六味地黄丸中成分在健康大鼠和肾阴虚大鼠体内药动学研究
|
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。
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.
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。
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.
引文
1曼军,六味地黄丸.北京：北京科学技术出版社[M],2004：5
2赵广文.六味地黄丸现代研究的文献分析.现代中西医结合杂志,2006,15(24)：3410-3411
3文红梅,过科家,杏文龙,等.六味地黄方在大鼠及家兔体内的吸收入血成分的研究[J].南京中医药大学学报,2007,23(2)：96-100
4杨胜,张永祥,吕晓东,等.六味地黄汤醇溶部分免疫调节活性成分的药理学导向分离评价[J].中药药理与临床,2000,16(3)：1-3
5艾厚喜,李蕾,许栋明,等.莫诺苷对局灶性脑缺血再灌注大鼠皮层总抗氧化能力影响[J].中国康复理论与实践,2009,15(9)：833-834
6张永梅,梁红,杨玉梅,等。丹皮酚对小鼠免疫功能的影响[J].包头医学院学报,2003,19(4)：261-263
7郭丽丽,周勇,王旭丹,等.山茱萸分离提取物对脾淋巴细胞增殖的影响[J].解放军药学学报,2002,18(4)：357-359
8赵武述,张玉琴,赵世萍,等.山茱萸成分的免疫活性研究[J].中草药,1990.21：17-19
9徐飞,尹蓉莉,赖庆宽,等.不同提取工艺的六味地黄复方提取物对肾阴虚模型小鼠的药理作用比较[J].中国药房,2007,18(6)：418-420
10王喜军,张宁,孙晖,等.六味地黄丸血中移行成分对氢化可的松致大鼠肾虚动物模型的保护作用[J].中国实验方剂学杂志,2008,14,(2)：33-37
11王秀荣,张永红,王秀英.六味地黄汤对糖皮质激素肾阴虚模型免疫功能的影响[J].河北医科大学学报,2005,26(11)：451-454
12夏清山,张旭升.六味地黄丸在化疗中增效减毒作用的临床观察[J].湖北中医杂志,2005,27(11)：14-15
13聂伟,张永祥,茹祥斌,等.六味地黄汤活性部位CA4的抗肿瘤作用研究[J].中草药,1999,30：121-123
14戴冰,肖子曾,刘磊,等.六味地黄丸入血成分莫诺苷对大鼠前脂肪细胞增殖与分化的影响[J].中国临床药理学与治疗学,2007,12(11)：1245-1249
15强世平,赵华.六味地黄汤对老龄小鼠血清SOD和LPO的影响[J].中医药研究,1998,14(6)：42-43
16郭立玮.中药药物动力学方法与应用(M),北京：人民卫生出版社,2002,70-73
17刘荣霞,果德安.液质联用技术(LC／MS)在中药现代研究中的应用[J].世界科学技术-中医药现代化,2005,7(5)：32-40
18曾苏.药物代谢学[M].浙江：浙江大学出版社.2004.
19王广基,刘晓东,柳晓泉.药物代谢动力学[M].北京：化学工业出版社,2005.
20赵春丽,刘建芳,刘会臣.中药药物动力学研究进展[J].解放军药学学报,2003,19,(2)：125-128
21李小娜,王巧,张兰桐,等.山茱萸中马钱苷在大鼠体内的药物动力学研究[J].药物分析杂志,2007,27(1)：4-7
22谢跃生,张振清,阮金秀.小鼠血浆中马钱素的高效液相色谱测定法及药代动力学[J].药学学报,2002,37(7)：548-550
23 HaoWu, Zhenyu Zhu, Guoqing Zhang, et al. Comparative pharmacokinetic study of paeoniflorin after oral administration of pure paeoniflorin, extract of Cortex Moutan and Shuang-Dan prescription to rats[J]. Journal of Ethnopharmacology. 2009,125(3):444-449
24 Chang-hongWang, RuiWang, Xue-mei Cheng, et al. Comparative pharmacokinetic study of paeoniflorin after oral administration of decoction of Radix Paeoniae Rubra and Radix Paeoniae Alba in rats[J].Journal of Ethnopharmacology,2008,117:467-472
