[生物医药论文精品]苦参方成分分析及配伍机制研究

1苦参方成分分析及配伍机制研究摘要目的对中医治疗过敏性皮肤病的传统方剂苦参方有效部位成分进行定性定量分析，研究方剂配伍对有效成份的影响。方法采用LC/MS、GC/MS联用技术确定苦参方的有效成分；采用HPLC色谱法对苦参方生物碱含量进行定量分析，考察方剂配伍后的成分变化。结果苦参方挥发油中的主要成分有胡薄荷酮、薄荷酮等，所含苦参生物碱主要有苦参碱（MAT）、氧化苦参碱（OMT）、槐果碱（SOP）、氧化槐果碱（OSP）；苦参方的方剂配伍后有效成分发生了转化，氧化苦参碱部份转化成苦参碱，氧化槐果碱部位转化为槐果碱，挥发油在配伍后成分及含量也有所变化。结论确定了苦参方的有效成份及各成分的含量，明确了该方剂配伍后的成分改变。关键词苦参方；苦参；荆芥；苦参碱；氧化苦参碱；槐果碱；氧化槐果碱；挥发油；含量测定；配伍变化COMPOSITIONANALYSISANDTRANSFORMATIONMECHANISMSTUDYDUETOCOMPATIBILITYOFTHERECIPEOFKUSHENABSTRACTOBJECTIVEQUALITATIVEANDQUANTITATIVEANALYSISFOREFFECTIVECOMPONENTSINATRADITIONALANTIANAPHYLAXISCHINESEMEDICINEOFKUSHENWASESTABLISHED,ANDTHECONTENTSCHANGEOFTHEACTIVECOMPOSITIONWASTESTIFIED,WHICHWASDUETOCOMPATIBILITYBETWEENSOPHORAFLAVESCENSAITANDSCHIZONEPETATENUIFOLIABRIQMETHODSTHEEFFECTIVECOMPONENTSWERECONFIRMEDBYLC/MSANDGC/MSHPLCWASUTILIZEDTOQUANTITATETHEALKALOIDS,SOASTOINVESTIGATETHECHANGEOFACTIVECOMPOSITIONSCONTENTRESULTSTHEMAINCOMPONENTSOFSCHIZONEPETATENUIFOLIABRIQVOLATILEOILWERELMENTHONEANDPULEGONE,ETCTHEALKALOIDSEXTRACTINGFROMTHERECIPEINCLUDEDMATRINE,OXYMATRINE,SOPHOCARPINEANDOXYSOPHOCARPINETHECONTENTOFALKALOIDSCHANGEDBECAUSEOFCOMPATIBILITY,OXYMATRINETRANSFORMEDTOMATRINEANDOXYSOPHOCARPINETRANSFORMEDTOSOPHOCARPINEPARTLY,THEINGREDIENTSANDCONTENTOFVOLATILEOILWEREDIFFERENTBEFOREANDAFTERCOMPATIBILITYCONCLUSIONTHEINGREDIENTS,CONTENTANDTRANSFORMATIONWEREIDENTIFIED,SOTHETRADITIONALRECIPEWOULDBEAPPLIEDMOREDEFINITE,REASONABLEANDSCIENTIFICKEYWORDSRECIPEOFKUSHEN；SOPHORAFLAVESCENSAIT；SCHIZONEPETATENUIFOLIABRIQ；MATRINE；OXYMATRINE；SOPHOCARPINE；OXYSOPHOCARPINE；VOLATILEOIL；ASSAY；COMPATIBILITY；TRANSFORMATION苦参方由苦参、荆芥两味药组成，始见于太平惠民和剂局方，原方为治疗疥癣、湿疹和皮肤瘙痒的有效方剂。现代药理研究证明，苦参中苦参型生物碱和荆芥中荆芥挥发油均具有良好的调节免疫和抗过敏作用12。但G19283G7411G1209G7481，该方剂有效部位成分及含量G7422G14033明确，G7424研2究采用G3822G12193分析G6175G8585，确定了该方剂有效部位的具G1319成分及含量，G5194证G4466了方剂中挥发油和生物碱在苦参G994荆芥配伍后发生了成分的改变和转化，G1363G17837G980中药传统方剂的含G1053G7368G2164明确、G12197G4410、G2524理。1仪器和材料挥发油测定G3132（G990G9035G11439惠G10639G10839G1214G3132G2390）、10AVPG20652效G9094G11468色谱G1214（G7097G7424G4719G8953）、1100SERIESLC/MSD（G13666G3281AGLIENT）、6890NGC5973NMSD（G13666G3281AGILENT）。