Studies On The Quality Control Methods And Pharmacokinetics Of Relative Active Ingredients Of Dioscorea Nipponica Makino

Dioscorea nipponica Makino is one of the commonly used traditional Chinese medicines (TCM). To investigate the optimal extraction solvent, extracts of Dioscorea nipponica Makino were prepared by different methods. The effects of different extracts on the coagulate time, the total cholesterol level, the triglyceride level, the HDL-C and LDL-C level, and the SOD and LPO level of hyperlipemia rats were studied as the evaluation standard. As a result, 65% alcohol was selected as the optimal extraction solvent. To select the active fraction, the various polarity fractions from 65% alcohol extract were investigated in pharmacological experiments. Finally, the chloroform fraction and the n-butanol fraction were found to be significantly active by the initial pharmacodynamic experiments.Six active constituents of Dioscorea nipponica Makino were isolated and purified by various chromatographic techniques and identified with spectroscopy data and physical-chemical properties. They were diosgenin,β-sitosterol, dioscin, benzoic acid, protodioscin, and trillin. After pharmacodynamic studies, the four steroid compounds possessed the activities on reducing blood fat, anti-coagulate, and anti-free radical, among them the effects of dioscin and prodioscin were more significant.In this study, a RP-HPLC method for the determination of protodioscin in Dioscorea was established for the first time. The separation was carried out on an Xterra^TM C₁₈ column (250 mm×4.6 mm, 5μm) with the mobile phase consisting of acetonitrile-water (27:73, v/v). The calibration curve was linear over the range of 0.02～0.6 mg·mL^-1 (r= 0.9999, n = 6). The average recovery was 98.0% (RSD = 1.5%, n = 9). Besides, a RP-HPLC method was established for the determination of dioscin in Dioscorea. The linear range of dioscin was 0.1～0.8 mg·mL^-1 (r = 0.9999). The average recovery was 97.9% (RSD = 2.0%). The contents of protodioscin and dioscin in five plants from different origins of Dioscorea, which were recorded in ChP2005, were determined respectively in the same manner as the former chromatography conditions. It was concluded that the two methods were simple, accurate and specific, which provided a basis for the quality evaluation of Dioscorea nipponica Makino and Dioscorea.A LC-ESI-MS-MS method was first established to determine the protodioscin in rat plasma. The trillin was used as internal standard. Protodioscin were determined after acetonitrile-mediated plasma protein precipitation. Analytes were detected in multiple reaction monitoring mode (MRM) with m/z 1032.6 (precursor ion) to m/z 869.7 (production) for protodioscin; m/z 577.1 (precursor ion) to m/z 253.1 (product ion) for trillin (internal standard). The assay was shown to be linear over the range of 20～125000 ng·mL^-1 (r = 0.9970) with a lower limit of quantification of 20 ng·mL^-1. The method was shown to be reproducible and reliable with intra-day precision below 7.8%, inter-day precision below 6.8%, accuracy within±1.8%, and mean extraction recovery was 83.5% (RSD = 5.9%). After intravenous administrations (0.5 mg·kg^-1, 1.0 mg·kg^-1 and 3.0 mg·kg^-1) for each rat, the plasma concentration-time curve of protodioscin was plotted and the pharmacokinetic parameters were calculated, they were: t_1/2, 78 min, 58 min, and 27 min; C_max, 70μg·mL^-1, 116μg·mL^-1, and 378μg·mL^-1; k_e, 0.0089 min^-1, 0.012 min^-1, and 0.029 min^-1; V_d, 53.3 mL·kg^-1, 45.9 mL·kg^-1, and 9.32 mL·kg^-1; Cl, 0.796 mL·min^-1·kg^-1, 0.444 mL·min^-1·kg^-1, and 0.263 mL·min^-1·kg^-1; AUC_0-t, 732μg·min·mL^-1,1406μg·min·mL^-1, and 4196μg·min·mL^-1; AUC_0-∞, 785μg·min·mL^-1, 1673μg·min·mL^-1, and 4406μg·min·mL^-1。It was shown that AUC_0-t increased while dose increased and the Cl and V_d decreased while the dose increased, it could be the character of dose-dependability.A LC-ESI-MS-MS method was first developed for the determination of trillin in plasma and applied to its pharmacokinetic study in mice after intravenous administration. The ginsenoside Rh2 was used as internal standard. The methanol was used as extraction solvent and plasma protein precipitation. Analytes were detected in positive multiple reaction monitoring mode (MRM) with m/z 577.6 (precursor ion) to m/z 253.6 (product ion) for trillin; m/z 621.6 (precursor ion) to m/z 603.7 (product ion) for ginsenoside Rh2 (internal standard). The assay was shown to be linear over the range of 2.0～200μg·mL^-1 (r = 0.9964) with a lower limit of quantification 2.0μg·mL^-1. It has been shown to be reproducible and reliable with intra-day precision blow 3.3%, inter-day precision blow 5.1%, accuracy within±3.0%, and mean extraction recovery was 83.9% (RSD = 3.9%). After intravenous administration, the mean concentration-time profile reached the peak concentration (C_max= 130.1μg·mL^-1) and at first the clearance of trillin was fast and then was slower (t_1/2 = 5.56 h).The LC-ESI-MS-MS assay was first developed for the determination of trillin in mouse tissues. The ginsenoside Rh2 was used as the internal standard (IS). The assay showed the good linearity in tissues: heart: 1,0～100μg·mL^-1 (r = 0.9962), liver: 2.0～400μg·mL^-1(r = 0.9964), spleen: 1.0～100μg·mL^-1(r = 0.9985), lung: 1.0～300μg·mL^-1 (r = 0.9919), kidney: 1.0～100μg·mL^-1(r = 0.9976), and brain: 1.0～100μg·mL^-1 (r = 0.9948), the lower limits of quantification were 1.0μg·mL^-1, 2.0μg·mL^-1, 1.0μg·mL^-1, 1.0μg·mL^-1, 1.0μg·mL^-1, and 1.0μg·mL^-1, respectively. It had been shown to be reproducible and reliable with intra-day precision below 4.8%, inter-day precision below 7.9%, and accuracy within±11%. The mean recoveries were 94.2% (RSD = 2.4%), 72.0% (RSD = 4.0%), 77.7% (RSD = 3.5%), 85.7% (RSD = 2.8%), and 74.1% (RSD = 3.9%). After intravenous administrations, the concentrations in different tissue increased immediately. It suggested that the distribution of trillin was wide and rapid. AUC_0-t of trillin was in a descending order of lung, spleen, kidney, heart, liver and brain. The content in lung was more than in others. Trillin wasn't detected in brain and it was indicated that trillin couldn't pass through the blood-brain barrier easily to act on the central nervous system.Under the direction of theory and clinical practice of TCM, the primary approach on material foundation of medicinal effectiveness of Dioscorea nipponica Makino was put in practice and the quality control indices of Dioscorea nipponica Makino were selected by combining science of Chinese material medica, analytical chemistry, pharmacology, and computer technique. Meanwhile, the extraction processes and quality control methods for Dioscorea were optimized and developed. Pharmacokinetic studies of protodioscin and trillin were carried out, and also the tissue distribution of trillin was studied after intravenous administration, respectively. It took a limited view of the dynamic process in-vitro of active components in Dioscorea nipponica Makino and provided the basis for the approach of mechanism of new drug action, further it provided an exploration for the modernization of TCM.