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1 南京中医药大学学报 2017 年 7 月第 33 卷第 4 期 J NanjingUnivTraditChinMedVol.33No.4Jul 实验研究复合磷脂组成对香附四物汤活性成分透过脂质体人工皮肤膜行为的影响汤清涵, 张会, 沈菁菁, 江雯怡, 沈昊雯, 刘露, 马敏, 顾薇, 陈军 * ( 南京中医药大学药学院, 江苏南京 ) 摘要 : 目的考察蛋磷脂 (EPC) 与氢化大豆磷脂 (HSPC) 构成的复合磷脂组成对香附四物汤活性成分透过脂质体人工皮肤膜行为的影响方法用薄膜分散法制备复合磷脂脂质体, 脂质体通过冻融循环工艺改性聚碳酸酯膜制得脂质体人工皮肤膜, 细胞膜电阻仪测定膜电阻, 并且考察了香附四物汤 5 种活性成分阿魏酸芍药苷芍药内酯苷四氢非洲防己碱延胡索乙素透过不同复合磷脂组成脂质体人工皮肤膜的行为通过差示扫描量热法 (DSC) 分析复合磷脂之间的相互作用机制结果复合磷脂比例对脂质体人工皮肤膜的电阻起到调节作用, 在 EPC HSPC=3 1 摩尔比时达到最大, 此时屏障功能最强, 香附四物汤活性成分在此复合磷脂比例下的脂质体人工皮肤膜上透过量最少在 EPC HSPC=3 1~1 3 的范围内, 脂质体人工皮肤膜的透过量随 HSPC 的增加而增大 DSC 检测结果说明在 EPC HSPC 比例为 3 1 时 EPC 与 HSPC 融合为均一相结论应用复合磷脂比例可以有效控制脂质体皮肤膜的屏障功能, 从而有可能用于模拟不同状态下的真实皮肤以考察中药活性组分的透皮行为关键词 : 复合磷脂 ; 脂质体 ; 人工皮肤膜 ; 体外透皮吸收 ; 香附四物汤中图号 :R285.5 文献标志码 :A 文章编号 : (2017) DOI: /j.issn InfluenceofPhospholipidCompositiononthePermeationBehaviorsofActiveIngredientsfromXiang Fu Si WuDecoctionThrough Liposome BasedArtificialSkin Membrane TANGQing han ZHANG Hui SHEN Jing jing JIANG Wen yi SHEN Hao wen LIU Lu MA Min GU Wei CHEN Jun * SchoolofPharmacy NanjingUniversityofChineseMedicine Nanjing China ABSTRACT OBJECTIVE Toinvestigatetheefectsofthecompositionofeggphosphatidylcholine EPC andhydrogenated soybeanphosphatidylcholine HSPC onthepermeationbehaviorsofactiveingredientsfrom Xiang Fu Si WuDecoction XF- SWD throughliposome basedartificialskinmembrane LASM METHODS Liposomeswithdiferentphospholipidcompositionswerepreparedbyfilm dispersionmethod Thenylonmembranewascombinedwithliposomesbyfreezing thawingcycle toobtainlasmanditselectricalresistancevaluewasmeasuredbyacelmembraneresistancemeter TheLASM withdiferentphospholipidcompositionswasappliedforthemeasurementofskinpermeationbehaviorsoffiveactiveingredients ferulic acid paeoniflorin albiflorin tetrahydrocolumbamineandtetrahydropalmatine from XFSWD Interactionmechanismbetween EPCandHSPCwasinvestigatedbydiferentialscanningcalorimetry DSC analysis RESULTS Phospholipidcomposition playedaregulatoryrolefortheelectricalresistanceoflasm ThehighestbarrierfunctionofLASM wasobtainedatthemolar ratioofepc HSPC=3 1 resultinginthelowestpermeationvaluesoftheactiveingredientsfrom XFSWD ThepermeabilityincreasedwiththeincreaseofHSPCinLASMintherangeofthemolarratioofEPC HSPC =3 1~1 3 Additionaly DSCanalysisrevealedthatEPCandHSPCcouldbecombinedintoonehomogenousphaseatthemolarratioofEPC HSPC = 3 1 CONCLUSION ThebarrierfunctionofLASMcanbeefectivelycontroledbythemodificationofphospholipidcomposition facilitatingthesimulationofrealskinbarriertoinvestigatetheskinpermeationbehaviorsoftheactivecomponentsfrom traditionalchinesemedicine 收稿日期 : ; 修稿日期 : 基金项目 : 江苏省中医药管理局科研专项课题 (ZX2016D1); 江苏省中药资源产业化过程协同创新中心重点项目 (ZDXMHT 1 15); 江苏省大学生创新训练计划重点项目 ( Z); 江苏省高校优势学科资助项目 (2014); 南京中医药大学中药学优势学科专项培育基金项目作者简介 : 汤清涵 (1996-), 女, 江苏泰州人, 南京中医药大学 2014 级本科生 * 通信作者 :chenjun75@163.com

