| Gushudan prescription is formed according to "The Basic Concept of Establishing Platforms for Operational Techniques in Systems of Elaborately Selecting Small Prescriptions of Traditional Chinese Medicine" and "The Database Management System of PUJIFANG". The prescription is composed of Herba Epimedii, Fructus Cnidii, Rhizoma Drynariae and Radix Salviae Miltiorrhizae. It is a kidney-tonifying and bone-strengthening prescription used for the treatment of osteoporosis. It has been demonstrated in our laboratory that Gushudan prescription has the activity of promoting osteoblastic bone formation, and the determination and pharmacokinetic study of some active constituents have been carried out.In this study, a high performance liquid chromatography (HPLC) method has been developed for the fingerprint analysis of Gushudan prescription. The HPLC assay was performed on a DiamonsilTM C18 column. A gradient elution of 20 mmoL·L-1 aqueous sodium dihydrogen phosphate and acetonitrile was used. The column temperature was maintained at 25℃. Detection wavelength was 270 nm and the flow rate was 1.0 mL·min-1. Gushudan prescription was disassembled into 11 recipes according to the design of decomposed recipes. The fingerprint analysis of the 11 recipes was carried out by the HPLC method and their proliferative activity of osteoblast-like UMR106 cells was assyed. Correlation analysis was applied to correlate the peak area and the activity data. Among 18 peaks in the fingerprint chromatogram of Gushudan prescription, 9 peaks were correlated with the proliferative activity, which were suggested to be the material basis of Gushudan prescription promoting osteoblastic activity. By the correlation analysis between the composition of each recipe and the activity data, the relationship of herbal drugs in the prescription was consistent with the traditional interpretation.The serum pharmacochemistry study of Gushudan prescription was carried out using HPLC-UV and UPLC-MS methods to analyze the compound in rat's serum after oral administration of extracts of Gushudan and its principle drug Herba Epimedii. The condition of HPLC analysis was the same as above. The UPLC-MS analysis was performed on an ACQUITY UPLCTM BEH C18 column using a linear gradient elution of 0.1% aqueous acetic acid and acetonitrile. The column temperature was maintained at 40℃. The flow rate was set at 0.25 mL·min-1. The mass spectrometer was operated with an ESI source in the positive ionization mode scanning from 110 to 1000 amu. Four constituents in Gushudan prescription and two metabolites were detected in rat's serum after administration of Gushudan extract. Among the four constituents, there were three identified as epimedin B, icariin and osthole. Those constituents and metabolites were supposed to produce a marked effect in vivo.A spectrophotometric method has been developed for the determination of total flavonoids and total coumarins in Gushudan prescription. The content of total flavonoids was determined with AlCl3 complexation spectrophotometry at 414 run using icariin as reference, and the content of total coumarins was determined with ultraviolet spectrophotometry at 320 nm using osthole as reference. A good linearity between absorbance and the concentration was found over the range of 10-40μg·mL-1 for the total flavonoids and 2-12αg·mL-1 for the total coumarins. The average recoveries were 98.3% with RSD of 1.1% and 99.5% with RSD of 1.9%, respectively. The content of total flavonoids was 2.32% and the content of total coumarins was 2.05%. The methods were simple and accurate, and could be used as one of quality control methods of Gushudan prescription.ALC-MS/MS method was developed for simultaneous determination of icariin, icarisideⅡand osthole in rat plasma. With carbamazepine as the internal standard, plasma samples were prepared by protein precipitation. Detection was performed by means of electrospray ionization (ESI) mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves of icariin, icarisideⅡand osthole were obtained over the concentration range of 2.0-200.0 ng·mL-1, 2.0-200.0 ng·mL-1 and 2.0-500.0 ng·mL-1, respectively. The intra-and inter-day precisions (RSDs) were not above 14.1%, and the accuracy (RE) was from-6.0% to 9.0%. The recoveries were all above 80%.The pharmacokinetic study of icariin, icarisideⅡand osthole in rats after oral administration of Gushudan extract was investigated. The Cmax of icariin and icarisideⅡwere 101.82 ng·mL-1 and 46.28 ng·mL-1, respectively. The Tmax were 0.16 h and 0.24 h, respectively. The t(1/2) were both 0.28 h. And the AUC0-∞ were 23.20 ng·mL-1·h and 16.32 ng·mL-1·h, respectively. Icariin and icarisideⅡappeared to be absorbed and eliminated fast in vivo. The Cmax,Tmax, t1/2 and AUC0-∞ of osthole were 671.73 ng·mL-1, 0.70 h, 4.83 h, 1813 ng·mL-1·h, respectively. Osthole appeared to be absorbed fast but eliminated slowly in vivo, and double peaks were observed in the plasma concentration-time curves of osthole.Under the direction of the theory and clinical practice of Traditional Chinese Medicine, utilizing the techniques of Chinese material medica science, analytical chemistry, pharmacokinetics and chemometrics, the material basis of Gushudan prescription promoting osteoblastic activity was investigated in the present study. This work provided an exploration for the research and development of Gushudan prescription.
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