【作者】 李娴；

【导师】 郭瑞臣；

【作者基本信息】 山东大学 ， 药理学， 2010， 硕士

【摘要】 目的：1.测定六味地黄丸中丹皮酚、马钱苷、莫诺苷和芍药苷含量。2.提取和分离山茱萸中马钱苷和莫诺苷。3.建立灵敏度高、专属性强、高效稳定的HPLC-MS法,测定大鼠血浆中马钱苷、莫诺苷和芍药苷的浓度。4.进行健康大鼠单次灌胃莫诺苷粗品、马钱苷和莫诺苷混合物、六味地黄丸后马钱苷、莫诺苷、芍药苷的药代动力学研究,比较马钱苷和莫诺苷在不同组药代动力学差异。5.进行肾阴虚模型大鼠单次和多次灌胃六味地黄丸药动学和药效学研究。方法：1.采用HPLC-DAD法测定六味地黄丸中丹皮酚、马钱苷、莫诺苷和芍药苷含量。六味地黄丸经处理后,以乙腈(A)-0.15%磷酸(B)梯度洗脱,采用Agilent ZORBAX C18色谱柱(4.6×250mm,5μm)分离,流速1mL·min-1,DAD检测,波长238nm,柱温30℃；进样量10gL。2.称取山茱萸原药材400g,经浸泡、煎煮、滤过、浓缩,得山茱萸提取液约100mL；提取液结合AB-8大孔树脂柱富集苷类、硅胶柱层析分离纯化,采用HPLC-DAD法检测分离产物。3.血浆样品经乙酸乙酯提取、吹干、复溶后,以甲醇-0.02%甲酸(28：72,V／V)为流动相,采用Wondasil C18(150 mm×4.6 mm,5μm)色谱柱分离,流速0.7mL·min-1；柱温30℃；进样量10μL；ESI离子源,负离子模式,雾化压力50 psi,干燥气(N2)流速9 L·min-1,干燥气温度为350℃,毛细管电压4000 V,单离子反应监测(SIM)方式,监测离子m／z 435.2(马钱苷)、m／z451.2(莫诺苷)、m／z525.2(芍药苷)和m／z327.1(内标),马钱苷、莫诺苷和内标碎片电压均为110V,芍药苷碎片电压为100V。4.18只健康雄性Wister大鼠,随机分为3组,即莫诺苷组、马钱苷和莫诺苷混合组、六味地黄丸组,分别灌胃给予莫诺苷粗品(20mg·kg-1,以莫诺苷计)、马钱苷和莫诺苷混合物(15 mg·kg-1和20mg·kg-1)、10 g·kg-1六昧地黄丸,留取血浆样本,采用HPLC-MS法检测马钱苷、莫诺苷和芍药苷的药物浓度。采用DAS2.0实用药代动力学程序非房室模型计算药代动力学参数,评价不同形式给药后马钱苷和莫诺苷健康大鼠药代动力学差异。5.12只健康雄性Wister大鼠随机分为两组,皮下注射氢化可的松注射液造模后,分别单次和多次灌胃给予六味地黄丸混悬液,留取血浆和血清样本,采用HPLC-MS法检测血浆马钱苷和莫诺苷药物浓度,采用全自动酶标仪测定血清中SOD和AKP值。采用DAS2.0实用药代动力学程序非房室模型计算药代动力学参数,分析药动学特征,并与药效学指标相结合分析。结果：1.HPLC-DAD法测定六味地黄丸中丹皮酚、马钱苷、莫诺苷和芍药苷的含量,丹皮酚、马钱苷、莫诺苷和芍药苷分别在0.5～20、0.25-10、0.5-20和O.15-6μg·mL-1范围内线性关系良好,且同一厂家不同批次六味地黄丸四种成分含量相对稳定。2.山茱萸经提取和分离后,通过HPLC-DAD测定,得浓度为71.7 mg·mL-1的莫诺苷粗品6.0mL,及马钱苷和莫诺苷(马钱苷72.5mg·mL-1,莫诺苷96.5 mg·mL-1)混合物7.0mL。3.HPLC-MS法测定大鼠血浆中马钱苷、莫诺苷和芍药苷的浓度,马钱苷、莫诺苷和芍药苷5-1000ng·mL-1浓度范围内线性关系良好,回归方程分别为Y=0.0171X+0.0026,(r=0.99573),Y=0.0072X+0.0044, (r=0.99751), Y=0.0095X-0.0122,(r=0.99334).最低定量限均为5ng·mL-1,绝对回收率分别大于77%、69%和78%,相对回收率范围为85%-120%,批内变异均小于10%,批间变异均小于15%,马钱苷、莫诺苷和芍药苷在血浆中-20℃冷冻24小时和7天及反复冻融条件下稳定。4.马钱苷和莫诺苷在莫诺苷组、马钱苷和莫诺苷组药时曲线均呈单峰吸收；马钱苷和莫诺苷在六味地黄丸组,药时曲线呈双峰现象；在六味地黄丸大鼠血浆中未能检测到所有时间点的芍药苷浓度,因此未能进行药代动力学分析。用非房室模型计算药代动力学参数,莫诺苷在莫诺苷组、马钱苷和莫诺苷组、六味地黄丸组中主要药代动力学参数分别为MRT0-t(2.392±0.734)h、(2.768±0.819)h和(7.813±1.556)h,t1/2(1.485±0.624)h. (1.932±0.848)h和((7.71±5.756)h,Tmax(1.417±0.904)h.(1.375±0.44)h和(2.917±1.828)h,Cmax(423.12±52.641)ng·mL-1、(520.622±72.215)ng·mL-1和(336.162±69.765)ng·mL-1, AUC0-t(1267.791±326.319)ng·mL-1·h. (1610.961±550.065)ng·mL-1·h和(2622.371±864.174)ng·mL-1·h,AUC0～∞(1284.826±328.657) ng·mL-1·h.(1670.588±620.716)ng·mL-1·h和(46574.198±45140.607)ng·mL-1·h.马钱苷在马钱苷和莫诺苷组、六味地黄丸组中主要药代动力学参数分别为MRT0-t(2.599±1.037)h和(6.042±0.644)h,MRT0～∞(2.699±1.123)h和(7.033±1.03)h,t1／2(1.586±1.18)h和(4.398±1.6336)h,Tmax(1.458±0.401)h和(2.083±1.021)h,Cmax(658.597±85.48)ng·mL-1和(369.314±53.452)ng·mL-1,AUCo-24(1825.4±635.706)ng·mL-1·h和(2275.174±553.825)ng·mL-1·h,AUC0～∞(1832.324±643.67)ng·mL-1·h和(2343.478±601.313)ng·mL-1·h。