《葛根实验研究》PPT课件.ppt

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1、,中药葛根药学实验研究The Pharmaceutical Experimental Study of Puerariae Labatae Radix,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

2、验研究The Pharmaceutical Experimental Study of Puerariae Labatae Radix,葛根与粉葛的真实性鉴定性状鉴定 Macroscopic identification显微鉴定 Microscopic identification薄层色谱鉴别 TLC identification葛根总黄酮提取纯化过程的研究提取纯化工艺 Extraction and purification process各提取物含量测定 Extracts quantity determination葛根总黄酮的含量测定样品制备 Sample preparation标准曲线的

3、建立 Standard curve精密度试验 Precision test稳定性试验 Stability test重复性试验 Repetition test加样回收率试验 Recovery test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,Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix,性状鉴定 Macroscopic identification观察四种药材的性状，包括色泽、形状、大小、外皮

4、、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Microscopic identification,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。,Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix,显微

5、鉴定Microscopic identification,蔡菁 2010491017,1号粉末特征不明显,Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix,2号粉末有淀粉粒、石细胞、晶鞘纤维、具缘纹孔导管等特征,bordered pitted vessels,stone cells,crystal fibres,starch granules,显微鉴定Microscopic identification,Authentic identification of Puerariae L

6、abatae Radix and Puerariae Thomsonii Radix,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,crystal fibres,bordered pitted vessels,stone cells,starch granules,显微鉴定Microscopic identification,Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix,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,

7、crystal fibres,crystal fibres,starch granules,bordered pitted vessels,stone cells,bordered pitted vessels,stone cells,starch granules,显微鉴定Microscopic identification,Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix,薄层色谱鉴别 TLC identification,254nm紫外灯下观察到的薄层色谱图 365nm紫外灯

8、下观察到的薄层色谱图,Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix,Studies on extraction and purification process of total flavonoids from Puerariae Lobatae Radix,Extraction and purification processWater extractionAlcohol precipitationMacroporous resin CC,Structural formula

9、 of Puerarin,Extraction and purification process,Flavonoids content analysis,Studies on extraction and purification process of total flavonoids from Puerariae Lobatae Radix,Comparison of different extraction process,Studies on extraction and purification process of total flavonoids from Puerariae Lo

10、batae Radix,Extraction and purification process,Sample preparationStandard curvePrecision testStability testRepetition testRecovery testAssay,Determination of total flavonoids in Puerariae Lobatae Radix,Standard curve,The effect of puerarin concentration on absorbance,Standard curve,Determination of

11、 total flavonoids in Puerariae Lobatae Radix,Precision test,Results of precision test,Determination of total flavonoids in Puerariae Lobatae Radix,Stability test,Results of stability test,Determination of total flavonoids in Puerariae Lobatae Radix,Repetition test,Results of repetition test,Determin

12、ation of total flavonoids in Puerariae Lobatae Radix,Recovery test,Results of recovery test,Determination of total flavonoids in Puerariae Lobatae Radix,Assay,Determination of samples,Determination of total flavonoids in Puerariae Lobatae Radix,Antipyretic effect of different extraction from Puerari

13、ae Lobatae Radix on endotoxin-induced fever in rabbits,Antipyretic effect of different extraction from Puerariae Lobatae Radix on endotoxin-induced fever in rabbits,Time-body temperature curve,Time-temperature difference curve,Dissolution test of Yufengningxin tablets and Yufengningxin dropping pill

14、s,Standard curve,Determination of the dissolution,Dissolution of Yufengningxin tablets,T50=65min,Td=80min,Dissolution test of Yufengningxin tablets and Yufengningxin dropping pills,Determination of the dissolution,Dissolution of Yufengningxin dropping pills,T50=1min,Td=4min,Dissolution test of Yufen

15、gningxin tablets and Yufengningxin dropping pills,Dissolution of Yufengningxin tablets and dropping pills,Dissolution test of Yufengningxin tablets and Yufengningxin dropping pills,Conclusion and Discussion,1、Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii RadixSample 1 w

16、as counterfeit,sample 2 was Yufeng ningxin tablet,sample 3 was superiority of Puerariae Labatae Radix,sample 4 was Puerariae Thomsonii Radix,and sample 5 was inferiority of Puerariae Labatae Radix.2、Studies on extraction and purification process of total flavonoids from Puerariae Lobatae RadixExtrac

17、t yield rate of water extraction was 23.4%,and that of water extraction and alcohol precipitation was 13.4%,which were both higher than the macroporous resin method with the 6%rate.But the purity quotient of macroporous resin extraction was 26%,which was higher than the water extraction with the 5.4

18、%value and the alcohol precipitation with the 15.8%value.,Conclusion and Discussion,3、Determination of total flavonoids in Puerariae Lobatae RadixThe regression equation of standard curve was y=72.629 x+0.0291,r=0.9959 and the linear range was 0.194 to 0.733.The RSD value of precision test was 0.09%

19、,which showed a good precision of UV analyzer and the machine can be used in following tests.The RSD value of stability test was 0.035%,indicating that the absorbance of sample changed little as time went by and it was stable enough to be used to calculate the content of total flavonoids.The RSD val

20、ue of repetition test was 6.02%,so the absorbance of the 6 samples varied,that is to say the total flavonoids in each sample variedThe RSD value of recovery was 7.77%,so the recovery rates varied and the operation may not appropriate.In the determination,content of total flavonoids in Puerariae Loba

21、tae Radix,Puerariae Thomsonsi Radix and Yufengningxin tablet were 5.3575%，4.812%，8.385%respectively.,Conclusion and Discussion,4、Antipyretic effect of different extraction from Puerariae Lobatae Radix on endotoxin-induced fever in rabbitsThe results of negative control group were consistent with the

22、 expectation,for the temperature was increasing after modeling,which indicated the success of modeling.The value of positive control groups rocketing after 120min.Figures of P.L water-extraction decreased for a period 90min after modeling and then increased to an higher value than basic temperature.

23、The temperature of P.L resin-extraction kept going up until 120min and at that time the negative control group also presented a tendency of dropping back5、Dissolution test of Yufengningxin tablets and Yufengningxin dropping pillsT50 and Td of Yufengningxin dropping pills were 1min and 4min respectiv

24、ely,and T50 and Td of Yufengningxin tablets were 65min and 80min respectively,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-1 Ang,葛根素对压力超负荷大鼠心室重构的影响,实验材料仪器：RM 6280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Image Pro pl

25、us version 5.0 Media CyberneticsSynergy HT多功能酶标仪DL 250RC2L 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(collagen IV,Col IV)ELISA试剂盒，血管紧张素(angiotensin II，AngII)放射免疫分析药盒实验动物：SD大鼠，雄性，体重220250 g,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125 mg/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

26、卡托普利组：ig卡托普利水溶液40 mg/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(LVW

27、 I)，全心质量指数(HW I)。,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Image Pro plus 5.0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

28、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200 mg，加入2 m l生理盐水在冰浴环境中快速匀浆，3500 r/min离心10 min，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A

29、ng II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Ang II的含量表示。,葛根素对压力超负荷大鼠心肌细胞Ang II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!,