2 372 南京中医药大学学报 2017 年 7 月第 33 卷第 4 期 KEY WORDS complexphospholipidcomposition liposomes artificialskin membrane invitropermeationbehavior Xiang Fu Si WuDecoction Transdermaldrugdelivery TDD ofersnumerousadvantagesoveralternativeroutesofadministration including theavoidanceofgastrointestinalside efects hepaticfirst passefectsandreducedvariabilityinabsorptionaswelas lowerfluctuationsinplasmadruglevels Itshouldbenoted thattddisalsooneofthemostcommonlyusedadministrationroutesintraditionalchinesemedicine TCM Xiang Fu Si WuDecoction XFSWD createdbylian fuliangintheqingdynastyofchina isafamousancient prescriptionintcm Itiscomposedofsevencrudeherbs includingangelicasinensis Oliv Diels Ligusticumchuanxiong Hort Paeonialactiflora Pal Rehmanniaglutinosa Libosch Cyperusrotundus L Aucklandialappa DecneandCordalisyanhusuo W T Wang InChina ithas beenwidelyusedtotreatsyndromeofstaticbloodingynecologydiseasesinclinicalpracticeforhundredsofyears At present theactivefractionsofxfswdhavebeenadministeredbytddrouteastransdermalpatch 1 Furthermore theinvitro percutaneouspermeationbehaviorsofthe main activeingredientsfrom XFSWDhavebeenintensivelyinvestigated 2 Toevaluatethepenetrationenhancingefects thein vitropermeationstudiesaregeneralyperformedwiththeusageoffranzdifusionceldeviceandexcisedskin However theuseofisolatedskinfrom humanoranimaloriginhasa numberofdisadvantagessuchashighintra andinter individualvariations Consequently theartificialskin membranes thatcouldofersimpleandreproduciblealternativestohumanoranimalskinhaveatractedlotsofinterestinthepharmaceuticalsaswelascosmeticindustrials 3 Theartificial skinmembraneshavealsobeenappliedintheevaluationof skinpermeationbehaviorsofactiveingredientsinthefieldof TCM 4 Itiswelknownthattheskinhasexcelentbarrierwhich mainlyresidesintheuppermostlayeroftheepidermis and thestratumcorneum SC severelylimitsthetransportation ofmostdrugsthroughskin Recently thenovelliposome basedartificialskin membrane LASM model mimicking bothstructureandcompositionofthescbarrierwasdevelopedandprovedtobemorecostefectiveandeficientthan thestrat M TM modelfortheevaluationofactiveingredients from XFSWD 5 Inthepresentpaper theefectofliposomalphospholipidcompositiononthebarrierfunctionoflasm wasfurther investigated Previously liposomescomposedoftwophospholipidswithdiferentphasetransitiontemperatures Tm havebeenprovedtopossesshigherstabilityofresultantlipid vesicles 6 Therefore itishypothesizedthatthebarrier functioncanbeefectivelycontroledbythe modificationof phospholipidcomposition 1 Materialsandmethods 1 1 Materials Eggphosphatidylcholine EPC hydrogenatedsoybean phosphatidylcholine HSPC andcholesterol CHOL were purchasedfrom A V T PharmaceuticalLtd Shanghai China Ferulicacid paeoniflorin albiflorin tetrahydrocolumbamine and tetrahydropalmatine Purity > 98% werepurchasedfrom NanjingSpring & AutumnBiological EngineeringCo Ltd Nanjing China Cultureinserts d = 6 5 mm andplates werepurchasedfrom CorningInc New York NY USA TheNylonfilters 0 45μm pore size waspurchasedfrom Merck Milipore Darmstadt Germany HPLC gradeacetonitrile wasobtainedfrom Tedia CompanyInc Fairfield OH USA Alotherreagents