| Tang-Min-Ling-Wan (TMLW), consisted of Rhizoma Coptidis, Radix et Rhizoma Rhei, Radix Scutellariae, Radix Paeoniae Alba, Fructus Aurantii Immaturus, Radix Bupleuri, Rhizoma Pinelliae, Fructus Crataegi, Fructus Mume, Radix Trichosanthis, is one of Traditional Chinese Medicine formula for the treatment of type 2 diabetes mellitus and diabetic complications. In this paper, preparation procedure, quality control method and pharmacokinetics on a few active constitutes of TMLW were studied.The preparation procedure of TMLW was established using different kinds of methods and techniques. The optimal extraction process of 8 kinds of material including Paeonia lactiflora, et al was studied by Doehlert design with total amount of multicomponents from TMLW as indices. Three factors selected in water extraction were duration of extraction, times of extraction and the solvent consumption. Data was dealed with statistical software, such as SPSS 13.0, SigmaPlot 10.0, LINGO 9.0, et al. Concentration of alcohol and volume of extracting solution were tested to optimize the refining process. The optimal extraction process of Rhizoma Coptidis and Radix Scutellariae was studied by orthogonal design with total amount of alkaloids and flavones as indices.The HPLC-DAD-MS/MS method was developed for the qualitative characterization of the mafor constituents in TMLW. Total of 54 peaks in HPLC-DAD and HPLC-MS (TIC) including monoterpene glucosides, flavones, alkaloids, saikosaponins, anthraquinones, et al were unambiguously or tentatively identified by comparing their retention times, UV spectra and MS data with authentic compounds and those reported by literature. Furthermore, the fragmentation pathway of these compounds were investigated and summarized.HPLC quantitative analysis methods were established for assessing the quality of TMLW. Under the gradient elution mode, an HPLC-UV method was developed for simultaneous determination of 4 alkaloids and 10 flavones:naringin, hesperidin, baicalin, neohesperidin, wogonoside, coptisine, jatrorrhizine, palmatine, berberine, naringenin, baicalein, wogonin, chrysin and oroxylin A. Under the gradient elution mode, the simultaneous quantification of cinnamic acid, naringenin, hesperetin, aloeemodin, SSb1, emodin, chrysophanol and physcion was carried out by using HPLC with UV wavelength switch. Also under the gradient elution mode, a UV wavelength switch-HPLC method was developed for the simultaneous quantification of gallic acid, protocatechin, albiflorin, paeoniflorin and benzoic acid. The method were rapid, accurate and could be readily utilized as quality control methods of TMLW.A simple and sensitive method for simultaneous determination of naringin, hesperidin, neohesperidin, baicalin and wogonoside in rat plasma by using HPLC-MS/MS was developed and validated. Liquritin was selected as an IS. Plasma samples were extracted by SPE. Detection was performed by MRM mode using ESI in negative mode for naringin, hesperidin, neohesperidin and IS, and in positive mode for baicalin and wogonoside. The calibration curves were linear over a range of 2.580～516.0 ng·mL-1 for naringin, 0.5250～105.0 ng·mL-1 for hesperidin,5.040～1008 ng·mL-1 for neohesperidin, 68.10～1.362×104 ng·mL-1 for baicalin, and 4.420～844 ng·mL-1 for wogonoside. The LLOQ was 0.5160 ng·mL-1,0.5250 ng·mL-1,0.5040 ng·mL-1,0.3400 ng·mL-1 and 0.3680 ng·mL-1, respectively. The intra-and inter-day precision (RSD) were less 11%, and the accuracy (RE) ranged from-5.1% to 6.7%. The recoveries of the analytes and IS were above 77%. The corresponding pharmacokinetic parameters were obtained. Cmax, T1/2 and AUC0-∞for naringin were 281.1 ng·mL-1,8.2 h and 517.6 ng·h·mL-1, respectively; for hesperidin were 37.24 ng·mL-1,6.7 h and 91.57 ng·h·mL-1; for neohesperidin were 440.4 ng·mL-1,8.7 h and 868.4 ng·h·mL-1; for baicalin were 7138 ng·mL-1,4.9 h and 2.929×104 ng·h·mL-1; for wogonoside were 530.7 ng·mL-1,5.9 h and 2163 ng·h·mL-1.A rapid analytical method for determining 4 alkaloids and 5 flavones in rat plasma by HPLC-MS/MS was established. Daidzein and tetrahydropalmatine were selected as internal standards. Plasma samples were extracted with acetone, and detccted by MRM using ESI in positive mode. The calibration curves were linear over a range of 