葛根實驗研究

1、中藥葛根藥學(xué)實驗研究The Pharmaceutical Experimental Study of Puerariae Labatae Radix 實驗人員：馬丹萌，朱思然，胡少杰，楊梟，鄭喆姣葛根的現(xiàn)代研究葛根的藥學(xué)實驗研究葛根素對壓力超負(fù)荷大鼠心室重構(gòu)的影響（拓展）目錄葛根的現(xiàn)代研究葛根的現(xiàn)代研究化學(xué)成分黃酮苷類、三萜類、香豆素類等含測方法高效液相色譜法紫外-可見分光光度法中藥指紋圖譜提取純化工藝溶劑提取法微波輔助萃取法超聲提取法大孔樹脂吸附法離子交換纖維法配位體色譜法藥理作用解熱、降血壓、降血糖、抗心肌缺血、腦保護(hù)等藥物制劑滴丸、膠囊、片劑、顆粒、眼用凝膠劑等葛根的藥學(xué)實驗研究The

2、Pharmaceutical Experimental Study of Puerariae Labatae Radix葛根與粉葛的真實性鑒定性狀鑒定 Macroscopic identification顯微鑒定 Microscopic identification薄層色譜鑒別 TLC identification葛根總黃酮提取純化過程的研究提取純化工藝 Extraction and purification process各提取物含量測定 Extracts quantity determination葛根總黃酮的含量測定樣品制備 Sample preparation標(biāo)準(zhǔn)曲線的建立 Stand

3、ard curve精密度試驗 Precision test穩(wěn)定性試驗 Stability test重復(fù)性試驗 Repetition test加樣回收率試驗 Recovery test樣品的含量測定 Assay不同工藝野葛提取物對內(nèi)毒素所致發(fā)熱家兔體溫的影響愈風(fēng)寧心片、滴丸溶出度比較葛根的藥學(xué)實驗研究實驗流程Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix性狀鑒定 Macroscopic identification觀察四種藥材的性狀，包括色澤、形狀、大小、外皮、橫切面、氣味、重量、

4、質(zhì)地、是否具有粉性等，并與藥典中記載的特征對比，分辨出野葛、粉葛及偽品。1號2號3號4號5號顯微鑒定Microscopic identification用鑷子取少量1號粉末樣品置于載玻片上，并滴加蒸餾水，蓋上蓋玻片，制成水裝片；再用鑷子取少量1號粉末樣品置于載玻片上，滴加水合氯醛溶液，將載玻片置于酒精燈上灼燒至微沸，再滴加1-2滴甘油，蓋上蓋玻片，制成水合氯醛裝片。2-5號粉末進(jìn)行相同操作。Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix顯微鑒定Microscopic identi

5、fication蔡菁 20104910171號粉末特征不明顯Authentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix2號粉末有淀粉粒、石細(xì)胞、晶鞘纖維、具緣紋孔導(dǎo)管等特征bordered pitted vesselsstone cellscrystal fibresstarch granules顯微鑒定Microscopic identificationAuthentic identification of Puerariae Labatae Radix and Puerariae Tho

6、msonii Radix4號粉末有清晰淀粉粒，且多為單粒，還有石細(xì)胞、晶鞘纖維和具緣紋孔導(dǎo)管crystal fibresbordered pitted vesselsstone cellsstarch granules顯微鑒定Microscopic identificationAuthentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix 3號粉末 5號粉末均有清晰的淀粉粒、石細(xì)胞、晶鞘纖維、具緣紋孔導(dǎo)管及六角形具緣紋孔crystal fibrescrystal fibresstarch gra

7、nulesbordered pitted vesselsstone cellsbordered pitted vesselsstone cellsstarch granules顯微鑒定Microscopic identificationAuthentic identification of Puerariae Labatae Radix and Puerariae Thomsonii Radix薄層色譜鑒別 TLC identification 254nm紫外燈下觀察到的薄層色譜圖 365nm紫外燈下觀察到的薄層色譜圖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：葛根素對壓力超負(fù)荷大鼠心室重構(gòu)的影響葛根素的心血管藥理作用降血壓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、室重構(gòu)心肌肥大心臟重量增加心肌細(xì)胞外間質(zhì)膠原增生和纖維化心腔容積增大心臟質(zhì)量指數(shù)LVW /BWHW /BW血流動力學(xué)因素神經(jīng)內(nèi)分泌因素TGF-1 Ang 葛根素對壓力超負(fù)荷大鼠心室重構(gòu)的影響實驗材料儀器：RM 6280型多道生理信號采集處理系統(tǒng)電子天平OlympusXB51系統(tǒng)顯微鏡圖像分析系統(tǒng)Image Pro plus version 5.0 Media CyberneticsSynergy HT多功能酶標(biāo)儀DL 250RC2L 離心機F6/10手持式超細(xì)均漿機藥物與試劑：葛根素，卡托普利，戊巴比妥鈉，青霉素，IV型膠原(collagen IV, Col IV) ELISA試劑盒，血管緊張

34、素 (angiotensin II，AngII) 放射免疫分析藥盒實驗動物：SD大鼠，雄性，體重220250 g方法與結(jié)果動物建模模型組：腹主動脈不完全結(jié)扎假手術(shù)組：分離腹主動脈后不結(jié)扎分組與給藥葛根素小劑量組：ig葛根素125 mg/kg葛根素中劑量組：ig葛根素250mg/kg葛根素大劑量組：ig葛根素500mg/kg卡托普利組：ig卡托普利水溶液40 mg/kg假手術(shù)組和模型組:ig蒸餾水。方法與結(jié)果檢測指標(biāo)血壓、心率:25%烏拉坦腹腔注射麻醉大鼠，分離頸總動脈，插入聚苯乙烯導(dǎo)管，連接八導(dǎo)生理記錄儀記錄動脈收縮壓( SBP )、動脈舒張壓(DBP)、心率(HR)等。組別劑量SBP(mmH

35、g)DBP(mmHg)HR(bpm)模型組-假手術(shù)組-卡托普利組40mg/kg葛根小劑量組125mg/kg葛根中劑量組250mg/kg葛根大劑量組500mg/kg葛根素對壓力超負(fù)荷大鼠血壓、心率的影響（s，n=8）方法與結(jié)果檢測指標(biāo)心臟質(zhì)量指數(shù)：血壓、心率測定完成后，快速打開胸腔取心臟，去除心房組織，分離左、右心室，用冰冷的生理鹽水漂洗去血，濾紙吸干表面水分，電子天平準(zhǔn)確稱取左心室重量和全心重量。計算左心室重量/體重(LVW /BW )比，全心重量/體重(HW /BW )比，分別記為左心室質(zhì)量指數(shù)(LVW I)，全心質(zhì)量指數(shù)(HW I)。組別劑量LVWI(mg/g)HWI(mg/g)模型組-假手術(shù)組-卡托普利組40mg/kg葛根小劑量組125mg/kg葛根中劑量組250mg/kg葛根大劑量組500mg/kg葛根素對壓力超負(fù)荷大鼠心臟質(zhì)量指數(shù)的影響（s，n=