《葛根实验研究》PPT课件.ppt

,中药葛根药学实验研究ThePharmaceuticalExperimentalStudyofPuerariaeLabataeRadix,实验人员：马丹萌，朱思然，胡少杰，杨枭，郑喆姣,葛根的现代研究葛根的药学实验研究葛根素对压力超负荷大鼠心室重构的影响（拓展）,目录,葛根的现代研究,葛根的现代研究,化学成分黄酮苷类、三萜类、香豆素类等含测方法高效液相色谱法紫外-可见分光光度法中药指纹图谱提取纯化工艺溶剂提取法微波辅助萃取法超声提取法大孔树脂吸附法离子交换纤维法配位体色谱法药理作用解热、降血压、降血糖、抗心肌缺血、脑保护等药物制剂滴丸、胶囊、片剂、颗粒、眼用凝胶剂等,葛根的药学实验研究ThePharmaceuticalExperimentalStudyofPuerariaeLabataeRadix,葛根与粉葛的真实性鉴定性状鉴定Macroscopicidentification显微鉴定Microscopicidentification薄层色谱鉴别TLCidentification葛根总黄酮提取纯化过程的研究提取纯化工艺Extractionandpurificationprocess各提取物含量测定Extractsquantitydetermination葛根总黄酮的含量测定样品制备Samplepreparation标准曲线的建立Standardcurve精密度试验Precisiontest稳定性试验Stabilitytest重复性试验Repetitiontest加样回收率试验Recoverytest样品的含量测定Assay不同工艺野葛提取物对内毒素所致发热家兔体温的影响愈风宁心片、滴丸溶出度比较,葛根的药学实验研究实验流程,AuthenticidentificationofPuerariaeLabataeRadixandPuerariaeThomsoniiRadix,性状鉴定Macroscopicidentification观察四种药材的性状，包括色泽、形状、大小、外皮、横切面、气味、重量、质地、是否具有粉性等，并与药典中记载的特征对比，分辨出野葛、粉葛及伪品。,1号,2号,3号,4号,5号,显微鉴定Microscopicidentification,用镊子取少量1号粉末样品置于载玻片上，并滴加蒸馏水，盖上盖玻片，制成水装片；再用镊子取少量1号粉末样品置于载玻片上，滴加水合氯醛溶液，将载玻片置于酒精灯上灼烧至微沸，再滴加1-2滴甘油，盖上盖玻片，制成水合氯醛装片。2-5号粉末进行相同操作。,AuthenticidentificationofPuerariaeLabataeRadixandPuerariaeThomsoniiRadix,显微鉴定Microscopicidentification,蔡菁2010491017,1号粉末特征不明显,AuthenticidentificationofPuerariaeLabataeRadixandPuerariaeThomsoniiRadix,2号粉末有淀粉粒、石细胞、晶鞘纤维、具缘纹孔导管等特征,borderedpittedvessels,stonecells,crystalfibres,starchgranules,显微鉴定Microscopicidentification,AuthenticidentificationofPuerariaeLabataeRadixandPuerariaeThomsoniiRadix,4号粉末有清晰淀粉粒，且多为单粒，还有石细胞、晶鞘纤维和具缘纹孔导管,crystalfibres,borderedpittedvessels,stonecells,starchgranules,显微鉴定Microscopicidentification,AuthenticidentificationofPuerariaeLabataeRadixandPuerariaeThomsoniiRadix,3号粉末5号粉末均有清晰的淀粉粒、石细胞、晶鞘纤维、具缘纹孔导管及六角形具缘纹孔,crystalfibres,crystalfibres,starchgranules,borderedpittedvessels,stonecells,borderedpittedvessels,stonecells,starchgranules,显微鉴定Microscopicidentification,AuthenticidentificationofPuerariaeLabataeRadixandPuerariaeThomsoniiRadix,薄层色谱鉴别TLCidentification,254nm紫外灯下观察到的薄层色谱图365nm紫外灯下观察到的薄层色谱图,AuthenticidentificationofPuerariaeLabataeRadixandPuerariaeThomsoniiRadix,StudiesonextractionandpurificationprocessoftotalflavonoidsfromPuerariaeLobataeRadix,ExtractionandpurificationprocessWaterextractionAlcoholprecipitationMacroporousresinCC,StructuralformulaofPuerarin,Extractionandpurificationprocess,Flavonoidscontentanalysis,StudiesonextractionandpurificationprocessoftotalflavonoidsfromPuerariaeLobataeRadix,Comparisonofdifferentextractionprocess,StudiesonextractionandpurificationprocessoftotalflavonoidsfromPuerariaeLobataeRadix,Extractionandpurificationprocess,SamplepreparationStandardcurvePrecisiontestStabilitytestRepetitiontestRecoverytestAssay,DeterminationoftotalflavonoidsinPuerariaeLobataeRadix,Standardcurve,Theeffectofpuerarinconcentrationonabsorbance,Standardcurve,DeterminationoftotalflavonoidsinPuerariaeLobataeRadix,Precisiontest,Resultsofprecisiontest,DeterminationoftotalflavonoidsinPuerariaeLobataeRadix,Stabilitytest,Resultsofstabilitytest,DeterminationoftotalflavonoidsinPuerariaeLobataeRadix,Repetitiontest,Resultsofrepetitiontest,DeterminationoftotalflavonoidsinPuerariaeLobataeRadix,Recoverytest,Resultsofrecoverytest,DeterminationoftotalflavonoidsinPuerariaeLobataeRadix,Assay,Determinationofsamples,DeterminationoftotalflavonoidsinPuerariaeLobataeRadix,AntipyreticeffectofdifferentextractionfromPuerariaeLobataeRadixonendotoxin-inducedfeverinrabbits,AntipyreticeffectofdifferentextractionfromPuerariaeLobataeRadixonendotoxin-inducedfeverinrabbits,Time-bodytemperaturecurve,Time-temperaturedifferencecurve,DissolutiontestofYufengningxintabletsandYufengningxindroppingpills,Standardcurve,Determinationofthedissolution,DissolutionofYufengningxintablets,T50=65min,Td=80min,DissolutiontestofYufengningxintabletsandYufengningxindroppingpills,Determinationofthedissolution,DissolutionofYufengningxindroppingpills,T50=1min,Td=4min,DissolutiontestofYufengningxintabletsandYufengningxindroppingpills,DissolutionofYufengningxintabletsanddroppingpills,DissolutiontestofYufengningxintabletsandYufengningxindroppingpills,ConclusionandDiscussion,1、AuthenticidentificationofPuerariaeLabataeRadixandPuerariaeThomsoniiRadixSample1wascounterfeit,sample2wasYufengningxintablet,sample3wassuperiorityofPuerariaeLabataeRadix,sample4wasPuerariaeThomsoniiRadix,andsample5wasinferiorityofPuerariaeLabataeRadix.2、StudiesonextractionandpurificationprocessoftotalflavonoidsfromPuerariaeLobataeRadixExtractyieldrateofwaterextractionwas23.4%,andthatofwaterextractionandalcoholprecipitationwas13.4%,whichwerebothhigherthanthemacroporousresinmethodwiththe6%rate.Butthepurityquotientofmacroporousresinextractionwas26%,whichwashigherthanthewaterextractionwiththe5.4%valueandthealcoholprecipitationwiththe15.8%value.,ConclusionandDiscussion,3、DeterminationoftotalflavonoidsinPuerariaeLobataeRadixTheregressionequationofstandardcurvewasy=72.629x+0.0291,r=0.9959andthelinearrangewas0.194to0.733.TheRSDvalueofprecisiontestwas0.09%,whichshowedagoodprecisionofUVanalyzerandthemachinecanbeusedinfollowingtests.TheRSDvalueofstabilitytestwas0.035%,indicatingthattheabsorbanceofsamplechangedlittleastimewentbyanditwasstableenoughtobeusedtocalculatethecontentoftotalflavonoids.TheRSDvalueofrepetitiontestwas6.02%,sotheabsorbanceofthe6samplesvaried,thatistosaythetotalflavonoidsineachsamplevariedTheRSDvalueofrecoverywas7.77%,sotherecoveryratesvariedandtheoperationmaynotappropriate.Inthedetermination,contentoftotalflavonoidsinPuerariaeLobataeRadix,PuerariaeThomsonsiRadixandYufengningxintabletwere5.3575%，4.812%，8.385%respectively.,ConclusionandDiscussion,4、AntipyreticeffectofdifferentextractionfromPuerariaeLobataeRadixonendotoxin-inducedfeverinrabbitsTheresultsofnegativecontrolgroupwereconsistentwiththeexpectation,forthetemperaturewasincreasingaftermodeling,whichindicatedthesuccessofmodeling.Thevalueofpositivecontrolgroupsrocketingafter120min.FiguresofP.Lwater-extractiondecreasedforaperiod90minaftermodelingandthenincreasedtoanhighervaluethanbasictemperature.ThetemperatureofP.Lresin-extractionkeptgoingupuntil120minandatthattimethenegativecontrolgroupalsopresentedatendencyofdroppingback5、DissolutiontestofYufengningxintabletsandYufengningxindroppingpillsT50andTdofYufengningxindroppingpillswere1minand4minrespectively,andT50andTdofYufengningxintabletswere65minand80minrespectively,拓展：葛根素对压力超负荷大鼠心室重构的影响,葛根素的心血管药理作用,降血压改善心肌缺血抗心律失常,CHF？,原理和思路,慢性心力衰竭（CHF）,心室重构,心肌肥大心脏重量增加心肌细胞外间质胶原增生和纤维化心腔容积增大,心脏质量指数,LVW/BWHW/BW,TGF-1Ang,葛根素对压力超负荷大鼠心室重构的影响,实验材料仪器：RM6280型多道生理信号采集处理系统电子天平OlympusXB51系统显微镜图像分析系统ImageProplusversion5.0MediaCyberneticsSynergyHT多功能酶标仪DL250RC2L离心机F6/10手持式超细均浆机药物与试剂：葛根素，卡托普利，戊巴比妥钠，青霉素，IV型胶原(collagenIV,ColIV)ELISA试剂盒，血管紧张素(angiotensinII，AngII)放射免疫分析药盒实验动物：SD大鼠，雄性，体重220250g,方法与结果,动物建模模型组：腹主动脉不完全结扎假手术组：分离腹主动脉后不结扎分组与给药葛根素小剂量组：ig葛根素125mg/kg葛根素中剂量组：ig葛根素250mg/kg葛根素大剂量组：ig葛根素500mg/kg卡托普利组：ig卡托普利水溶液40mg/kg假手术组和模型组:ig蒸馏水。,方法与结果,检测指标血压、心率:25%乌拉坦腹腔注射麻醉大鼠，分离颈总动脉，插入聚苯乙烯导管，连接八导生理记录仪记录动脉收缩压(SBP)、动脉舒张压(DBP)、心率(HR)等。,葛根素对压力超负荷大鼠血压、心率的影响（s，n=8）,方法与结果,检测指标心脏质量指数：血压、心率测定完成后，快速打开胸腔取心脏，去除心房组织，分离左、右心室，用冰冷的生理盐水漂洗去血，滤纸吸干表面水分，电子天平准确称取左心室重量和全心重量。计算左心室重量/体重(LVW/BW)比，全心重量/体重(HW/BW)比，分别记为左心室质量指数(LVWI)，全心质量指数(HWI)。,葛根素对压力超负荷大鼠心脏质量指数的影响（s，n=8）,方法与结果,检测指标左室心肌胶原含量的测定：常规制作病理切片进行苦味酸天狼猩红染色。在OlympusXB51型显微镜下观察并摄片(400)，用ImageProplus5.0图像分析系统，分别测量、型胶原纤维的含量(、型胶原面积/总面积)。在常规显微镜下测量心肌胶原容积分数(CVF)和血管胶原容积分数(PVCA)。CVF=胶原面积/总面积，PVCA=小动脉管腔周围胶原面积/动脉管腔面积。每一标本均随机取3个视野测量,计算其均值。,葛根素对压力超负荷大鼠心脏质量指数的影响（s，n=8）,方法与结果,检测指标心肌细胞生长因子TGF-1含量的测定取大鼠的左室心肌组织约200mg，加入2ml生理盐水在冰浴环境中快速匀浆，3500r/min离心10min，取上清液既得10%匀浆，用ELISA法测定心肌细胞中TGF-1的含量，具体操作均按ELISA试剂盒说明书进行，用酶标仪于波长450nm处上读取各孔的OD值，以标准品的OD值为纵坐标，浓度为横坐标，绘制标准曲线。,葛根素对压力超负荷大鼠心肌细胞TGF-1含量的影响（s，n=8）,方法与结果,检测指标心肌细胞血管紧张素(Ang)含量的测定取0.1%的心肌组织匀浆，采用放射免疫分析法（radioimmunoassay，RIA）测定匀浆中AngII的浓度，具体操作按放免药盒说明书进行，结果以每毫克蛋白中AngII的含量表示。,葛根素对压力超负荷大鼠心肌细胞AngII含量的影响（s，n=8）,研究展望,首先，十分感谢崔亚军老师给了我们这样一次机会，让我们将三年所学知识进行串联与融合，以更从容和自信的姿态应对即将来临的毕业课题。其次，非常感谢带教的王冰老师在整个综合实验过程中给予我们细心和耐心的指导，这是我组最终顺利完成综述、实验、论文、拓展、答辩等各个环节的最大助力。同时，也要感谢其他带教老师和同学对我组综合实验提供的帮助，你们及时指出了许多错误，弥补了我组自身的不足，并让我们少走了许多弯路。最后，感谢我们小组的五位成员对本次综合实验的全程参与，每一位成员的辛劳付出与团结协作是顺利完成此次综合实验的坚实基础。在此，特向以上成员致以崇高的敬意和衷心地感谢！,致谢,谢谢!,