| Chinese herb preparations, the organic combinations of various crude drugs under the guidance of the theory of traditional Chinese medicine （TCM） science, have played an indispensable role in the prevention and treatment of diseases in China for centuries. Sanwu-Huangqin-Tang （SHT） is a well known TCM prescription used for alleviating dysphoria and pyrexia of females after parturition. It originated from Qianjing Fang, a great pharmaceutical book written by physician Si-miao Sun during the Tang dynasty. The prescription composed of three commonly used Chinese herbs, Huangqin （Radix Scutellariae, 6.0g）, Kushen （Radix Sophoraeflavescentis, 6.0 g）, Dihuang （Radix Rehmanniae, 12.0 g）, and was extracted with water. In the modern clinical practice of TCM science, the formula has been widely used as a remedy for the treatment of fever, hepatitis, nephritis, tetter and cancer. In this paper the chemical constituents of its compositive herb, antibacterial activity, quality control method and pharmacokinetics on a few active constitutes of Sanwu-Huangqin-Tang were studied.The chemical constituents of Scutellaria baicalensis Georgi and Sophora flavescens Air., which were the principal and ministerial drugs of Sanwu-Huangqin-Tang, were systematically studied and 24 compounds in Scutellaria baicalensis Georgi, 15 compounds in Sophora flavescens Ait.. were isolated and purified by silica gel, polyamide, sephadex LH-20, ODS and PHPLC column chromatography. Utilizing chemical and spectroscopic methods （UV, IR, NMR, MS）, the structures for total of 37 compounds were fully characterized. These compounds all belonged to flavonoids except one wasβ-sitosterine. Six of these constituents were isolated from Sophora flavescens Ait. for the first time.The methods for HPLC-fingerprint analysis of Scutellaria baicalensis Georgi and Sophora flavescens Ait. were established. Firstly, HPLC methods were applied to separate flavonoids in Huangqin, flavonoids and alkaloids in Kushen collected from different habitat to get chemical data, which were used for hierarchical clustering analysis and similarity analysis of these herbs. 21 batches of Scutellaria baicalensis Georgi and 24 baches of Sophoraflavescens Air. samples were classified into three and two groups based on the results of cluster analysis.The superior samples were selected to develop the common recognition pattern and or to generate the representative characteristic chromatograms. The similarity analysis methods for quality assessment of herbs were developed by cosine, crrelation and euclidean. Based on the results of similarity, the evaluation criterions for quality contral of the two herbs were established. Furthermore, the typical samples of Huangqin and Kushen were detected by HPLC-DAD-ESI-MS/MS under the negative and positive ion mode, and the main common peaks of in the fingerprint chramatograms were identified.The HPLC-DAD-ESI-MS/MS methods were developed for the qualitative characterization of flavonoids in Scutellaria baicalensis Georgi and Sophoraflavescens Ait.. Total of 44 flavonoids （20 in Huangqin and 24 in Kushen） including flavones, flavaones, isoflavones, chalcones, pterocarpan et al were unambiguously or tentatively identified by comparing their retention times （tR）, UV and. MS spectra with, authecitc compounds and those reported by literature. Furthermore, the fragmentation path way of these compounds on negative and positive ESI ionization mode were investigated and summarized. Meanwhile the possible flavonoids presented in Sophora flacescens Ait. that have not been reported were presumed according to their UV spectra and MS fragments.The present study provided an approach to rapidly characterize bioactive constituents in Scutellaria baicalensis Georgi and Sophora flavescens Air..HPLC-UV quantitative analysis methods were established for assessing the quality of the formular Sanwu-Huangqin-Tang and its compositive herbs. Under the isocratic elution mode, ion pair chromatography and ion suppression chromatography were developed for determination of matrine, oxymatrine