25 Xiaona Li, Qiao Wang, Xiaowei Zhang, et al. HPLC study of pharmacokinetics and tissue distribution of morroniside in rats[J]. Journal of Pharmaceutical and Biomedical Analysis,2007,45:349-355
26孙言才,孙国平,沈玉先,等.丹皮酚在小鼠体内的药动学研究[J].中国医院药学杂志,2006,26(5)：543-545
27马丽焱,缪剑华,许旭东,等.丹皮酚在清醒大鼠体内的药动学和绝对生物利用度[J].时珍国医国药,2009,20(2)：413-414
28高会丽,于成谣,李连达.中药复方配伍规律研究[J].中国实验方剂学杂志, 2006,12(9)：60-63
29吴秀华,申屠建中,史美浦.细胞色素P450酶系和药物的不良反应[J].药学专论,2001,10(11)：18-21
30赵子剑,刘敏,张恩户.中药证治药动学研究进展[J].陕西中医学院学报,2005,8(1)：63-64
31封吉化,黄熙,任平.证治药动学研究进展[J].武警医学院学报,2003,13(12)：154-156
32王明艳,赵凤鸣,蔡宝昌.山茱萸及其活性成分的药效研究概述[J].中华中医药学刊,2008,26(7)：1419-1421
33王祝举,唐力英,赫炎.牡丹皮的化学成分和药理作用[J].国外医药·植物药分册,2006,21(4)：155-159
34唐益华,肖凯华.RP-HPLC法测定浓缩六味地黄丸中马钱苷含量[J].中国药事,2007,21(10)：834-835
35曾常青,曾宇,赵越,等.六味地黄汤中4种有效成分的含量测定[J].中国医院药学杂志,2007,27(8)：1034-1036
36王喜军,张宁,孙晖.六味地黄丸指纹图谱的研究[J].中国中药杂志,2004,29(10)：1004-1005
37杨建芳,路福平,高文远,等.山茱萸的化学、药理及开发应用研究进展[J].现代医学生物进展.2006,6(12)：127-131
38李小娜,霍长虹,殷玮,等.山茱萸中马钱苷和莫诺苷对照品的制备[J].中草药,2006,37(8)：1168-1170
39周剑,丁玉峰.大孔吸附树脂分离中草药有效成分的应用[J].中国医院药学杂志,2006,26(1)：69-71
40张蔚君,朱莉,董晓莉,等.大孔吸附树脂在中草药有效成分提取中的应用及其影响因素[J].空军总医院学报,2006,22(2)：96-98
41潘璐琳,欧阳臻,杨雨,等.葛根及复方中葛根素在大鼠体内的药动学研究[J].中国药学杂志,2009,44(9)：703-705
42洪战英,乐健,范国荣,等.延胡索伍用白芷对延胡索乙素药动学的影响[J].中国药学杂志,2009,44(8)：620-623
43杨祖贻,裴瑾,刘荣敏,等.赤芍胡椒复方中胡椒对芍药苷血药浓度的影响.中国药科大学学报,2004,35(6)：558-561
44魏玉辉,秦红岩,段好刚,等.六味地黄丸对大鼠肝微粒体代谢酶P450活性的影响[J].中国医院药学杂志,2008,28(19)：1665-1669
45韩国柱,中草药药代动力学,[M].北京：中国医药科技出版社,1992.。
46王广基,刘晓东,柳晓泉.药物代谢动力学[M].北京：化学工业出版社,2005.
47黄林清,徐传福.萘丁美酮片药物动力学和生物利用度[J].中国医院药学杂志,1992,12(4)：151-154
48阳长明,侯世祥,孙毅毅,等.宝心微丸中肉桂酸大鼠体内药动学研究[J].中草药,2001,32(7)：616-618
49阴健,任天池,曹春林.黄芩甙及其在清开灵注射液中的药代动力学研究[J].中国实验方剂学杂志,1998,4(4)：31-33
50任平,黄熙,张莉,等.麻醉犬心房内注射复方川芎汤后血清中川芎嗪药时曲线双峰与血流动力学效应的关系[J].第四军医大学学报,2000,21(7)：157-160
51付晓伶,方肇勤.阴虚证动物模型的造模方法及评析[J].上海中医药大学学报,2004,18(2)：51-54
52孙敬方.动物实验方法学[M].北京：人民卫生出版社,2002.490
53郭金瑞,严惠芳.近20年临床肾阴虚证客观指标研究概况[J].陕西中医函授,2001,3,36-37
54严惠芳,马居里.对肾阴虚证动物实验研究现状分析与思考[J].陕西中医学院学报,2003,26(6)：65-69