对各组莫诺苷药代动力学参数进行统计学分析,除Tmax外,其他药代动力学参数六味地黄丸组与马钱苷和莫诺苷混合组和莫诺苷组均有统计学差异,但马钱苷和莫诺苷混合组与莫诺苷组药代动力参数均无统计学差异。马钱苷和莫诺苷混合组、六味地黄丸组中莫诺苷药代动力学参数MRT0-24、MRT～∞、t1／2、Tmax均比莫诺苷组滞后,AUC0-24、AUCo-明显增加。六味地黄丸组与马钱苷和莫诺苷组比较,马钱苷的药代动力学参数MRT0-24和MRT0～∞有统计学差异,其他参数虽然有增高趋势,但无统计学差异。本结果从药动学角度说明六味地黄丸组方配伍影响马钱苷和莫诺苷的吸收,进一步验证六味地黄丸组方配伍的合理性,为深入研究组方原理打下基础。5.莫诺苷在肾阴虚模型大鼠体内单次和多次给药后主要药动学参数分别为MRT0-24(8.337±0.848)h和(7.592±1.105)h,MRT0～∞(13.149±3.947)和(9.875±3.291)h,t1／2(8.728±2.282)h和(6.078±2.293)h,Tmax(2.167±0.408)h和(3.333±0.516)h,Cmax(287.013±58.389)ng·mL-1和(240.275±34.891)ng·mL-1,AUC0-24(2852.796±430.373)ng·mL-1·h和(2225.752±535.232)ng·mL-1·h,AUC0～∞(3311.084±349.092)ng·mL-1·h和(2463.095±694.01)ng·mL-1·h。马钱苷在肾阴虚模型大鼠体内单次和多次给药后主要药动学参数分别为MRT0-24(6.471±0.957)h和(7.246±1.732)h,MRT0～∞(8.257±2.852)h和(11.048±7.256)h,t1／2(4.852±1.772)h和(5.676±5.84)h,Tmax(2.5±2.145)h和(3.5±0.548)h,Cmax(287.607±24.486)ng·mL-1和(262.314±24.972)ng·mL-1, AUC0-24 (1956.656±130.427)ng·mL-1·h和(1638.489±196.911)ng·mL-1·h,AUC0～∞(2027.44±173.357) ng·mL-1·h和(2070.227±447.341)ng·mL-1·h.马钱苷和莫诺苷在大鼠体内单次和多次给药后药动学参数经统计学分析,均无统计学差异。可能原因一方面说明在体内无蓄积,另一方面给药间隔相对其半衰期较长有关。SOD和AKP测定结果表明六味地黄丸可升高SOD值、降低AKP值。多次给药比单次给药后血清SOD值大,但无统计学差异；多次给药比单次给药后血清AKP值小,具有统计学差异。结论：1.同一厂家六味地黄丸中四种成分含量相对稳定。2.从山茱萸中提取和分离莫诺苷粗品与马钱苷和莫诺苷混合物可满足药代动力学实验的要求。3.六味地黄丸中其他配伍药材促进马钱苷和莫诺苷在大鼠体内的吸收。健康大鼠单次灌胃后,莫诺苷药代动力学参数除Tmax外,其他参数六味地黄丸组与马钱苷和莫诺苷混合组和莫诺苷组均有统计学差异,AUC增加,t1/2延长。与混合组相比,六味地黄丸组马钱苷的药代动力学参数MRT0-24和MRT0～∞延长,有统计学差异。4.肾阴虚大鼠单次和多次灌胃六味地黄丸后,马钱苷和莫诺苷药动学参数均无统计学差异；血清AKP值有统计学差异。更多还原

【Abstract】 Objective:1. To establish an HPLC method for the simultaneous determination of paeonol, loganin, morroniside and paeoniflorin in Liuwei Dihuang pills.2. To isolate and purify loganin and morroniside from the extract of Fructus Corni.3. To establish sensitive and selective HPLC-MS methods for the determination loganin, morroniside and paeoniflorin in rats plasma.4.To study the pharmacokinetics of loganin, morroniside and paeoniflorin after administration of Liuwei Dihuang pills, mixture containing loganin and morroniside, morroniside, and compare pharmacokinetic parameters of loganin and morroniside.5. To study the pharmacokinetics and pharmacodynamics of loganin and morroniside after single and multi-doses Liuwei Dihuang pills on kidney-yin deficiency rats.Methods:1. An HPLC method was established for the simultaneous determination of paeonol, loganin, morroniside and paeoniflorin in Liuwei Dihuang pills. A C18 column was used with the mobile phase of acetonitrile-0.1% phosphoric acid for gradient elution at a flow rate of 1 mL·min-1 and at the detection wavelength of 238 nm. The temperature of column was 30℃. 10μL was injected to analyze.2. 