葛根实验研究

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验研究The

2、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标准曲线的建立 Stand

3、ard 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、横切面、气味、重量、

4、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显微鉴定Microscopic identi

5、fication蔡菁 20104910171号粉末特征不明显Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix2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 vesselsstone cellscrystal fibresstarch granules显微鉴定Microscopic identificationAuthentic identification of Puerariae Labatae Radix and Puerariae Tho

6、msonii Radix4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 fibresbordered pitted vesselsstone cellsstarch granules显微鉴定Microscopic identificationAuthentic 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crystal fibrescrystal fibresstarch gra

7、nulesbordered pitted vesselsstone cellsbordered pitted vesselsstone cellsstarch granules显微鉴定Microscopic identificationAuthentic 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紫外灯下观察到的薄层色谱图Authentic identification of Puerariae

8、Labatae Radix and Puerariae Thomsonii RadixStudies on extraction and purification process of total flavonoids from Puerariae Lobatae RadixExtraction and purification processWater extractionAlcohol precipitationMacroporous resin CCStructural formula of PuerarinExtraction and purification processFlavo

9、noids content analysisExtraction methodTotal volume(ml)Sampling volume(ml)DilutionAbsorbanceContent(g/ml)Total flavonoids(mg)Water extraction68052500.4786.1811.051Alcohol precipitation208510000.5827.6131.583Macroporous resin CC10055000.3464.3630.218Studies on extraction and purification process of t

10、otal flavonoids from Puerariae Lobatae Radix Comparison of different extraction processExtraction methodTotal flavonoids content%Yield%Total flavonoids recovery%Water extraction11.924.25.4Alcohol precipitation19.013.415.8Macroporous resin CC14.16.026.0Studies on extraction and purification process o

11、f total flavonoids from Puerariae Lobatae RadixExtraction and purification processSample preparationStandard curvePrecision testStability testRepetition testRecovery testAssayDetermination of total flavonoids in Puerariae Lobatae RadixStandard curveMeasurementFoldConcentration(mg/ml)Absorbance1400.0

12、0250.1942250.0040.3183200.0050.41412.50.0080.6335100.0100.733The effect of puerarin concentration on absorbanceStandard curveDetermination of total flavonoids in Puerariae Lobatae RadixPrecision testMeasurementAbsorbanceAverage absorbanceRSD%10.4480.44780.0920.44730.44840.44850.44860.448Results of p

13、recision testDetermination of total flavonoids in Puerariae Lobatae RadixMeasurementSampling time(min)AbsorbanceAverage absorbanceRSD%100.5150.51820.0421200.51831800.52042100.52052400.51862700.518Stability testResults of stability testDetermination of total flavonoids in Puerariae Lobatae RadixMeasu

14、rementWeight(mg)Sample concentration(mg/ml)AbsorbanceContent(mg/ml)Concentration(%)Average concentration(%)RSD%10.10030.050150.4000.0051110.183011.07836.0320.09990.049950.4200.0053810.770830.10030.050150.4490.0057811.525440.10020.050100.4670.0060312.035950.10040.050200.4410.0056711.294860.10000.0500

15、00.4160.0053310.6600Repetition testResults of repetition testDetermination of total flavonoids in Puerariae Lobatae RadixMeasurementSampling weight(g)Originalweight(mg)Added Weight(mg)AbsorbanceMeasured Weight(mg)Recovery(%)Average Recovery(%)RSD(%)10.05045.554.440.4110.49 111.3 107.697.7720.05044.4

16、40.40910.46 110.6 30.05065.550.47212.20 119.8 40.05005.550.43711.23 102.4 50.05026.660.48912.66 106.8 60.05016.660.46111.89 95.3 Recovery testResults of recovery testDetermination of total flavonoids in Puerariae Lobatae RadixSamplePuerariae Thomsoni Radix Puerariae Lobatae RadixYufengningxin Tablet

17、121212Weight(g)0.10050.10010.09990.10000.05010.0502Absorbance02930.4660.4200.4160.3400.329Content(%)3.6156.0095.3885.3278.5448.226Average(%)4.8125.35758.385AssayDetermination of samplesDetermination of total flavonoids in Puerariae Lobatae RadixAntipyretic effect of different extraction from Puerari