55戴冰,双华,刘磊,等.六味地黄丸对糖皮质激素阴虚大鼠血清中马钱苷浓度的影响[J].湖南中医药大学学报,2007,27(2)：35-37
56高月,吕戟军,刘永学,等.中药复方物质基础的研究[J].中国新药杂志,2000,9(5)：307-308
2赵广文.六味地黄丸现代研究的文献分析.现代中西医结合杂志,2006,15(24)：3410-3411
3文红梅,过科家,杏文龙,等.六味地黄方在大鼠及家兔体内的吸收入血成分的研究[J].南京中医药大学学报,2007,23(2)：96-100
4杨胜,张永祥,吕晓东,等.六味地黄汤醇溶部分免疫调节活性成分的药理学导向分离评价[J].中药药理与临床,2000,16(3)：1-3
5艾厚喜,李蕾,许栋明,等.莫诺苷对局灶性脑缺血再灌注大鼠皮层总抗氧化能力影响[J].中国康复理论与实践,2009,15(9)：833-834
6张永梅,梁红,杨玉梅,等。丹皮酚对小鼠免疫功能的影响[J].包头医学院学报,2003,19(4)：261-263
7郭丽丽,周勇,王旭丹,等.山茱萸分离提取物对脾淋巴细胞增殖的影响[J].解放军药学学报,2002,18(4)：357-359
8赵武述,张玉琴,赵世萍,等.山茱萸成分的免疫活性研究[J].中草药,1990.21：17-19
9徐飞,尹蓉莉,赖庆宽,等.不同提取工艺的六味地黄复方提取物对肾阴虚模型小鼠的药理作用比较[J].中国药房,2007,18(6)：418-420
10王喜军,张宁,孙晖,等.六味地黄丸血中移行成分对氢化可的松致大鼠肾虚动物模型的保护作用[J].中国实验方剂学杂志,2008,14,(2)：33-37
11王秀荣,张永红,王秀英.六味地黄汤对糖皮质激素肾阴虚模型免疫功能的影响[J].河北医科大学学报,2005,26(11)：451-454
12夏清山,张旭升.六味地黄丸在化疗中增效减毒作用的临床观察[J].湖北中医杂志,2005,27(11)：14-15
13聂伟,张永祥,茹祥斌,等.六味地黄汤活性部位CA4的抗肿瘤作用研究[J].中草药,1999,30：121-123
14戴冰,肖子曾,刘磊,等.六味地黄丸入血成分莫诺苷对大鼠前脂肪细胞增殖与分化的影响[J].中国临床药理学与治疗学,2007,12(11)：1245-1249
15强世平,赵华.六味地黄汤对老龄小鼠血清SOD和LPO的影响[J].中医药研究,1998,14(6)：42-43
16郭立玮.中药药物动力学方法与应用(M),北京：人民卫生出版社,2002,70-73
17刘荣霞,果德安.液质联用技术(LC／MS)在中药现代研究中的应用[J].世界科学技术-中医药现代化,2005,7(5)：32-40
18曾苏.药物代谢学[M].浙江：浙江大学出版社.2004.
19王广基,刘晓东,柳晓泉.药物代谢动力学[M].北京：化学工业出版社,2005.
20赵春丽,刘建芳,刘会臣.中药药物动力学研究进展[J].解放军药学学报,2003,19,(2)：125-128
21李小娜,王巧,张兰桐,等.山茱萸中马钱苷在大鼠体内的药物动力学研究[J].药物分析杂志,2007,27(1)：4-7
22谢跃生,张振清,阮金秀.小鼠血浆中马钱素的高效液相色谱测定法及药代动力学[J].药学学报,2002,37(7)：548-550
23 HaoWu, Zhenyu Zhu, Guoqing Zhang, et al. Comparative pharmacokinetic study of paeoniflorin after oral administration of pure paeoniflorin, extract of Cortex Moutan and Shuang-Dan prescription to rats[J]. Journal of Ethnopharmacology. 2009,125(3):444-449
24 Chang-hongWang, RuiWang, Xue-mei Cheng, et al. Comparative pharmacokinetic study of paeoniflorin after oral administration of decoction of Radix Paeoniae Rubra and Radix Paeoniae Alba in rats[J].Journal of Ethnopharmacology,2008,117:467-472
25 Xiaona Li, Qiao Wang, Xiaowei Zhang, et al. HPLC study of pharmacokinetics and tissue distribution of morroniside in rats[J]. Journal of Pharmaceutical and Biomedical Analysis,2007,45:349-355