100mL extract of Fructus Corni was isolated and purified by macroporous resin and silica gel column, and the contents of loganin and morroniside identified by HPLC-DAD.3. The plasma samples were extracted by ethyl acetate and separated on a Wondasil C18 column (150mm×4.6mm,5μm), eluted with the mobile phase of methanol -0.02% formic acid (28:72, V/V) at a flow rate of 0.7 mL·min-1. The temperature of column was 30℃. 10μL was injected to analyze. Mass spectrometer was operated using electronic spray ionization (ESI) with negative ionization mode at following parameters:nebulizer gas of 50 psi, spray gas of 9 L·min-1. The turbo ion spray source temperature was set at 350℃, and the capillary voltage was 4000V. The determination was performed by single ion monitoring (SIM) and ion mass spectrum (m/z) of 435.2,451.2,525.2 and 327.1 for loganin, morroniside, paeoniflorin and internal standard (I.S.), respectively. The fragmentor of paeoniflorin was 100V and the others were all 110V.4.18 Wistar rats were divided into three groups randomly and received an intragastric administration of 20 mg·kg-1 morroniside, loganin and morroniside (15 mg·kg-1 and 20 mg·kg-1),10 g·kg-1 Liuwei Dihuang pills. The plasma samples were collected as scheduled and analyzed by HPLC-MS. The pharmacokinetic parameters of loganin, morroniside and paeoniflorin were calculated by non-compartment model.5.12 Wistar rats were divided into two groups randomly and subcutaneously injected with hydrocortisone inducing kidney-yin deficiency, received an intragastric administration of a single dose and multiple doses Liuwei Dihuang pills 10 g·kg-1. The plasma and serum samples were collected as scheduled and analyzed by HPLC-MS and Bio-Tek. The pharmacokinetic parameters of loganin and morroniside were calculated by non-compartment model, and evaluated the dependablity of pharmacokinetic parameters and pharmacodynamics (SOD and AKP).Result:1.The linear range of the calibration curve for determination of paeonol, loganin, morroniside and paeoniflorin in Liuwei Dihuang pills by HPLC-DAD method was 0.5～20,0.25～10,0.5～20 and 0.15～6μg·mL-1, and the four constituents in Liuwei Dihuang pills were stable.2. The concentration of morroniside was 71.7 mg-mL-1, and the mixture of loganin and morroniside were 72.5mg-mL-1 and 96.5 mg-mL-1, respectively.3. The linear range of the calibration curve for determination of loganin, morroniside and paeoniflorin in plasma by HPLC-MS method were 0.5～1000 ng-mL-1, and the regression equations were Y=0.0171X+0.0026 (r=0.99573), Y=0.0072X+0.0044 (r=0.99751), Y=0.0095X-0.0122 (r=0.99334), respectively. The low limit of quantitation (LLOQ) were all 5 ng-mL-1. The absolute recoveries were more than 77%,69% and 