidesetc[ 3, 15] .Cucurbitacins, belongingtotetra-cyclictriterpenoids, hasbeenisolatedfromvariouskindsofplantfamilies, mushroomandevenshel-lessmarinemolusks.Theyarenotedfortheircytotoxicbehaviorandananticancerdrugdevelopmentper-spective.Besidesanticanceractivity, cucurbitacinsal-soexhibitedfurtherwiderangingpharmacologicalefectsinvitroandinvivo, suchaspurgative, anti-
inflammatory, andanti-fertilityactivities[ 17] .Manycucurbitacinshavebeenreportedwithan-ti-HIV-1 activitybefore[ 18] .HemslecinsAandBas
twocucurbitacinswerefirstlyisolatedfromH.amabi-lisin1975andaremaincomponentsofpilsofaChi-nesetraditionalmedicine, Xue-dan-su-pian, whichhavebeenusedfortherapyofbacilarydysentery, en-teritis, bronchitisinChina.BothhemslecinsAandBpossessantibacterialactivities, buttheyareantagonis-ticatahighconcentration, andtheyalsohaveinhibi-toryefectsonEpstein-Barrvirus(EBV)activationinducedbythepromotionof12-o-tetradecanoylphor-bol-13-acetate(TPA)[ 6-8] .HemslecinsBalsoexhibi-tedremarkableantitumorpromotionefectonmouseskintumorpromotioninvivo.Two-stagecarcinogene-sisexperimentandtheefectofhemslecinAagainst
infectiousdiseaseshasbeendoneinclinicaltrial[ 9] .Inthepresentstudy, wefoundthathemslecinsAandBhadmoderateanti-HIV-1 activitieswithselec-tiveindexabove20 inassaysofsyncytiaformationandp24 antigenproduction.Theycanblockthetransmissionofvirusthroughcel-to-celfusion, butcannotinhibittheactivitiesofHIV-1PR, RTandtheviralreplicationinchronicalyinfectedH9 cels,whereastheycanneitherejectzincfromNCp7.TheseresultssuggestthathemslecinsAandBarepotentialanti-HIV-1 compoundsandtheiractionmechanismmaybecorelatedwithvirusentry.Fromtheresultsofdiferentpretreatmentinco-culture, wespeculatethathemslecinsAandBcanblockorcompetethebindingsiteofHIV-1 ormodulateexpressionofmolecularwhichisnecesaryforHIV-1bindingtoreduceentryofvirus.Furthermore, whentakingthestructuresofcompoundsandbioactivitiesintoaccounttogether, theresultsimplythatthefunctionalityofacetylgroupcanenhancetheanti-HIV-1 activitiesofthiskindcom-
TIANRen-Rong, etal:Anti-HIV-1 ActivitiesofHemslecinsAandB
218 ChinJNatMed May2008 Vol.6 No.3 2008年 5月 第 6卷 第 3期
pound.Althoughthepilsofxue-dan-suhavebeenusedfortherapyofbacilarydysentery, enteritis, bronchitis
inChina[ 6-8] , itsanti-HIVactivityhavenotbeenre-ported.Inthisstudy, wefoundthattwomaincompo-nentsofpilsofxuedansu, hemslecinsAandB, withinhibitoryefectsonHIVentry.TheynotonlygreatlyexpandtheprecautionaryandtherapeuticoptionsforthetreatmentofHIV/AIDS, butalsomayhighlightthetherapeuticoptionsforthetreatmentofopportun-isticinfectioninAIDSpatients.Inconclusion, hems-lecinsAandBarepotentialanti-HIV-1 compoundsandtheiractionmechanismmaybecorelatedwithvirusentry.
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雪胆素 A和 B的体外抗 HIV-1活性
田仁荣 1, 5 ,陈剑超1, 4 ,张高红 1 ,邱明华2 ,王云华 1, 4 ,杜 丽 3, 4 ,沈 旭 3 ,刘廼发 5 ,郑永唐1, ＊
1中国科学院昆明动物研究所动物模型和人类疾病机理重点实验室分子免疫药理学实验室 ,昆明 650223;
2中国科学院昆明植物研究所植物化学与西部植物资源可持续利用国家重点实验室 ,昆明 650204;
3中国科学院上海药物研究所新药研究国家重点实验室 , 上海 201203;
4中国科学院研究生院 , 北京 100049;
5兰州大学生命科学学院 ,兰州 , 730000
【摘 要】 目的:研究从药用植物金佛山雪胆分离的雪胆素 A和雪胆素 B两个三萜类化合物的体外抗 HIV活性。
方法:应用合胞体抑制实验 、p24抗原产生的抑制实验 、慢性感染细胞和正常细胞间的细胞融合抑制实验等技术检测化合
物的体外抗 HIV-1活性;利用 HIV-1逆转录酶 、蛋白酶抑制实验 , NCp7锌离子逐出实验探讨化合物的作用机制。结果:雪
胆素 A和雪胆素 B在体外有较好的抑制 HIV-1活性 , 其活性主要表现为:(1)抑制 HIV-1诱导合胞体形成 , EC50值分别为
3.09 μg·mL-1和 2.53μg·mL-1;(2)抑制 HIV-1急性感染的 C8166细胞 p24抗原产生 , EC50值分别为 3.97μg·mL-1和 18.
90μg·mL-1;(3)抑制 HIV-1慢性感染 H9与正常 C8166细胞间融合 , EC50分别为 1.76μg·mL-1和 11.95μg·mL-1。雪胆
素 A和雪胆素 B对 HIV-1逆转录酶 、蛋白酶 、NCp7锌离子逐出均没有抑制作用。 雪胆素 A对 HIV-1整合酶有微弱的结
合活性 , 而雪胆素 B对 HIV-1整合酶没有结合活性。在共培养实验中 , 雪胆素 A和雪胆素 B预处理 C8166细胞组比未经
预处理细胞组能够更有效的抑制 HIV-1活性。结论:化合物雪胆素 A和雪胆素 B体外有较好的抗 HIV-1活性 ,可能主要
作用于 HIV-1病毒进入细胞阶段。
【关键词】 金佛山雪胆;雪胆素;三萜类化合物;抗 HIV;天然产物
【基金项目】 国家 “十五”科技攻关计划(2004BA719A14);云南省科技攻关计划(2004NG12);中国科学院知识创新
工程重要方向(KSCX1-YW-R-4)项目