| GuizhiFuling Capsule (GF Capsule), a widely used traditional Chinese Medicine (TCM)formula,has a broad spectrum of applications in treatment of gynecological diseases. In this study, chemical basis, in vitro and in vivo correlation of the composition, quality control method, and pharmacokinetics GF Capsule were studied systematically as follows:1. By the sovereign drug poria in the GF Capsule as the research object, with thin layer chromatography, fast column chromatography, and semi-preparative HPLC led to the isolation and purification of 9 compounds. On the basis of physicochemical properties and spectroscopic analysis including 1D, 2D NMR, MS, their structures were identified as follows:Dehydrotumulosic acid, 3-Epi-dehydro-tumulosic acid, Polyporenic acid C,Dehydropachymic acid, 3-Keto-6-hydroxyl- Dehydrotumulosic acid,Tumulosic acid,3?-(4-hydroxybenzoyloxyl )-Dehydrotumulosic acid, Pachymic acid,3?-benzoyloxyl-Dehydrotumulosic acid. Among them, 3a-benzoyloxyl-Dehydrotumulosic acid was new compound.2. A high performance liquid chromatographic tandem trap mass spectrometric(HPLC-TRAP -MS/MS) method combined with liquid-liquid extraction sample preparation was developed for screening and identification of the main constituents of Cinnamomi Ramulus, Poria, Moutan cortex, Persicae semen, Paeoniae radix alba and GF Capsule. 17 compounds were detected, of which were identified including Cinnamic acid from Cinnamomi Ramulus, 3-Keto-6-hydroxyl- Dehydrotumulosic acid, 3-Epi-dehydro-tumulosic acid, Tumulosic acid, Polyporenic acid C, Dehydrotumulosic acid, Dehydropachymic acid,and Pachymic acid from Poria, Gallic acid, Benzoic acid, Paeonol, Oxypaeoniflorin,Paeoniflorin, Benzoyloxypaeoniflorin, and Benzoylpaeoniflorin from Moutan cortex,Amygdalin from Persicae semen, Albiflorin from Paeoniae radix alba. The HPLC-TRAP-MS/MS method was also applied to screen the constituents in rat plasma after oral administration of GF Capsule. 7 compounds were detected in rat plasma, including 3-Keto-6-hydroxyl- Dehydrotumulosic acid, 3-Epi-dehydro-tumulosic acid, Tumulosic acid,Polyporenic acid C, Dehydrotumulosic acid, Pachymic acid and Amygdalin. On the basis of the blood composition, 4 triterpene acids were identified in the target organs (uterus, ovary)samples, including 3-Epi-dehydro-tumulosic acid, Tumulosic acid, Polyporenic acid C, and Dehydrotumulosic acid.3. A high performance liquid chromatographic tandem mass spectrometric (HPLC-MS)method was developed and validated to simultaneously determine 7 compounds in Poria,pieces, intermediate, and GF Capsule including 3 -Epi-dehydro-tumulosic acid,3-Epi-dehydro-tumulosic acid, Tumulosic acid, Polyporenic acid C, Dehydrotumulosic acid,Dehydropachymic acid, and Pachymic acid. Separations were carried out with a Kromasil C18 reversed-phase column (150 mm×4.6 mm, 5?m). The mobile phase consisted of acetonitrile-5 mM ammonium acetate solution. A gradient elution program was used. Mass data were acquired using a mono-quadrupole mass spectrometer coupled with an electrospray ionization source (ESI) in the negative ion mode with a curved desolvation line (CDL)temperature of 200?, a heat block temperature of 200?, and a detector voltage of 1.75 kV.All the analytes showed good linearity (n = 6, r> 0.9995) in a relatively wide concentration range. The recoveries for the quantified compounds were observed in the range of 94.8?103.3 % with RSD values less than 5.0 %. The established method was applied to determine the amounts of the 7 compouds in 10 batchs of Poria, pieces, intermediate, and GF Capsule.The