78%, respectively. The relative recoveries were between 85% and 120%. Intra-day RSD and inter-day RSD were less than 10% and 15%, respectively. Loganin, morroniside and paeoniflorin in plasma were stable when frozen at-20℃for 24 hours and seven days and were also stable after two freeze-thawing cycles.4. Loganin and morroniside were two peaks in the concentration time curves in Liuwei Dihuang pills group and one peak in morroniside and mixture containing loganin and morroniside group. The plasma concentration of paeoniflorin was not detected in all scheduled time. Pharmacokinetic parameters were calculated by non-compartment model. The main parameters of morroniside in morroniside, mixture containing loganin and morroniside, Liuwei Dihuang pills group were shown as follows: MRT0-t(2.392±0.734)h, (2.768±0.819)h and (7.813±1.556)h, t1/2 (1.485±0.624) h, (1.932±0.848)h and (7.71±5.756)h, Tmax(1.417±0.904)h, (1.375±0.44)h and (2.917±1.828)h, Cmax (423.12±52.641)ng·mL-1, (520.622±72.215)ng·mL-1 and (336.162±69.765)ng·mL-1, AUC0-t(1267.791±326.319)ng·mL-1·h, (1610.961±550.065) ng·mL-1·h and (2622.371±864.174)ng·mL-1·h, AUC0-∞(1284.826±328.657)ng·mL-1·h, (1670.588±620.716)ng·mL-1·h and (46574.198±45140.607)ng·mL-1·h. The main parameters of loganin in mixture containing loganin and morroniside, Liuwei Dihuang pills group were shown as follows:MRT0-24(2.599±1.037)h and (6.042±0.644)h, MRT0-∞(2.699±1.123)h and (7.033±1.03)h, t1/2 (1.586±1.18) h and (4.398±1.6336)h, Tmax(1.458±0.401)h and (2.083±1.021P)h, Cmax(658.597±85.48)ng·mL-1 and (369.314±53.452)ng·mL-1, AUC0-24(1825.4±635.706)ng·mL-1·h and (2275.174±553.825)ng·mL-1·h, AUC0-∞(1832.324±643.67)ng·mL-1·h and (2343±601.313)ng·mL-1·h. The pharmacokinetic parameters of morroniside in Liuwei Dihuang pills group were statistically significant differences in parameters including the MRT, Cmax, t1/2, AUC compared with morroniside and mixture containing loganin and morroniside group. But the pharmacokinetic parameters of morroniside were no statistically significant differences in parameters between morroniside and mixture containing loganin and morroniside group. In particularly, on the rats administrated Liuwei Dihuang pills, the Tmax of morroniside was delayed, MRT and t1/2 were long, the AUC was increased compared with the rats administrated morroniside and mixture containing loganin and morroniside. The pharmacokinetic parameters of loganin were statistically significant differences in parameters including the MRT0-24 and MRT0-∞ in the two groups. The results indicated that the other herbs in Liuwei Dihuang pills improved the absorption of morroniside significantly. The influence