wereusedcommercialyavailableandofanalyticalgrade 1 2 Preparationofliposomes Liposomeswerepreparedbyfilm dispersionmethod 5 Thecalculatedamountsofphosphatidylcholine PC namely EPC HSPC orthemixtureofepcand HSPCatdiferent molarratio 77% w w andchol 23% w w were dissolvedinanhydrousethanolinaround botomflask The solventwasevaporatedusingarotaryevaporatortoform a thinlayeroflipid Theround botomflaskwasmaintainedo- vernightinvacuumtoremovetracesofethanolbeforehydra- tion withphosphatebufer PBS ph7 4 containing10% vv ethanoltoobtaina6% w v liposomaldispersion TheliposomaldispersionwasthensonicatedbyusingaJY92 Ⅱ probeultrasonicator XinzhiBiotechnology Co Ltd Ningbo China toobtainsmal SL 200~300nm and large LL >500nm liposomes respectively TheparticlesizevaluesofSLandLL weremeasuredin triplicatebydynamiclightscatering usingazetapotential Analyzer MalvernInstrumentInc Malvern UK 1 3 Preparationofliposome basedartificialskinmembrane LASM LASM werepreparedaspreviouslydescribed 5 Cultureinserts d=0 65mm bearingthe0 45μm nylonfilter werepreparedin housebyfusionofsuchafiltertothebase

3 汤清涵等复合磷脂组成对香附四物汤活性成分透过脂质体人工皮肤膜行为的影响第 4 期 373 ofthecultureinsertatmoderatepressureforabout30sat 150 AnaliquotoftheSLsuspension 100μL wasadded toeachcultureinsert Theinsertswereplacedintowelsof 24 welcultureplates coveredwithalidandcentrifugedat 600 gfor10minat25 ina5810rcentrifuge Eppendorf Corp Hamburg Germany performedtwicewithchange ofdirectionofinsertsinbetween Thereafter insertswere heatedat50 for45 mintoevaporatetheethanol water mixtureaswelastopromotecoatingofthefilterwithliposomes Afterinsertshadatainedroomtemperature another aliquotofllsuspension 100μL wasaddedtosetleonthe topofthefilterandcentrifugedat600 gfor10min The remainingsupernatant wasremovedcarefuly folowedby threefreeze thawcycles whereinsertswerefrozenat -80 for1handthawedat65 for0 5hundervacuum LASMcooperatedwith24 welcultureplateswasstoredat4 beforeuse 14 Electricalresistancemeasurement TheelectricalresistanceofLASM wasmeasuredbymillicel ERS 2Volt ohm meter Milipore Bilerica MA USA after1hhydrationbyph 7 4PBS theconductivity solution Theresistance values were normalized to Ω cm 2 7 Theexperimentswereperformedinquintuple wasinjected Thecumulativeamount Q ofdrugpermeatingthrough aunitareaofskinwasplotedagainsttime Steadystateflux 15 PermeationstudiesusingLASM LASM wascooperatedwith24 welcultureplateswith anefectivepermeationareaof0 33cm 2 Permeationstudies wereperformedafterloadingtheinserts with150μl drug solution theconcentrationsofferulicacid paeoniflorin albiflorin tetrahydrocolumbamine and tetrahydropalmatine were and100 0μg ml respectively dissolvedin 20% ethanol water and placing theminseparateacceptorcompartmentscontaining600μl 20% ethanol water Thepermeationexperimentwascarried outatroomtemperaturewithoutagitationandinsertswere movedtowelscontaininganequalquantityoffreshbuferat hofstudy Attheendofthepermeationexperiment samples 200 μl from each acceptor compartmentwereanalyzedby HPLCto