26孙言才,孙国平,沈玉先,等.丹皮酚在小鼠体内的药动学研究[J].中国医院药学杂志,2006,26(5)：543-545
27马丽焱,缪剑华,许旭东,等.丹皮酚在清醒大鼠体内的药动学和绝对生物利用度[J].时珍国医国药,2009,20(2)：413-414
28高会丽,于成谣,李连达.中药复方配伍规律研究[J].中国实验方剂学杂志, 2006,12(9)：60-63
29吴秀华,申屠建中,史美浦.细胞色素P450酶系和药物的不良反应[J].药学专论,2001,10(11)：18-21
30赵子剑,刘敏,张恩户.中药证治药动学研究进展[J].陕西中医学院学报,2005,8(1)：63-64
31封吉化,黄熙,任平.证治药动学研究进展[J].武警医学院学报,2003,13(12)：154-156
32王明艳,赵凤鸣,蔡宝昌.山茱萸及其活性成分的药效研究概述[J].中华中医药学刊,2008,26(7)：1419-1421
33王祝举,唐力英,赫炎.牡丹皮的化学成分和药理作用[J].国外医药·植物药分册,2006,21(4)：155-159
34唐益华,肖凯华.RP-HPLC法测定浓缩六味地黄丸中马钱苷含量[J].中国药事,2007,21(10)：834-835
35曾常青,曾宇,赵越,等.六味地黄汤中4种有效成分的含量测定[J].中国医院药学杂志,2007,27(8)：1034-1036
36王喜军,张宁,孙晖.六味地黄丸指纹图谱的研究[J].中国中药杂志,2004,29(10)：1004-1005
37杨建芳,路福平,高文远,等.山茱萸的化学、药理及开发应用研究进展[J].现代医学生物进展.2006,6(12)：127-131
38李小娜,霍长虹,殷玮,等.山茱萸中马钱苷和莫诺苷对照品的制备[J].中草药,2006,37(8)：1168-1170
39周剑,丁玉峰.大孔吸附树脂分离中草药有效成分的应用[J].中国医院药学杂志,2006,26(1)：69-71
40张蔚君,朱莉,董晓莉,等.大孔吸附树脂在中草药有效成分提取中的应用及其影响因素[J].空军总医院学报,2006,22(2)：96-98
41潘璐琳,欧阳臻,杨雨,等.葛根及复方中葛根素在大鼠体内的药动学研究[J].中国药学杂志,2009,44(9)：703-705
42洪战英,乐健,范国荣,等.延胡索伍用白芷对延胡索乙素药动学的影响[J].中国药学杂志,2009,44(8)：620-623
43杨祖贻,裴瑾,刘荣敏,等.赤芍胡椒复方中胡椒对芍药苷血药浓度的影响.中国药科大学学报,2004,35(6)：558-561
44魏玉辉,秦红岩,段好刚,等.六味地黄丸对大鼠肝微粒体代谢酶P450活性的影响[J].中国医院药学杂志,2008,28(19)：1665-1669
45韩国柱,中草药药代动力学,[M].北京：中国医药科技出版社,1992.。
46王广基,刘晓东,柳晓泉.药物代谢动力学[M].北京：化学工业出版社,2005.
47黄林清,徐传福.萘丁美酮片药物动力学和生物利用度[J].中国医院药学杂志,1992,12(4)：151-154
48阳长明,侯世祥,孙毅毅,等.宝心微丸中肉桂酸大鼠体内药动学研究[J].中草药,2001,32(7)：616-618
49阴健,任天池,曹春林.黄芩甙及其在清开灵注射液中的药代动力学研究[J].中国实验方剂学杂志,1998,4(4)：31-33
50任平,黄熙,张莉,等.麻醉犬心房内注射复方川芎汤后血清中川芎嗪药时曲线双峰与血流动力学效应的关系[J].第四军医大学学报,2000,21(7)：157-160
51付晓伶,方肇勤.阴虚证动物模型的造模方法及评析[J].上海中医药大学学报,2004,18(2)：51-54
52孙敬方.动物实验方法学[M].北京：人民卫生出版社,2002.490
53郭金瑞,严惠芳.近20年临床肾阴虚证客观指标研究概况[J].陕西中医函授,2001,3,36-37
54严惠芳,马居里.对肾阴虚证动物实验研究现状分析与思考[J].陕西中医学院学报,2003,26(6)：65-69
55戴冰,双华,刘磊,等.六味地黄丸对糖皮质激素阴虚大鼠血清中马钱苷浓度的影响[J].湖南中医药大学学报,2007,27(2)：35-37
56高月,吕戟军,刘永学,等.中药复方物质基础的研究[J].中国新药杂志,2000,9(5)：307-308
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |