Studies On The Quality Control And Pharmacokinetics Of Apocynum Venetum L.

Apocynum venetum L. (Apocynaceae family) is one of the commonly used Traditional Chinese Medicines (TCM). The tea of the leaves of Apocynum Venetum L. called Luobumaye in Chinese and Rafuma in Japanese has been used widely in traditional Chinese medicine since ancient time. The leaves of Apocynum Venetum L. are the investigative target. In this thesis, the chemical constituents, quality control methods and the pharmacokinetics of Apocynum venetum L. were investigated in detail.The chemical constituents of Apocynum venetum L. were systematically studied and 9 compounds were purified with silica gel, thin-layer chromatography, polyamide column chromatography and MPLC methods. Utilizing chemical and spectroscopic methods (UV, NMR, MS), the structures of 9 compounds were fully characterized as quercetin, quercitrin, kaempferol, quercetin-3-O-β-D-glucopyranoside,β-sitosterol, chlorogenic acid, hyperoside, quercetin-3-O-rutinoside and succinic acid. These compounds were suitable as standard substances for quantitative and pharmacokinetic purposes.The method of HPLC fingerprint analysis was established for the quality control of Apocynum venetum L.. All of the 15 samples were selected to develop the common recognition pattern and to generate the representative characteristic chromatograms. The mobile phase was acetonitrile-0.1% glacial acetic acid solution by gradient elution with Zorbax Eclipse SB-C18 (250 mm×4.6 mm,5mm) column. Isoquercitrin was used as the reference. Fourteen common peaks of the fingerprints were marked, and five of them were identified to be chlorogenic acid, hyperoside, isoquercitrin, quercetin and quercitrin. The similarity was calculated. Based on the results of similarity, the evaluation criterion for quality control of the Apocynum venetum L. was established.The CGC chromatographic fingerprints were established for evaluating the quality of Apocynum venetum L.. Separation was achieved on VF-5MS Factor Four Capillary column (30m×0.25mm i.d.,0.25μm film thickness). Paeonol was used as the reference. Twenty six common peaks of the fingerprints were marked. The similarity was calculated. Based on the results of similarity, the evaluation criterion for quality control of the Apocynum venetum L. was established. The developed method was reasonable and feasible to describe and evaluate the quality of unknown samples based on the chemical data. The methodology might well be used to assess the quality of other herbs. It was necessary that the quality of Apocynum venetum L. should be evaluated by the two fingerprints.The chemical compositions of essential oils from Apocynum venetum L. have been studied by GC-MS method. Seventy five chromatographic peaks were detected. The chemical compositions of essential oil from Apocynum venetum L.. have been studied by GC-MS. Sixty constituents of the essential oils were identified. The compound of essential oils was retrieved in NIST standard spectrum. At the same time they were compared with EPA/NIH standard spectrum of mass spectrum.HPLC fingerprint of the plasma samples was firstly developed after oral administration of Apocynum Venetum L. extract. With the same chromatographic condition, plasma samples obtained at 0.5 h after oral administration of Apocynum Venetum L. extract were analyzed. Sixteen chromatographic peaks were detected from the plasma samples. Compared with the fingerprints in vitro, eight peaks were identified to be from Apocynum Venetum L.. And five peaks were quercetin, quercitrin, chlorogenic acid, hyperoside, quercetin-3-O-β-D-glucopyranoside. The HPLC fingerprint of the plasma samples shows that the flavones constituents were absorbed quickly.The determination of hyperoside and isoquercitrin was conducted by RP-HPLC using a Zorbax-C18(4.6mm×150mm,5μm) column. The mobile phase was acetonitrile-1.0% acetic acid solution (14:86) with the flow rate of 0.8 mL-min-1, the detection wavelength was 360 nm and the column temperature was set at 30℃. In HPLC analysis, the linear ranges of hyperoside and isoquercitrin were 0.02434-0.4868μg and 0.02756-0.5512μg, respectively. The average recovery of hyperoside and isoquercitrin were 98.7%(RSD=1.1%) and 99.8% (RSD=1.3%), respectively. Under the gradient elution mode, an HPLC-UV method was developed for the simultaneous quantification of those five compounds:chlorogenic acid, hyperoside, isoquercitrin, quercetin and quercitrin. The chromatographic separation was performed on a Zorbax SB-C18 column (250mm×4.6mm i.d.,5μm) with a gradient of acetonitrile and water containing 0.1% glacial acetic acid, at a flow rate of 1.0 ml·min-1 detected at 360 nm. Five regression equations showed good linear relationships (r2> 0.999) between the peak areas of each marker and concentrations. The recoveries, measured at three concentration levels, varied from 97.6% to 101.8%. The results showed that the herb content of different source is different. The results indicated that the developed assay method was rapid, accurate, reliable and could be readily utilized as a quality control method for Apocynum Venetum L.Hyperoside and isoquercitrin are two major bioactive flavones isolated from Apocynum Venetum L. leaves. Modern pharmacological studies have proved their various activities. They are the representative and specific ingredients of Apocynum Venetum L. leaves and their extract. So hyperoside and isoquercitrin are chosen as biomarkers in the quality control of Apocynum Venetum L. leaves. A HPLC-MS/MS method was firstly developed and validated for simultaneous quantification of hyperoside and isoquercitrin in rat plasma, which isolated the isomer by gradient elution and detected them by Tandem MS. The study of its pharmacokinetics after oral administration of 4.8 g·kg-1 Apocynum Venetum L. leaves extract, 43.5 mg-kg-1 of hyperoside and 44.6 mg-kg-1 of isoquercitrin to Wistar rats respectively. Following simple deproteinization by acetonitrile, the analytes were separated on an Agilent C18 column with the mobile phase consisted of water containing 0.1% glacial acetic acid and acetonitrile (83:17, v/v). The eluant was detected by tandem mass spectrometry with the multiple reaction monitoring (MRM) mode using the transition of m/z 463â†'300 for hyperoside and isoquercitrin, and m/z 415â†'267 for puerarin (internal standard) in negative ion mode. The method was linear over the concentration range from 3.91 to 195.5 ng-mL"1 for hyperoside and from 3.89 to 194.5 ng-mL-1 for isoquercitrin. The lower limit of quantification was 3.91 ng-mL-1 and 3.89 ng-mL-1 for hyperoside and isoquercitrin, respectively. The validated method was successfully applied for the pharmacokinetic study of hyperoside and isoquercitrin after oral administration Apocynum Venetum L. extract and hyperoside, isoquercitrin only in rat. The T12 of hyperoside was 2.5±0.6 h and 2.4±2.0 h respectively. The Cmax of hyperoside was 153.7±8.9 ng-mL-1 and 132.6±10.3 ng-mL"1 respectively. The Tmax of hyperoside was 0.5±0.0 h and 0.3±0.0 h respectively. The AUCo-t of hyperoside was 320.5±28.9 ng-h-mL’and 174.9±24.8 ng-h-mL1 respectively. The AUC0-∞of hyperoside was 339.2±38.2 ng-h-mL-1’and 194.1±35.4 ng-h-mL-1 respectively. The T12 of isoquercitrin was 2.7±2.0h and 2.2±1.5 h respectively. The Cmax of isoquercitrin was 147.7±9.2 ng-mL-1 and 142.0±13.4 ng-mL-1 respectively. The Tmax of isoquercitrin was 0.5±0.0 h and 0.4±0.1 h respectively. The AUC0-t of isoquercitrin was 317.4±14.1 ng-h-mL-1 and 172.1±21.1 ng-h-mL-1 respectively. The AUC0-∞of isoquercitrin was 330.5±7.9 ng-h-mL-1 and 184.5±28.8 ng-h-mL-1 respectively, which suggest a better absorption of isoquercitrin when Apocynum Venetum L. extract was administrated compared with the administration of isoquercitrin only. There are significant differences among the pharmacokinetic parameters.Combining the utilization of phytochemistry, pharmaceutical analysis, pharmacognosy, pharmacology and pharmacokinetics, the constituents and quality assessment standards for Apocynum Venetum L. were studied, and the pharmacokinetics of this medicinal herb and its active constituents were also investigated. This research provided a beneficial exploration for the modernization of the herb.