| Forsythia suspensa is the dried fruit of Forsythia suspensaVahl, which belongs to the family of Oleaceae. Forsythia suspensa produced in Hebei province has good quality. It has heat-clearing, detoxicating and detumescence effects, it also could remove stasis. A number of compounds including phenylethanoid glycosides, lignans, flavonoids, terpenes, and volatile oils had been isolated from the plant,in which phenylethanoid glycosides and lignans are the bioactive components. Because of the difference of geography and growth environment, Forsythia suspensa from different places are different on the chemical composition and contents, that leads to the instability of the quality. In our study, we established HPLC and HPCE fingerprints of Forsythia suspensa from Hebei Province, providing scientific method for the quality control of Forsythia suspensa. We also developed HPLC, HPCE and HPLC-ESI-MS method to determine the contents of the bioactive components in order to provide more basis for the quality control of Forsythia suspensa.Trollius ledebouri is the dried flower of Trollius chinensis Bunge, it has antibiosis and anti-virus effects, and it is widely used for the treatment of common cold, fervescence, chronic tonsillitis, acute tympanitis, urinary tract infection, and so on. Pharmacological studies suggest that flavonoid glycosides are the main bioactive components in Trollius ledebouri. Based on the results we obtained before, in the present study, a HPLC-ESI-MS method was developed and validated for the investigation of the pharmacokinetic profile of orientin-2″-O-β-L-galactopyranosyl, orientin and vitexin following the oral administration of Trollius ledebouri extract. The study provided basis for the further research of Trollius ledebouri. Part one Study on HPLC fingerprints of Forsythia suspensa from Hebei ProvinceObjective: To establish HPLC-PDA fingerprints of Forsythia suspensa from Hebei Province, get reference fingerprint and compare the fingerprints of Forsythia suspensa collected from different producing areas. To determine the contents of forsythin and rutin in each batch of Forsythia suspensa, so as to establish a sensitive and specific method for controlling the quality of Forsythia suspensa.Methods: The chromatographic procedure was carried out with DiamonsilTMC18 (250 mm×4.6 mm,5μm) and a mixture consisting of acetonitrile and 0.1% phosphoric acid was used as mobile phase following the gradient elution, the temperature of column was 30 ?C. The detection wavelength was set at 235 nm, the flow rate was 0.8 mL/min. In the determination of forsythin and rutin, the detection wavelength was 210 nm and 254 nm, respectively, the flow rate was 1.0 mL/min.Results: The mutual mode of HPLC-PDA fingerprints was set up, the semblances to the crude drugs of different producing areas were compared,and the contents of forsythin and rutin were determined in 29 batches of Forsythia suspensa. It is indicated that Forsythia suspensa produced in Hebei province were of good quality, and semblances of Shanxi and Henan were also higher. There were big discrepancy on the semblances of Forsythia suspensa from different parts of the herb. The contents of forsythin and rutin in leaf and flower were much higher than those in fruit.Conclusion: We have established HPLC-PDA fingerprints of Forsythia suspensa from Hebei Province. The operation of the mothod is simple, quick, accurate and can be used for the identification and quality control of Forsythia suspensa. Furthermore, the contents of forsythin and rutin in leaf and flower are much higher than those in fruits, so whether the leaf and flower can take the place of fruit needs more study and research.Part two Study on HPCE fingerprints of Forsythia suspensa from Hebei Province Objective: To establish HPCE fingerprints of Forsythia suspensa from Hebei Province, get reference fingerprint and compare the fingerprints of Forsythia suspensa collected from different producing areas and different parts of the plant. To develop a CZE method for the determination of phillyrin, phillygenin, (+)-epipinoresinol-4'-O-glucoside, (+)-pinoresinol-β-D-glucoside and (+)-epipinoresinol in different parts of Forsythia suspensa.Methods: Capillary electrophoresis was performed using a fused silica capillary column (75μm×60 cm, an effective length of 30 cm). The running buffer was composed of 50 mmol/L borax (adjust to pH 9.90 with 0.1 mol/L NaOH). The applied voltage was 15 kV and the temperature was 20℃. The detection wavelength was 214 nm.Results: The mutual mode of HPCE fingerprints was set up with 12 common peaks. The fingerprints of Forsythia suspensa from Hebei province had high similarity, Forsythia suspensa from Shanxi and Henan were also of good quality. The chemical composition in different parts of the herb had big differences. In the experiment of determination, the five components were seperated sucessfully within 12.5 min. The contents of phillyrin, phillygenin, (+)-epipinoresinol-4'-O-glucoside and (+)-pinoresinol-β-D-glucoside in leaf and flower are much higher than those in fruit.Conclusion: We have established HPCE fingerprints of Forsythia suspensa from Hebei Province