of the drug-drug interaction on the pharmacokinetics of loganin and morroniside is an important topic for further studies.5. The main parameters of morroniside after administration of a single dose and multiple doses Liuwei Dihuang pills on kidney-yin deficiency rats were shown as follows:MRT0-24(8.337±0.848)h and (7.592±1.105) h, MRT0～∞(13.149±3.947) and (9.875±3.291) h, t1/2 (8.728±2.282) h and (6.078±2.293)h, Tmax(2.167±0.408)h and(3.333±0.516)h, Cmax(287.013±58.389)ng·mL-1 and (240.275·34.891)ng·mL-1, AUC0-24 (2852.796±430.373)ng·mL-1·h and (2225.752±535.232) ng·mL-1·h, AUC0～∞(3311.084±349.092) ng·mL-1·h and (2463.095±694.01) ng·mL-1·h. The main parameters of loganin after administration of a single dose and multiple dose Liuwei Dihuang pills on kidney-yin deficiency rats were shown as follows:MRT0-24(6.471±0.957)h and (7.246±1.732)h, MRT0-∞(8.257±2.852)h and (11.048±7.256)h, t1/2 (4.852±1.772) h and (5.676±5.84)h, Tmax(2.5±2.145)h and (3.5±0.548)h, Cmax (287.607±24.486)ng·mL-1 and (262.314±24.972)ng·mL-1, AUC0.24 (1956.656±130.427)ng·mL-1·h and (1638.489±196.911)ng·mL-1·h, AUC0-∞(2027.44±173.357) ng·mL-1·h and (2070.227±447.341)ng·mL-1·h. No significant differences were observed in the pharmacokinetic parameters after a single dose of Liuwei Dihuang pills compared with multiple doses, which were possibly related that the drug did not accumulate after multiple doses; the other reason may be the time of interval doses was longer compared with t1/2. Compared the pharmacodynamic parameters of the two groups, AKP has statistically significant difference in two groups, while SOD no significant difference.Conclusion:1.The content uniformity of four constituents in Liuwei Dihuang pills were excellent.2. The contents of loganin and morroniside extracted from Fructus Corni could be used for the pharmacokinetic test on rats.3. The other herbs in Liuwei Dihuang pills improved the absorption of loganin and morroniside. It may be due to the influence of the drug-drug interaction. The pharmacokinetic parameters including the t1/2, MRT, Cmax, AUC of morroniside in Liuwei Dihuang pills group were statistically significant different compared with morroniside and mixture containing loganin and morroniside groups. The pharmacokinetic parameters MRT0-24 and MRT0～∞of loganin were prolonged from 2.599±1.037h、2.699±1.123h to 6.042±0.644h,7.033±1.03h, which had statistical significant differences.4. No significant differences were observed in the pharmacokinetic parameters between a single dose and multi-doses of Liuwei Dihuang pills. The pharmacodynamic parameter of AKP had statistically significant differences in two groups.更多还原