10.14～5070 ng·mL-1 for wogonin,0.2380～119.0 ng·mL-1 for chrysin,1.014～507.0 ng·mL-1 for oroxylin A, 1.006～503.0 ng·mL-1 for naringenin,0.998～499.0 ng·mL-1 for hesperetin,1.010～505.0 ng·mL-1 for berberine,9.96×10-2～49.80 ng·mL-1 for coptisine,5.010×10-2～25.05 ng·mL-1 for jatrorrhizine, and 8.89×10-2～44.46 ng·mL-1 for palmatine. The LLOQ was 10.14 ng·mL-1,0.2380 ng·mL-1,1.014 ng·mL-1,1.006 ng·mL-1,0.998 ng·mL-1,1.010 ng·mL-1, 9.96×10-2 ng·mL-1,5.010×10-2 ng·mL-1 and 8.89×10-2 ng·mL-1, respectively. The intra- and inter-day precision (RSD) were less 15%, and the accuracy (RE) ranged from-7.5% to 4.5%. The recoveries of the analytes and IS were above 54%. Three or four peaks were observed in both individual and mean plasma-concentration curves of alkaloids. The corresponding pharmacokinetic parameters were obtained. Cmax, T1/2 and AUC0-∞for berberine were 156.2 ng-mL-1,7.3 h and 473.4 ng-h-mL-1, respectively; for coptisine were 16.94 ng·mL-1,6.5 h and 49.29 ng·h·mL-1; for jatrorrhizine were 7.884 ng·mL-1,7.2 h and 33.10 ng·h·mL-1; for palmatine were 16.52 ng-mL"1,6.8 h and 55.69 ng·h·mL-1. Wogonin, chrysin and oroxylin A absorped rapidly, and another small peak could be seen at 8 h to 12 h in the plasma concentration-time profile. The corresponding pharmacokinetic parameters were as followes:Cmax, T1/2 and AUC0-∞for wogonin were 1378 ng-mL"1,6.5 h and 4520 ng·h·mL-1, respectively; for chrysin were 19.47 ng·mL-1,6.6 h and 107.5 ng·h·mL-1; for oroxylin A were 155.9 ng·mL-1,7.6 h and 566.3 ng·h·mL-1. Flavanones aglycone of naringenin and hesperetin showed a much longer Tmax than other compounds. Cmax, T1/2 and AUC0-∞for naringenin were 133.8 ng·mL-1,4.1 h and 1007 ng·h·mL-1, respectively; for hesperetin were 190.3 ng·mL-1,3.4 h and 1507 ng·h·mL-1.A specific HPLC-MS/MS method was developed and validated for simultaneous determination of emodin, aloe-emodin, chrysophanol and physcion in rat plasma using 1,8-dihydroxyanthraquinone as IS. Plasma samples were extracted with ethyl ether, and detected by MRM using ESI in negative mode. The calibration curves were linear over a range of 1.925～385.0 ng·mL-1 for emodin,1.520～304.0 ng·mL-1 for aloe-emodin, 1.325～265.0 ng·mL-1 for chrysophanol, and 1.525～305.0 ng·mL-1 for physcion. The LLOQ was 1.925 ng·mL-1,1.520 ng·mL-1,1.325 ng·mL-1 and 1.525 ng·mL-1, respectively. The intra-and inter-day precision (RSD) were less 12%, and the accuracy (RE) ranged from-6.6% to 4.6%. The recoveries of the analytes and IS were above 52%. All the four analytes were slowly absorped rapidly and eliminated from plasma. The corresponding pharmacokinetic parameters were as followes:Cmax, T1/2 and AUC0-∞for emodin were 20.67 ng·mL-1,20.4 h and 193.6 ng·h·mL-1, respectively; for aloe-emodin were 29.05 ng·mL-1,22.4 h and 211.7 ng·h·mL-1; for chrysophanol were 82.63 ng·mL-1,24.2 h and 599.6 ng·h·mL-1; for physcion were 15.48 ng·mL-1,27.8 h and 183.3 ng·h·mL-1.An HPLC-MS/MS method was developed and validated for simultaneous determination of albiflorin and paeoniflorin in rat plasma using geniposide as an internal standard (IS). Plasma samples were extracted by solid-phase extraction (SPE). Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in positive ion mode. The calibration curves were linear over a range of 1.000～1000 ng·mL-1 for albiflorin and 2.000～2000 ng·mL-1 for paeoniflorin. The lower limit of quantification (LLOQ) for albiflorin and paeoniflorin was 1.000 ng·mL-1 and 2.000 ng·mL-1, respectively. The intra-and inter-day precision (RSD) were less 11% for both albiflorin and paeoniflorin, and the accuracy (RE) ranged from-4.9% to 2.4%. The recoveries of the analytes and IS were above 80%. After oral administration of TMLW, the mean plasma concentration-time curves were plotted and the pharmacokinetic parameters were calculated. The pharmacokinetic parameters were as followes:Cmax, T1/2 and AUC0-∞for albiflorin were 1117 ng·mL-1,2.6 h and 874.8 ng·h·mL-1, respectively; for paeoniflorin were 1591 ng·mL-1,2.2 h and 1467 ng·h·mL-1. These two analytes exhibited consistent tendencies in plasma concentration-time profiles and similar Tmax and T1/2 values. That these two compounds had the similar pharmacokinetic behavior could be tentatively explained based on their similar structures. |
PDF Full Text Request