and developed a CZE method for the determination of phillyrin, phillygenin, (+)-epipinoresinol-4'-O-glucoside, (+)-pinoresinol-β-D-glucoside and (+)-epipinoresinol. The method is simple, quick, accurate and can be used as a new means for the quality control of Forsythia suspensa. The medicinal value of leaf and flower need more research.Part three Simultaneous quantification of 14 constituents in Forsythia suspensa by HPLC-ESI-MSObjective: To establish a HPLC-ESI-MS method for simultaneous quantification of 14 constituents of Forsythia suspensa in different places of China and different parts of this herb. Methods: The optimal chromatographic conditions were achieved on a Kromasil C18 column (150×4.6 mm, 5μm) with gradient elution of methanol, acetonitrile and 0.1% formic acid in 27 min. The HPLC system was connected to 3200 Q TRAP LC/MS/MS System, a hybrid triple quadrupole/LIT (linear ion trap) mass spectrometer equipped with an ESI ion source (Applied Biosystems/MDS Sciex, USA). The turbo spray temperature was set at 600℃with a ionspray voltage -4500 V. Nebulizer gas (gas 1) 40 psi, heater gas (gas 2) 50 psi and curtain gas (CUR) 25 psi. Nitrogen was used in all cases. Detection was performed in negative ionization mode by monitoring the precursor-product combination in multiple reaction monitoring (MRM) mode.Results: All calibration curves showed good linear regression (r > 0.9990) within test ranges. Intra-day and inter-day variations were of 0.7%~4.3% and 1.1%~3.9% respectively, and overall recoveries were of 96.65%~101.2 % for the compounds analyzed. Forsythoside was the major compound in the fruits of Forsythia suspensa. The total contents of the 14 compounds in different places of China had a certain degree of difference. The contents of most compounds in leaves, stems and flowers were much higher than in fruits.Conclusion: We have firstly established a HPLC-ESI-MS method for simultaneous quantification of 14 constituents in Forsythia suspensa. It's a rapid analytical method, with a shorter run time, high specificity and sensitivity. With the pervasion of the LC-MS equipment, the proposed HPLC-ESI-MS method is a useful process for the quality control of Forsythia suspensa.Part four Study on pharmacokinetics of 3 components in rat plasma after oral administration of Trollius ledebouri extract by HPLC-ESI-MSObjective: To develop a HPLC-ESI-MS method for simultaneous determination of orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin in rat plasma for studying the pharmacokinetics of them after oral administration of Trollius ledebouri extract. Methods: After rats were oral administrated extract solution of 2 mL, blood samples were obtained from fossa orbitalis vein according to the specific schedule, 5,10,15,25,40,55,70,90,120,180,270 and 390 min and collected in heparinized centrifuge tube, respectively. Sample was pretreated by a single-step protein precipitation with methanol. Chromatographic separations were performed on a Waters SunFireTM C18 column (150 mm×4.6 mm, 5μm) protected by a C18 guard column (12.5 mm×4.6 mm, 5μm) at room temperature. The mobile phase consisted of methanol-0.01% formic acid using gradient elution (0-3 min, 30%-90% methanol; 8.1 min, 30% methanol). The flow rate was set at 0.7 mL/min. Aliquots of 5μL were injected into HPLC system for analysis. The HPLC system was connected to 3200 Q TRAP LC/MS/MS System, a hybrid triple quadrupole/LIT (linear ion trap) mass spectrometer equipped with an ESI ion source (Applied Biosystems/MDS Sciex, USA). The turbo spray temperature was set at 600℃with a ionspray voltage -5000 V. Nebulizer gas (gas 1) 40 psi, heater gas (gas 2) 40 psi and curtain gas (CUR) 25 psi. Nitrogen was used in all cases. Detection was performed in negative ionization mode by monitoring the precursor-product combination in multiple reaction monitoring (MRM) mode.Results: Calibration curves of orientin-2′′-O-β-L-galactop -yranosyl, orientin and vitexin showed good linear regression over the range 4.5-4200 ng/mL,6.5-6200 ng/mL and 2-800 ng/mL, respectively. The intra- and inter-day precisions (RSD) for the analysis of three analytes were between 1.7% and 10.8% with accuracies (RE) between -5.1% and 5.0%. The mean extraction recoveries were between 97.30% and 106.7%. The matrix effect were between 95.80% and 101.7%. The main pharmacokinetic parameters of orientin-2′′- O-β-L-galactopyranosyl, orientin and vitexin were as follows: T1/2 were 82.84±11.50, 82.57±7.61 and 71.16±8.57 min, AUC0?t were 285.4±29.3, 152.8±15.2 and 10.3±1.3μg min/mL, AUC0?∞were 313.8±27.8, 167.6±24.7 and 10.8±1.2μg min/mL, MRT0?∞were 148.68±7.69, 131.75±25.26 and 115.48±17.32 min. Conclusion: A HPLC-ESI-MS method has been developed for simultaneous determination of orientin-2′′-O-β-L- galactopyranosyl, orientin and vitexin in rat plasma following oral administration of Trollius ledebouri extract and the method was successfully applied to the pharmacokinetic studies of the three active components. The method has good precision and accuracy with high sensitivity and shorter time, it is fit to the needs of biological sample analysis. |
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