苦参、荆芥G20290G10267（G13475G12544G1120G1903医G3835G4410G19283G9035医G19510药G4410部G10591G5556G3778G6957G6492G18504定G12538G2524中G3281药G18682005G5192G10268要G8726）；苦参碱对G10043G2709（中G3281药G2709生物G2058G2709G7828定所，G6221G2507110780200402）；氧化苦参碱对G10043G2709（中G3281药G2709生物G2058G2709G7828定所，G6221G2507110805200306）；氧化槐果碱对G10043G2709（中G3281药G2709生物G2058G2709G7828定所，G6221G2507111652200301）；槐果碱G13443G2709（含量G30598，G990G9035G1505G13736生物G12197技G1856G2508）；G11014G18267（色谱G13443，SIGMA）；G1069G14108色谱G13443，MERCK；G1069G18267（药用，G990G9035G8716G10602G4466G1006有G19492G1856G2508）；G1866G1194G16809剂为分析G13443；G8712为G18337G14988G8712。2方法21苦参方有效部位的提取及样品制备G12228G2474苦参G2345味药G6122荆芥G2345味药G6122苦参、荆芥（G18337量G8616为21）配伍，G2164G1849药G74608G1505量于药G7460的G8712，G63652005G5192G10268中G3281药G1868的挥发油测定法（G19480G5417XD），进行G8712G14988G8680G14988G20323，G6922G19610挥发油，为G9141G21656色油G10378G9094G1319，G5483G10587为06ML/100G（G1209荆芥G20290G10267G17148量G16772），用于GC/MSG7828测。G8712G6564G9094过G9400，60G1069G18267G18267G8797过G9094G1209G19512G2447G15519G11345G17148及G3822G12970等G7446G17148，G6289G9400G2474G990G9177；G8712G14988G8785G14988G20323后的药G7460G8543G9207G21648G1505量的60的G1069G18267G3250G898133G4579G7114，G6564G24742G8437，G4570G6564G2474G9094G6289G9400，所G5483G9400G9094G994G18267G8797G9094G2524G5194，G8999缩G3250G6922G1069G18267，G8999缩G9094用G14988G20323G8712适当稀释，5000RPM离心10MIN，过G9400，G5483HPLC、LC/MS待测样G2709。22GC/MS鉴定挥发油主要成份G8680G11468色谱条件及G17148谱G7828测条件色谱柱HP5MS柱（298M025MM025M，AGILENT19091J433）；柱温初始温度60，G12095MIN1升至250，恒温10MIN，载G8680HE（99999），恒G89811MLMIN1，进样口温度230，进样量1L，分G8981G8616501，四极管温度150，离子源温度230，EI源，电子G14033量70EV，接口温度280。23LC/MS鉴定苦参方生物碱成分G9094G11468色谱条件色谱柱DIAMOSALC18TM柱（250MM46MM5M）；G8981动G11468G1069G14108G8712（G11014酸调PH26）298；G8981速1MLMIN1，G7828测温度25；G7828测波G19283220NM；进样量20L。3G17148谱G7828测条件电喷雾离子化（ESI）方式，G7828测离子为正离子，G17148量扫描范围M/Z150500，传输/裂解电压为70V。干燥G8680G1319（N2）G8981速10LMIN1，雾化压力40PSI。24HPLC定量测定苦参生物碱241色谱条件色谱柱DIAMOSALC18TM柱；G8981动G11468G1069G14108G8712（005三G1069胺，磷酸调PH35）2984；G8981速12MLMIN1，G7828测温度25；G7828测波G19283220NM；进样量20L。242标准曲线的G2058备精密G12228G2474苦参碱（MAT）、氧化苦参碱（OMT）、槐果碱（SOP）、氧化槐果碱（OSP）对G10043G2709适量，G1209G14988G20323G8712G2058备含MAT、OMT、SOP、OSP各为200GML1的储备G9094；精密量G247405ML、10ML、25ML、50ML、100ML、200ML、300ML、400ML、500ML贮备G9094用G14988G20323G8712定容于100ML容量瓶中，G5483G8999度范围为1100GML1的MAT、OMT、SOP、OSP标准溶G9094。在G990述色谱条件下，依G8437进样测定。G1209峰面积（A）对G8999度（C）作线性G3250归，G5483MAT的G3250归方程为A11188C18716，R09999；OMT的G3250归方程为A11167C24596，R09999；SOP的G3250归方程为A29409C29141，R09999；OSP的G3250归方程为A25737C19017，R09999，四G12193生物碱在1100GML1G8999度范围内，线性关系良好。最低G7828测G19492为10NGML1（S/N3）。3结果31荆芥挥发油主要成分分析及配伍变化由GC/MS分析荆芥G2345味药、苦参方（苦参、荆芥两味药）G8712G14988G8785G14988G20323法G6564G2474的挥发油成分，荆芥在G994苦参配伍前后挥发油成分及各成份G8616例都有所改变，含量最G20652的成分为G7481自荆芥的薄荷酮、胡薄荷酮、右旋柠檬烯等5，苦参挥发油成份6所占G8616例较少。32苦参方提取物生物碱成份分析及配伍变化321苦参方生物碱成份的确定苦参方G6564G2474物的HPLC色谱图显示G1866中主要含4G12193生物碱成分。G13475LC/MS分析，G17148谱图显示4个峰分别为苦参碱MH2491、MK2871；槐果碱MH2471、MK2851；氧化槐果碱MH2631、MK3011；氧化苦参碱MH2651、MK3031。G13475标准G2709HPLC图谱对G10043，可确定四G12193苦参类生物碱成分为苦参碱、氧化苦参碱、槐果碱、氧化槐果碱。322苦参方配伍生物碱成分变化分析G8616较苦参G2345味药和苦参方（苦参和荆芥两味药）的G6564G2474样G2709HPLC色谱图，见图1，G5194对各生物碱含量进行分析G8616较，结果见表1，发现苦参G994荆芥配伍后OMT和OSP成分明显减少，而MAT和SOP含量显著增G2164。4A11A3A0A3A0A3A0A5A5A3A0A2A4A6A7A8A9A10A11A12A13A14A15A16A8A9A10A11A13A14TAB1COMPARISONOFALKALOIDSCONTENTSBETWEENRADIXSOPHORAEFLAVESCENTISANDTHECOMPOUNDRECIPEOFHERBASCHIZONEPETAEANDRADIXSOPHORAEFLAVESCENTISA19A20MATA21A17A18A22A17A18A24A23A25A24A23OMTA21A17A18A22A17A18A24A23A25SOPA21A17A18A22A17A18A24A23A25A24A23OSPA21A17A18A22A17A18A24A23A25A24A23A26A27A28A29A300398100371067290103602241005790381900799A26A27A31A32A33A28A29A300537400393031150022605139007130197700224PA34001A35PA34005A35N64讨论苦参方是由苦参和荆芥两味中药组成的治疗过敏性皮肤病的有效传统方剂，G7424研究通过G3822G12193分析方法的采用G1363苦参方的有效部位各成分及组成G7368G2164明确，G19512挥发油外，苦参类生物碱主要G1209苦参碱、氧化苦参碱、槐果碱、氧化槐果碱为主，在LC/MS分析中还发现有少量槐定碱、槐胺碱等，但由于含量较低，没有对G1866进行分析，G7424研究对苦参方主要成份分析达到了定性、定量的目的，克服了传统方剂不确定性较G3835的不足。在研究中我们发现苦参G994荆芥配伍后挥发油成份及生物碱成份都有所变化，含量较G20652的几G12193挥发油成分中，异胡薄荷酮、丁香烯、棕榈酸在苦参G994荆芥配伍后含量有较G3835增G20652，左旋薄荷酮含量减少，右旋薄荷酮、胡薄荷酮变化不G3835，且在配伍后G980些成分消失，也有G980些新的成分G1998现，但在G5647挥发油中所占G8616例G5468G4579，所G1209G6984G1319影响不G3835。苦参G994荆芥配伍后各生物碱含量发生了明显的改变，分析两药配伍后可G14033发生成分的转化，苦参G994G1866G1194药G7460G3926G15515G5214子、G21656G7587、A361A37HPLCA38A39A40A39A40AA37A41A42A43A41A42A44BA37A45A46A47A48A49A46A47A44CA37A45A46A50A50A51A52A47A48A49A51A52A511A37A45A46A53A46A53A442A37A54A55A53A54A55A443A37A56A57A54A55A53A54A55A444A37A56A57A45A46A53A46A53A58FIG1HPLCCHROMATOGRAMASTANDARDPREPARATION,BEXTRACTOFRADIXSOPHORAEFLAVESCENTIS,CEXTRACTOFHERBASCHIZONEPETAEANDRADIXSOPHORAEFLAVESCENTIS1MATRINE,2SOPHOCARPINE,3OXYSOPHOCARPINE,4OXYMATRINEB1234A1234C34125G17722前G14621、G3332G21869等配伍G7114，氧化苦参碱转化为苦参碱的G5785G1929G5062有G6265G17959789，而G994荆芥配伍G7114两G13785的转化G7422见G6265G17959，槐果碱G994氧化槐果碱G1055G19400在配伍后发生转化G1146G7422见G6265G17959。G1186苦参类生物碱的化G4410结G7512及理化性G17148G7481G11487，苦参碱型生物碱均具有G1209苦参碱（G12628G12228M）为代表的G20604G7562结G7512，G4658于四G10627的G2957G3006G2890类，分子G20604G7562可G11487作G1120个G2957G3006G2890G10627的G12276G2524G1319，M分子式为C15H24N2O，OM是M的N2氧化物，分子式为C15H24N2O2，在G980定条件下M和OM可G11468G1126转化10，所G1209氧化苦参碱G994苦参碱，氧化槐果碱G994槐果碱G1055G19400的转化是可G14033的。中药配伍后成分的转化也是中药方剂G17789证用药治疗G11154病的G7693G7424所在，所G1209明确中药G7460有效成分和中药方剂配伍后成分的改变对于研究中药G19462治G11154病的原理是G19762G5132G18337要的，也是中药现代化的要G8726。REFERENCE1A59A60A61A59A62A63A61A64A65A66A67A68A69A70A71A72A73A74A75A76A77JA67A78A79A78A72A80A81A612003A6128A829A83A84801804A672A85A86A61A87A88A89A61A59A90A91A61A92A67A93A94A95A96A97A98A99A100A101A74A75JA67A102A103A103A104A105A106A107A1081998A10814A1096A110A1112426A1123A113A114A108A115A116A117A108A118A119A120A108A121A122A123A124A125A126A127A128A129A130A102A128A129A131A132A133A134A135A136A137A138A139JA112A103A140A141A142A105A143A144A1082007A1087A1092A110A111124126A1124A145A146A147A108A148A149A150A108A151A152A153A112HPLCA154A155A156A157A130A158A159A160A102A128A129A161A162A163A164A128A129A161A135A165A166JA112A102A167A103A1082005A10836A1093A110A111383384A1125A168A169A170A108A171A172A173A108A174A131A175A112A176A177A178A179A180A181A103A182