畢業(yè)論文HPLC法測(cè)定龍芪柔肝膠囊中丹參酮A的含量系別生物技術(shù)系班級(jí)中藥制藥2班姓名學(xué)號(hào)指導(dǎo)老師目錄摘要1前言11龍芪柔肝膠囊簡(jiǎn)介（3）12處方分析（3）13中藥藥理學(xué)分析（3）14小結(jié)（3）2正文21儀器與試藥（3）211儀器3212試藥322方法與結(jié)果3221對(duì)照品溶液的制備3222供試品溶液的制備4223色譜條件4224線(xiàn)性關(guān)系的考察4225精密度試驗(yàn)4226重復(fù)性試驗(yàn)4227穩(wěn)定性試驗(yàn)4228加樣回收率試驗(yàn)4229樣品的含量測(cè)定（4）3討論31檢測(cè)波長(zhǎng)的選擇（4）32色譜條件的選擇（4）33提取方法的選擇（4）331藥品預(yù)處理（5）332供試品溶液的提取（5）333供試品的篩選（5）4參考文獻(xiàn)（5）5致謝（5）HPLC法測(cè)定龍芪柔肝膠囊中丹參酮A的含量摘要目的建立HPLC法測(cè)定龍芪柔肝膠囊中丹參酮A含量的方法。方法選用ANJILENTC18柱（250MM46MM,5UM）,以甲醇水（2080）為流動(dòng)相，流速10MLMIN1,檢測(cè)波長(zhǎng)270NM,柱溫25。結(jié)果丹參酮IIA在169615264UGML間呈良好的線(xiàn)性關(guān)系R2099969,平均加樣回收率為10132（RSD28N5）；結(jié)論該方法快速、準(zhǔn)確、精密，可用于龍芪柔肝膠囊的質(zhì)量控制。關(guān)鍵字HPLC法；龍芪柔肝膠囊；丹參酮IIADETERMINATIONOFTANSHINONETONGAINTHELONGQICAPSULESBYHPLCABSTRACTOBJECTIVETOESTABLISHANHPLCMETHODFORTHETANSHINONETONGAINTHELONGQICAPSULESMETHODTHEANALYSISWASPERFORMEDONACOLUMNOFANJILENTC18（250MM46MM,5UM）THEMOBILEPHASECONSISTEDOFMETHANOLANDWATER（2080）WITHTHEFLOWRATEOF10MLMIN1THEDETECTIONWAVELENGTHWAS270NMANDTHECOLUMNTEMPERATUREWAS25RESULTPROMISINGLINEARITYWASOBTAINEDWITHINTHERANGEOF169615264UGMLFORTANSHINONETONGATHEAVERAGERECOVERYOFTANSHINONETONGAWASR2099969CONCLUSIONTHISMETHODISRAPIDANDACCURATEANDCANBEUSEDTOCONTROLTHEQUALITYOFTANSHINONETONGAKEYWORDSHPLCTANSHINONETONGALONGQICAPSULES1前言11龍芪柔肝膠囊簡(jiǎn)介龍芪柔肝膠囊為信陽(yáng)市中醫(yī)院院內(nèi)制劑，由黃芪、丹參、黨參、生地黃、北沙參、當(dāng)歸、地龍、醋白芍、鱉甲、黃精、五味子、靈芝、桃仁、紅花、炒穿山甲共15位中藥制備而成。主治慢性病毒性肝炎，肝纖維化、肝硬化等癥。具有益氣養(yǎng)陰，活血柔肝的功效。方中地龍有較強(qiáng)的異味，穿山甲屬名貴藥材以打粉入藥。因膠囊劑具有能夠掩蓋藥物不良臭味，載藥量大的特點(diǎn)，故選用膠囊劑4。12處方分析方中以黃芪、地龍為君藥，以丹參、當(dāng)歸、生地黃、北沙參、黨參為臣藥。方中黃芪甘溫入脾肺經(jīng)，補(bǔ)中氣、升清陽(yáng)，益肺氣、實(shí)皮毛，以扶正；地龍同入肝脾經(jīng)，息風(fēng)止痙，通絡(luò)，以開(kāi)血脈阻滯，二者共為君藥。丹參、當(dāng)歸并用，生血、活血且化瘀、通絡(luò)而不傷血；生地、北沙參并用，滋陰而又養(yǎng)陰；黨參補(bǔ)氣以行血；五者共為臣藥。取君二臣五，奇之制之意。五味子、醋白芍共同輔助君藥抑陽(yáng)斂陰，以柔肝；鱉甲、黃精、靈芝共用助君藥滋補(bǔ)肝陰。五者共為佐藥。桃仁、紅花活血祛瘀，穿山甲消腫排膿，三者共同引藥歸經(jīng)，均為使藥。本方為補(bǔ)中益氣湯（脾胃論）和補(bǔ)陽(yáng)還五湯（醫(yī)林改錯(cuò)）的復(fù)合方。