measuretheconcentrationsofthefiveactivecompounds AShimadzuHPLCsystem Kyoto Japan consistingof alc 20ATpumpandaSPD 20A UV VISdetectorwasused foranalysis The mobilephaseconsistedofacetonitrileand 005% aqueousformicacid vv Separationwas carriedoutat25 usingareverse phasec18column 5μm 46mm 250mm MerkCorp Darmstadt Germany Thedetectionwavelengthwassetat230nmandaflowrate of1 0 ml min wasemployed Asamplevolumeof20μL values Jss werecalculatedfromtheslopeofthelinearportionoftheplot TheobtainedJss wasthenusedtocalculate theapparentpermeabilitycoeficient Papp byuseofthefollowingequation Papp cm s =Jss Cd wherejss wastheobservedfluxrateatsteady state μgs andcd μg ml was theconcentrationofsolutionindonorchamber Thecumulativepercentagesofdrugspermeatingthroughtheskinat12h Q12% werecalculatedfromthedrugconcentrationsinthe receivercompartments 16 DeterminationofTm bydiferentialscanningcalorimetry DSC Liposomesofdiferentphospholipidcompositions were driedinvacuum for24htoremovesolvent DSCanalysis wascarriedouttodeterminethetm valuesofthedriedliposomesusingadscapparatus DSC200 NetZsch Germany 60~7 0mgofsolidsamplesweretransferredintoaluminumpans whichweresubsequentlysealedandanalyzed Thesampleswerescannedfrom25to200 at5 minas comparedtothereferenceemptypan 2 Results 21 Theparticlesizesofliposomes TheresultsoftheparticlesizesofSLandLLliposomes withdiferentphospholipidcompositions werepresentedin Table1 Table1 Particlesizesofliposomeswithdiferentphospholipid compositions x±s n=3 Phospholipidcomposition molarratio SL nm LL nm EPC 193 5± ±79 17 EPC HSPC= ± ±22 59 EPC HSPC= ± ±37 60 EPC HSPC= ± ±41 00 EPC HSPC= ± ±54 57 HSPC 279 9± ±105 8 Fig 1 ElectricalresistancemeasurementofLASM withdiferent phospholipidcompositions x±s n=5 22 Electricalresistancestudies AsshowninFig 1 theratiosofepcand HSPCobviouslyinfluencedtheelectricalresistancevaluesofthepreparedLASM The maximum membraneresistancevalueswasobtainedtobe 1988±

4 374 南京中医药大学学报 2017 年 7 月第 33 卷第 4 期 311 Ω cm 2 whenthemolarratioofepctohspcwassetat3 1 Itwasacceptedthatthebarrierfunctioncouldbeefectivelyevaluated bytheelectricalresistancemeasurementoftheartificialskin membranes Therefore thebarrierfunctionoflamscouldbeeasilycontroledbythe modificationofphospholipidcompositiontosimulate diferenttypesofskin 2 3 Permeationstudies In ordertofurtherinvestigatetherelationship betweenthe membraneresistanceandthebarrierfunctionofthemembrane the permeationstudieswerecarriedoutbylasmconsistingofepcand HSPCatthemolarratiosof and1 3 respectively Thepermeationbehaviorsoffiveactiveingredients ferulicacid paeoniflorin albiflorin tetrahydrocolumbamineandtetrahydropalmatine from XFSWD withdiferentphysicochemicalpropertieswere measuredandcompared TheresultswereshowninFig 2 Table2 andtable3 Itwas foundthatthemembranepermeabilityincreasedwiththeincreaseof HSPCinLASMinaccordancewiththedecreaseofelectricalresistancevalues Especialy theq% andpappvaluesofpaeoniflorinand albiflorinwereverylow Q%<1 75% Papp< cm s