18、ae Lobatae Radix on endotoxin-induced fever in rabbitsGroupMedicineDosage(g/kg)Basic body temperature()temperature difference = the measured body temperature -basic body temperature()30min60min90min120min150min180 min 210 minNegative controlNS/38.330.981.531.882.032.332.182.00Positive controlAnalgin

19、0.07838.720.850.680.350.381.351.651.05P.L. water-extractionPueraria lobata water extract6.2539.680.621.081.270.780.821.171.22P.L.resin-extractionPueraria lobata resin extract6.2538.990.991.311.291.941.861.801.36Antipyretic effect of different extraction from Puerariae Lobatae Radix on endotoxin-indu

20、ced fever in rabbitsTime-body temperature curveTime-temperature difference curveDissolution test of Yufengningxin tablets and Yufengningxin dropping pillsStandard curveDetermination of the dissolutionSampling Time Point（min）5101530456090120180Dilution Times（Fold）111244888Absorbance0.1470.2180.3060.3

21、100.3140.4550.3590.4360.493Concentration（mg/ml）1.82*10-32.72*10-33.85*10-37.81*10-31.58*10-22.30*10-23.61*10-24.40*10-24.98*10-2Cumulative Release（%）3.675.617.7215.6731.6846.0972.5388.24100.0Dissolution of Yufengningxin tabletsT50=65min , Td=80minDissolution test of Yufengningxin tablets and Yufengn

22、ingxin dropping pillsDetermination of the dissolutionSampling Time Point（min）5101530456090120180Dilution Times（Fold）11124 4 8 88Absorbance0.4310.4740.5040.5130.5030.5000.5010.5000.503Concentration（mg/ml）5.61*10-36.16*10-36.54*10-36.66*10-36.53*10-36.50*10-36.51*10-36.50*10-36.50*10-3Cumulative Relea

23、se（%）85.9194.33100.0102.0100.0100.0100.0100.0100.0Dissolution of Yufengningxin dropping pillsT50=1min , Td=4minDissolution test of Yufengningxin tablets and Yufengningxin dropping pillsDissolution of Yufengningxin tablets and dropping pillsDissolution test of Yufengningxin tablets and Yufengningxin

24、dropping pillsConclusion and Discussion1、Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii RadixSample 1 was 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 inferi

25、ority of Puerariae Labatae Radix. 2、Studies on extraction and purification process of total flavonoids from Puerariae Lobatae RadixExtract 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 met

26、hod 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% value and the alcohol precipitation with the 15.8% value.Conclusion and Discussion3、Determination of total flavonoids in Puerariae Lobatae RadixThe regressi

27、on 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% , 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

28、 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 value 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 r

29、ecovery was 7.77%, so the recovery rates varied and the operation may not appropriate.In the determination, content of total flavonoids in Puerariae Lobatae Radix, Puerariae Thomsonsi Radix and Yufengningxin tablet were 5.3575%，4.812%，8.385% respectively.Conclusion and Discussion4、Antipyretic effect

30、 of different extraction from Puerariae Lobatae Radix on endotoxin-induced fever in rabbitsThe results of negative control group were consistent with the expectation, for the temperature was increasing after modeling, which indicated the success of modeling.The value of positive control groups rocke

31、ting 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.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 dropp

32、ing back5、Dissolution test of Yufengningxin tablets and Yufengningxin dropping pillsT50 and Td of Yufengningxin dropping pills were 1min and 4min respectively, 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

33、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血流动力学因素神经内分泌因素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 plus 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

34、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。组别剂量SBP(mmH

35、g)DBP(mmHg)HR(bpm)模型组-假手术组-卡托普利组40mg/kg葛根小剂量组125mg/kg葛根中剂量组250mg/kg葛根大剂量组500mg/kg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 I)，全心质量指数(HW I)。组别剂量LVWI(mg/g)HWI(mg/g)模型组-假手术组-卡托普利组40mg/kg葛根小剂量组125mg/kg葛根中剂量组250mg/kg葛根大剂量组500mg/kg葛根素对压力超负荷大鼠心脏质量指数的影响（s，n=