一為補(bǔ)氣，一為活血祛瘀，二者并用以達(dá)到益氣養(yǎng)陰、活血柔肝的功效5。13中藥藥理學(xué)分析黃芪主含多糖類(lèi)和皂苷類(lèi)成分，能增強(qiáng)非特異性免疫功能，包括增加WBC和中性粒細(xì)胞、M吞噬功能。地龍?zhí)崛∥锟诜?，?duì)小鼠S180和H22均有抑瘤作用，并可增強(qiáng)5FU受體活化，從而增強(qiáng)免疫細(xì)胞的活性。丹參中主含丹參酮A，丹參酮A磺酸鈉可抑制心肌收縮力，降低竇房結(jié)自律性，能促進(jìn)肝、骨、皮膚等多種組織的修復(fù)與再生，其中促進(jìn)肝組織的修復(fù)與再生尤為顯著3。因此丹參酮A也是本藥的重要和關(guān)鍵性成份。14小結(jié)本文重點(diǎn)探索了HPLC法測(cè)定龍芪柔肝膠囊中丹參酮A含量時(shí)方法的建立及優(yōu)化，為全面評(píng)價(jià)該制劑質(zhì)量和完善本復(fù)方制劑的質(zhì)量提供實(shí)驗(yàn)依據(jù)。2正文21儀器與試藥211儀器AGILENT1200液相色譜儀美國(guó)AGILENT公司，AGILENT1200檢測(cè)器，AGILENT工作站；WATERS1525液相色譜儀（WATERS公司，WATERS2489柱溫箱BREEZE工作站）；普利賽斯雙量程電子天平美國(guó)塞利普斯公司；超純水機(jī)（基因型185OD重慶摩爾制水設(shè)備有限公司）；超聲儀（寧波新芝生物科技有限公司）。212試藥丹參酮A對(duì)照品（規(guī)格20MG支，110766200518，中國(guó)藥品生物制品檢定所）；甲醇（色譜純，美國(guó)AGILENT公司）；龍芪柔肝膠囊（信陽(yáng)市中醫(yī)院制劑室，規(guī)格60粒瓶，批號(hào)20110410、20110512、20110823）；水（信陽(yáng)市藥檢所中藥實(shí)驗(yàn)室制水機(jī)，一級(jí)水）；其他試劑均為分析純。22方法與結(jié)果221對(duì)照品溶液的制備取丹參酮A對(duì)照品106MG置25ML容量瓶中，精密稱(chēng)定，加甲醇定容。用1ML移液管移取配置好的對(duì)照品溶液，另置25ML容量瓶中，加甲醇定容，即得。配得的對(duì)照品溶液濃度為1696UGML222供試品溶液及陰性樣品的制備取本藥品5份，每份32G，精密稱(chēng)定。分別置同型號(hào)250ML具塞錐形瓶中，各加入25ML甲醇。精密稱(chēng)定。超聲提取30MIN。放至室溫，精密稱(chēng)定，用甲醇補(bǔ)足損失的量。過(guò)濾，取續(xù)濾液，即得。模擬處方的制備方法，制備不含丹參的陰性樣品，按供試品制備方法制備成陰性樣品溶液。表1取樣量超聲前超聲后補(bǔ)足后1號(hào)實(shí)驗(yàn)318731163690116326911636422號(hào)實(shí)驗(yàn)3177011229921123287111229313號(hào)實(shí)驗(yàn)319339430759429459430214號(hào)實(shí)驗(yàn)321881043507104334810435205號(hào)實(shí)驗(yàn)31842881713881548881748223色譜條件色譜柱AGILENTC18柱（250MM46MM,5UM）；流動(dòng)相以甲醇水（2080）為流動(dòng)相；流速10MLMIN1；檢測(cè)波長(zhǎng)270NM；柱溫25。理論板數(shù)按丹參酮A峰計(jì)算應(yīng)不低于2000。分離度15；龍芪柔肝膠囊中其他成分對(duì)丹參酮A含量的測(cè)定無(wú)影響。1對(duì)照品2供試品3缺丹參對(duì)照品224線(xiàn)性關(guān)系的考察精密吸取配置好的丹參酮A對(duì)照品溶液1UL、3UL、5UL、7UL、9UL注入高效液相色譜儀，依法測(cè)定，以峰面積積分值為縱坐標(biāo)（Y），以進(jìn)樣量（UG）為橫坐標(biāo)（X）,繪制標(biāo)準(zhǔn)曲線(xiàn)。結(jié)果表明丹參酮A在169615264UGML范圍內(nèi)呈良好的線(xiàn)性關(guān)系。