intheformertwoprescriptions EPC HSPC=3 1or1 1 whereastheywerehigh Q%>38 35 Papp> cm s in Table2 thethirdprescription EPC HSPC=1 3 Fig 2 Invitropermeationbehaviorsoffiveactiveingredientsfrom XFSWDthroughLASM withthreediferentphospholipid compositions n=5 ApparentpermeabilitycoeficientvaluesoffiveactiveingredientsfromXFSWDthroughLASM with diferentphospholipidcompositions n=5 Ingredient Mwa LogP a Pappcm s 10-7 EPC HSPC=3 1 EPC HSPC=1 1 EPC HSPC=1 3 ferulicacid paeoniflorin albiflorin tetrahydrocolumbamine tetrahydropalmatine Note a valuesofmwandlogpareobtainedfromthewebsiteofwww chemspidercom Table3 Q12% valuesoffiveactiveingredientsfromxfswdthroughlasm withdifferent ingredients phospholipidcompositions x±s n=5 Phospholipidcompositions EPC HSPC=3 1 EPC HSPC=1 1 EPC HSPC=1 3 ferulicacid ± ± ± 5 27 paeoniflorin 0 19 ± ± ± 2 19 albiflorin 0 19 ± ± ± 3 19 tetrahydrocolumbamine 7 95 ± ± ± 1 24 tetrahydropalmatine 8 13 ± ± ± 4 91 SkinpermeabilitycanbepredictedbasedontheLogP andmolecular weight Mw values 8 Ferulicacid with smalmolecularweightandrelativelylow polarityhadbeen provedtopossessthehighestskinpermeability However albiflorinandpeoniflorin withlarge Mw valuesandhigher polarity were found to have poor permeability through LASM Moreover itwasalsofoundthatthepermeabilityof tetrahydrocolumbamineandtetrahydropalmatine with the lowestpolarity wasdirectlyassociatedwiththephospholipid compositionoflasm 24 DeterminationofTm bydsc AsshowninFig 3 therewasnopeakinepc thetm valueofepc<0 ForCHOLand HSPC exothermic peaksweredeterminedtobe150 7 and124 2 respectively TheresultsofDSCanalysisforliposomeswithdiferent phospholipidcompositions wereshownin Fig 4 andthe measuredtmsofliposomeswithdiferentphospholipidcompositionswereshownin Table4 Itshouldbenotedthat therewasonlyoneendothermicpeak 50 7 whenthe

5 汤清涵等复合磷脂组成对香附四物汤活性成分透过脂质体人工皮肤膜行为的影响第 4 期 375 molarratioofepctohspcwassetat3 1 indicatingthat EPCand HSPC couldbecombinedintoonehomogeneous phase However whenthemolarratioofepctohspcwas decreasedto1 1or1 3 twoendothermicpeakswereappeared indicatingthatepcand HSPCcouldnotbecombinedintoonehomogeneousphase Therefore theresultsof DSCanalysiswereinaccordancewiththeresultsofpermeationstudies PhaseseparationresultedinhigherpermeabilityofLASM whenthemolarratioofepctohspcwassetat 1 1or1 3 Accordingly phasefusionofepc HSPC andcholresultedinlowestpermeabilityoflasm when themolarratioofepcto HSPC wassetat3 1andthe weightratioofpctocholwassetat77 23 Phospholipidcompositionplaysaregulatoryrolewithrespectto electricalresistanceoflasm ThehighestbarrierfunctionofLASM wasobtainedatthemolarratioof3 1betweenEPCandHSPC resultinginthelowestpermeationvaluesoftheactiveingredientsfrom XFSWD Thepermeabilityincreased withtheincreaseofhspcin LASM especialyintherangesofthemolarratiosofepc HSPCat 1 1and1 3 Sincetheskinisabarrierwithpronouncedvariations suchas theanatomicsiteandage Theaim ofthestudy wastodevelop LASM modelsabletopredictdrugpermeationinsituationswherewe