Studies On The Pharmacokinetic Of Constituents In Liuwei Dihuang Pills On Health And Kindey-Yin Deficiency Rats

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.