回歸方程為Y5759589X101938R2099969進(jìn)樣量進(jìn)樣量（G）保留時(shí)間峰面積面積1微升155112573000075463微升15537183679000082315微升15511383743000091797微升15504580916000097889微升1549678100300009191Y575958924336X10193821441R209996910000000000000000100000000002000000000030000000000400000000005000000000060000000000700000000008000000000090000000000000000002000004000006000008000010000012000014000016000018000進(jìn)樣量峰面積225精密度試驗(yàn)取中間上述25對(duì)照品注入高效液相，重復(fù)進(jìn)樣6次，每次進(jìn)樣5UL。記錄丹參酮A峰面積，結(jié)果丹參酮ARSD169N5226重復(fù)性試驗(yàn)取同一批號(hào)供試品，按23項(xiàng)下方法制備5份供試液，分別進(jìn)樣，測(cè)定峰面積，計(jì)算丹參酮A的平均含量為0081G1，其RSD267（N5）。227穩(wěn)定性試驗(yàn)取同一供試品溶液，分別在0、3、6、9H按上述色譜條件試驗(yàn)，記錄丹參酮A峰面積積分值，結(jié)RSD3；表明供試品溶液在9H內(nèi)基本穩(wěn)定。228加樣回收率試驗(yàn)精密稱(chēng)定已測(cè)定含量的樣品（1696UGML），共取5份，每份32G，按22項(xiàng)下方法制備供試品溶液。分別精密加入丹參酮A對(duì)照品儲(chǔ)備液1ML，進(jìn)樣。計(jì)算丹參酮A含量并計(jì)算回收率為10132RSD28N5。229樣品的含量測(cè)定取3批龍芪柔肝膠囊樣品，按上述供試品溶液的制備方法制備，并按上述色譜條件測(cè)定丹參酮A含量。結(jié)果0081G13討論31檢測(cè)波長(zhǎng)的選擇經(jīng)過(guò)AGILENT1200三維圖譜分析，發(fā)現(xiàn)在270NM,275NM處均有吸收峰，且吸收系數(shù)均較大，考慮到龍芪柔肝膠囊藥味比較復(fù)雜，對(duì)丹參酮A的檢測(cè)容易產(chǎn)生干擾，因此選擇檢測(cè)波長(zhǎng)為270NM。32色譜條件的選擇曾用甲醇水（2575）作為流動(dòng)相1，但丹參酮A峰形有拖尾，用于定量分析不準(zhǔn)確。經(jīng)過(guò)試驗(yàn)，調(diào)整流動(dòng)相為甲醇水（2080）。峰形明顯改善。影響峰形的因素是否還與其他因素（如PH）有關(guān)，尚需進(jìn)一步研究。33提取方法的選擇331藥品預(yù)處理取龍芪柔肝膠囊4份，每份32G，精密稱(chēng)定。分別置250ML具塞錐形瓶中。編號(hào)為回1、回2、超1、超2。各瓶精密加入甲醇50ML,稱(chēng)定重量。332供試品溶液的提取對(duì)回1進(jìn)行30MIN回流提取，對(duì)回2進(jìn)行45MIN回流提取。對(duì)超1進(jìn)行超聲30MIN超聲提取，對(duì)超2進(jìn)行45MIN超聲提取1。333供試品的篩選將上述四瓶供試品放至室溫，精密稱(chēng)定，補(bǔ)足損失的量，過(guò)濾。取續(xù)慮液，用HPLC進(jìn)行檢定。發(fā)現(xiàn)30MIN時(shí)即提取完全。且超聲提取優(yōu)于回流提取。4參考文獻(xiàn)1國(guó)家藥典委員會(huì)中華人民共和國(guó)藥典一部S北京中國(guó)醫(yī)藥科技出版社,20102張德欽中藥分析制劑技術(shù)北京中國(guó)中醫(yī)藥出版社3方泰惠吳清和中藥藥理學(xué)北京科學(xué)出版社4陳瓊中藥制劑技術(shù)北京中國(guó)農(nóng)業(yè)大學(xué)出版社5王均寧薛建國(guó)方劑學(xué)北京科學(xué)出版社6凌一揆中藥學(xué)上海上海科學(xué)技術(shù)出版社7唐麗琴，張善堂，陳象青，等HPLC法測(cè)定不同來(lái)源補(bǔ)骨脂中補(bǔ)骨脂素和異補(bǔ)骨脂素的含量M中國(guó)藥師，2011，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