experiencedacontroledbarrierfunctionoftheskin Theideawas thatifthelevelofdecreasedbarrierfunction wasinducedinacontroledmanner themodelcouldbeusedtomimicthecompromised skinbarrier 11 Forthefirsttime itwasfoundthatphospholipid compositionsignificantlyafectedthebarrierfunctionoftheresultant LASM models 4 Conclusions ThebarrierfunctionofLASM canbeefectivelycontroledby themodificationofphospholipidcomposition facilitatingthesimulationofthediferentrealskintoevaluatethepercutaneouspermeation Fig 3 DSCanalysisofpurephospholipidmaterials Fig 4 DSCanalysisforliposomeswithdiferentphospholipid compositions Table4 ComparisonofliposomalTmsdeterminedbyDSCanalysis Phospholipidcomposition Tms EPC - EPC HSPC= EPC HSPC= EPC HSPC= HSPC Discusion XFSWDisarepresentativeprescriptionintreatingprimarydysmenorrhea 9 Ferulic acid paeoniflorin albiflorin tetrahydrocolumbamineandtetrahydropalmatineare mainactivecomponents from XFSWD whichcanpenetratethroughratskin 10 PhysicochemicalpropertiesoffiveactivecompoundswerelistedinTable2 ThelogP oilwaterpartitioncoeficient andthemwvalueswerein therangesof-1 09~3 14and194 18~ respectively Asa generalruleofthumb chemicals witha molecular weightgreater than500donotpenetratetheskin 8 Thisupperlimitonmolecular sizemainlyresultsfromthephysicalarrangementoflipidsbetween adjacentcorneocytesofsc behavioroftheactiveingredients TheLASM modelshavethepotentialtoprovidepermeationpredictionsforthedevelopmentoftdd inthefieldoftcm References 1 PANY LIZH QIANDW etal EfectsofdiferentpenetrationenhancersonpharmacokineticsofactivecomponentsinXiangfuSiwutransdermal patchesj ChinJChin MaterMed ZHANG H ZHANG HQ JIANG QD etal Studyoninvitrotransdermalabsorptionpropertiesofactivefractionfrom XiangfuSiwuDecoction J ChinTraditHerbDrug FLATENGE PALACZ ENGESLANDA etal Invitroskinmodelsas atoolinoptimizationofdrugformulationj EurJPharmSci ZHUL FUC MAJ etal ComparisonoftransdermalpenetrationbehaviorsinvitroofDingguiHuoluopatchanditsrubberpasterJ Chin HospPharmJ ZHANG H ZHUX SHENJ etal Characterizationofaliposome based artificialskin membraneforinvitropermeationstudiesusingfranzdifusionceldevicej JLiposomeRes CHENJ PING QN CAIBC etal Studyoncompositephospholipidliposomescontaining9 nitrocamptothecin C The6thChinaPharmaceuticalAssociationSymposium Guangzhou KANZERJ THOI FLATENGE etal In vitropermeabilityscreening ofmeltextrudateformulationscontainingpoorlywater solubledrugcompoundsusingthephospholipidvesicle basedbarrierj JPharm Pharmacol BARTOSOVA L BAJGARJ Transdermaldrugdeliveryinvitrousing difusioncelsj CurrMedChem LIUP DUANJA HUAYQ EfectsofXiang Fu Si Wudecoctionandits maincomponentsfordysmenorrheaonuteruscontraction J JEthnopharmacol LIZH LIUP QIANDW etal Invitrotransdermaldeliveryoftheac- tivefractionofxiangfusiwudecoctionbasedonprincipalcomponentanal- ysisj ActaPharmSin ENGESLANDA SKALKO BASNETN FLATENGE Invitromodels toestimatedrugpenetrationthroughthecompromisedstratum corneum barrierj DrugDevIndPharm 编辑董宇

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