Hypericum Quality Control Methods And Composition Of Drug Activity

Hypericum japonicum Thunb. (Tianjihuang) has been used as a Chinese medicine herb for the treatment of bacterial diseases, infectious hepatitis, gastrointestinal disorder, internal hemorrhage and tumors. Previous phytochemical studies of Tianjihuang have demonstrated that it mainly contains flavonoids, phloroglucinol derivatives, lactones, xanthonoids, chromone glycosides and peptides. For many years, Tianjihuang ethanol extracts exhibit a good therapeutic effect in treating acute and chronic hepatitis. But there are few methods about quality evaluation of these constituents. Along with more and more extensive application of Tianjihuang, it is absolutely necessary and urgent to develop a novel quality standard to validly control its quality.For the lack of standards, three compounds were isolated from the whole plant of Tianjihuang by silica gel, Sephadex LH-20, and Polyacylamine chromatography and were identified by chemical reaction and spectral analysis.A simple reversed-phase liquid chromatographic method has been established for the simultaneous quantification of five bioactive flavonoids, i.e., taxifolin-7-O-rhamnoside, isoquercitrin, quercitrin, quercetin and kaempferol in Tianjihuang. Chromatographic separations were achieved on a Luna C₁₈ column (Phenomenex, 250 mm×4.6 mm, 5μm) with a gradient of methanol and 0.5 % aqueous acetic acid as mobile phase and UV detection at 350 nm. The assay was reproducible with overall intra- and inter-day variation of less than 4.8%. The mean recovery of the method was 99.8±2.5 %, with RSD less than 3.0%. Using the optimized method, 19 samples were analyzed. The results indicated that the developed HPLC assay could be readily utilized as a quality control method for Tianjihuang.A simple HPLC method was developed for determination of quercitrin and isoquercitrin in rat plasma. Reversed-phase HPLC was employed for the quantitative analysis by using kaempferol-3-O-β-D-glucopyranoside-7-O-α-L-rhamnoside as an internal standard. Following extraction from the plasma samples with ethyl acetate-isopropanol (95:5, v/v), these two compounds were successfully separated using a Luna C₁₈ column (Phenomenex, 250mm×4.6 mm, 5μm) with a isocratic elution of acetonitrile-0.5% aqueous acetic acid (17:83, v/v) as a mobile phase. The flow-rate was set at 1 mL·min^-1 and the eluent was detected at 350 nm for both quercitrin and isoquercitrin. The method was linear over the studied range of 50.30～6595 and 51.38～5635 ng·mL^-1 for quercitrin and isoquercitrin, respectively. The intra-day and inter-day precision of the analysis were better than 13.1 and 13.2 %, respectively. The low limit of quantitation for quercitrin and isoquercitrin in plasma were 50.30 ng·mL^-1 and 51.38 ng·mL^-1. The mean extraction recoveries were 73.2 and 60.6 % for quercitrin and isoquercitrin, respectively. The validated method was successfully applied to pharmacokinetic studies of the two analytes in rat plasma after the oral administration of Tianjihuang ethanol extract to rats. The pharmacokinetic results provide a firm basis for evaluating the clinical efficacy of Tianjihuang extract.