and oxysophocarpine in Sophora flavescens Air.. Under the gradient elution mode, The quantitative analysis methods for simultaneous determination of 10 flavonoids in Huangqin and 7 flavonoids in Kushen were performed by using HPLC technique. On the basis, HPLC-UV and HPLC-MS methods for simultaneous quantitfication of 6 main chemical compounds and 15 active compounds were established. The interaction of chemical constituents was observed when the herbs were used in compatibility. The results indicated that the developed method was rapid, accurate and could be readily utilized as quality control methods of Sanwu-Huangqin-Tang.A simple and sensitive LC-ESI-MS method has been developed for the determination of matrine, oxymatrine and oxysophorcarpine in plasma and applied for its pharmacokinetic study in rats after administration of Sanwu-Huangqin-Tang and Kushen decoction. Pseudoephedrine hydrochloride was selected as. internal standard. After extracted by chloroform, the organic phase was dried under the stream of nitrogen and the residue was reconstituted with mobile phase for analysis. The linear calibration curve of matrine was obtained in the concentration range of 10.0-5000 ng·mL-1, oxymatrine and oxysophocarpine in the concentration range of 2.0-1000 ng·mL-1. The assay had a lower limit of quantification of 10.0 ng·mL-1 for MT, 2.0 ng·mL-1 for OMT and 2.0 ng·mL-1 for OSP using 200μL of plasma. The within day precision and between day precision for matrine, oxymatrine and oxysophocarpine at low, medium and high concentrations were found to be less 10.1%. The recoveries of the three alkaloids and internal standard were above 83.9%. After Sanwu-Huangqin-Tang and Kushen decoction were orally administrated to rats, the mean plasma concentration-time curves were plotted and the pharmacokinetic parameters were calculated. The pharmacokinetic parameters for the three alkaloids after administration of Sanwu-Huanqin-Tang were as followes: Tmax, Cmax, T1/2 and AUC0-∞ for matrine were 1.92 h, 2.25×103 ng·mL-1, 12.4 h and 1.843×104 ng·h·mL-1, respectively; for oxymatrine were 1.58h, 157.8ng·mL-1, 2.55h and 473.9ng·h·mL-1; for oxysophocarpine were 1.00 h, 73.5 ng·mL-1, 2.45 h and 202.0 ng·h·mL-1, The pharmacokinetic parameters for the three alkaloids after administration of Kushen decoctions were as followes: Tmax, Cmax, T1/2 and A UC0-∞ for matrine were 2.08h, 2.53×103 ng·mL-1, 9.75 h and 2.269×104 ng·h·mL-1, respectively; for oxymatrine were 1.58h, 408.3 ng·mL-1, 3.44 h and 795.7 ng·h·mL-1; for oxysophocarpine were 1.42 h, 207.2 ng·mL-1, 2.68 h and 453.7 ng·h·mL-1.A simple and reliable method for simultaneous determination of 5,7,2’,6’-tetrahydroxyflavone, viscidulinⅢ, wogonin and oroxylin A in rat plasma by using HPLC-APCI/MS was developed and validated. 5,7,4’-trihydroxy-8-methoxyflavone was selected as internal standard. After extracted by mixture solvent of ether-N-hexane （4: 1） the organic phase was dried under the stream of nitrogen and the residue was reconstituted with mobile phase for analysis. The detection utilized selected ion monitoring at m/z 286.95 for 5,7,2’,6"-tetrahydroxyflavone, m/z 346.90 for viscidulinⅢ, m/z 284.95 for wogonin, m/z 284.95 for oroxylin A and m/z 301.05 for I.S. of 5,7,4’-trihydroxy-8-methoxyflavone, respectively. The assay showed good linear over the range of 2.0～1000.0 ng·mL-1 for 5, 7,2’, 6’-tetrahydroxyflavone, 5.0-2500 ng-mL-1 for viscidulinⅢ, wogonin and oroxylin A. Mean recoveries for the 4 detections and the internal standard were found to be above 82.7%. The HPLC method developed had been applied to determine the pharmacokinetics of 5,7,2’,6’-tetrahydrexyflavone, viscidulinⅢ, wogonin and oroxylin A in rat plasma after having taken Sanwu-Huangqin-Tang and Huangqin decoction via oral route. The pharmacokinetic behavior for the 4 flavonoids was found to be in line with the one-compartment model. A better absorption of wogonin and oroxylin A was founded when Sanwu-Huangqin-Tang was administrated compared with the administration of Huangqin decoctions. |
PDF Full Text Request