results indicated that the content of 7 compounds in Poria test ranged on average concentration: 77.45?139.2 %. In pieces: 73.11?145.1%. The results of statistical analysis for two sets of data showed that the content of 7 compounds changes between the two groups had no significant difference (P>0.05), the preparation and storage from Poria to pieces which did not affect its quality. In intermediate: 68.53?148.0 %. In GF Capsule: 83.4?123.0 %(RSD? 10.20?23.80 %). In this study,selected ion monitoring (SIM) mode of LC-MS Combined with the internal standard method to establish a simple, sensitive and specific method for the determination, which makes manufacturer is more convenient to monitor stability of production process and control quality of GF Capsule.4. A high performance liquid chromatographic-mass spectrometric (HPLC-MS) method was developed and validated for the simultaneous determination of Dehydro-tumulosic acid,Tumulosic acid, Polyporenic acid C in rat plasma for the first time. After addition of the internal standard Glycyrrhetinic acid,plasma samples were extracted by liquid-liquid extraction (LLE) method with ether and separated on an Kromasil C18 reversed-phase column(150 mm”4.6 mm, 5?m) using a mobile phase composed of acetonitrile-5 mM ammonium acetate solution (75:25, v/v) within a runtime of 12.5 min. The linear range was 20?1000 ng/mL for Dehydro-tumulosic acid,10?500 ng/mL for Tumulosic acid,11.5?575 ng/mL for Polyporenic acid C with the lower limit of quantitation of 20 ng/mL, 10 ng/mL and 11.5 ng/mL, respectively. The validated method was successfully applied to the comparative pharmacokinetic study of Dehydro-tumulosic acid, Tumulosic acid and Polyporenic acid C in rat plasma after oral administration of Poria extract and GF Capsule, respectively. After Poria extract and GF Capsule were orally treated,the Cmax of Dehydro-tumulosic acid was 486.9±50.5 ng/mL,417.6±22.4 ng/mL,AUC0-?,was2173±584 ngxh/mL,3425±696 ngxh/mL,T1/2 was2.5 ± 0.5 h,8.9± 2.7 h,respectively. the Cmax of Tumulosic acid was 417.3±41.5 ng/mL,414.3±53.3 ng/mL,AUC0-?, was2467± 641 ngxh/mL,5101±1211 ngxh/mL,T1/2 was4.2±1.4 h,10.6±2.3 h,respectively. the Cmxx of Polyporenic acid C was 311.5 ± 4.6 ng/mL,199.0 ± 25.4 ng/mL, AUC0-? was1111±288 ngxh/mL,1873±270 ngxh/mL, T1/2 was 2.3±0.7 h,7.9 ± 2.9 h,respectively. The results indicated that there were obvious differences in the pharmacokinetic behaviors after oral administration of Poria extract and GF Capsule to normal rat.5. To establish the pathological model of adult female rats with endometriosis, and for the first time to the system comparative pharmacokinetic studies of Dehydro-tumulosic acid,Tumulosic acid, and Polyporenic acid C in plasma after oral administration of GF capsule to normal rats and pathological model rat. The Cmax of Dehydro-tumulosic acid was 384.9 ±95.2 ng/mL,355 ± 156.7 ng/mL,AUC0-?- was 3127±1062 ngxh/mL,3307±565.1 ngxh/mL,T1/2 was 10.2±6.7 h,11.9 ±3.1 h,respectively. The Cmax of Tumulosic acid was 360.5±59.4 ng/mL,414.6±41.6 ng/mL,AUC0-? was 2216±732.7 ngxh/mL,5234±1440 ngxh/mL,T1/2 was 8.7±2.1 h,11.9±2.8 h,respectively. The Cmax of Polyporenic acid C was 193.3±66.3 ng/mL,291.8±46.9 ng/mL, AUC0-? was 1643±746 ngxh/mL, 3740±743 ngxh/mL, T1/2 was 11.8±7.7 h,12.2±4.2 h, respectively. The results showed that pharmacokinetic behavior of Dehydro-tumulosic acid between the two groups did not differ significantly, but AUC0-? of Tumulosic acid and Polyporenic acid C between the two groups showed significant differences. |
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