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The anti-hepatitis B virus (HBV) effects and its mechanisms of the ethanol extracts of Hypericum perforatum L. (EHP) in vitro were explored. HepG2 2.2.15 cells, a stable HBV-producing cell line, were cultured as the model system to observe the anti-HBV effect. The viral antigens of cellular secretion, HBsAg and HBeAg, were determined by enzyme linked immunosorbent assay (ELISA). The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR. In order to understand the mechanisms of the suppression of HBV replication, all HBV promoters (Cp, Xp, S1p, S2p and Fp) with luciferase reporter gene were transfected into HepG2 cells respectively. Then the activities of viral promoters were examined by luciferase reporter assay. It was found EHP effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, as well as the extracellular HBV DNA. And EHP could selectively inhibit the activity of HBV promoter Fp. Our data suggest that EHP exerts anti-HBV effects via inhibition of HBV transcription, which helps to elucidate the mechanism underlying the potential therapeutic value of EHP.
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OBJECTIVE: To establish an HPLC method for simultaneous determination of the contents of Chlorogenic Acid and Coffeic Acid in the Fruit of Chaenomeles. METHODS: HPLC was applied to determine and compare the contents of 11 batches of Chaenomeles medicinal materials collected at different time, prepared with different process and stored for different length of time, using Coffeic Acid and Chlorogenic Acid standard substances. The chromatogram conditions were as follows: Kromasil C18( 250mm? 4. 6mm, 5? m) column; mobile phase of ( A) acetonitrile, ( B) 0. 05% phosphoric acid; gradient el-ution ( 0～ 10min, 0% A～ 15% A, 100% B～ 85% B; 10～ 40min ( 15% A～ 30% A , 85% B～ 70% B) ; flow rate at 0. 8 mL? min- 1; detection wavelength at 325nm. RESULTS: The content of Chlorogenic Acid in batches 1 to 9 were respectively 0. 020% , 0. 035% , 0. 043% , 0. 051% , 0. 260% , 0. 104% , 0. 081% , 0. 056% , 0. 034% ; that of Coffeic Acid were respectively 0. 005% , 0. 006% , 0. 008% , 0. 010% , 0. 051% , 0. 024% , 0. 020% , 0. 015% , 0. 007% . The contents of Chlorogenic Acid and Coffeic Acid in batch 8’ ( burn- prepared) were respectively 0. 045% and 0. 010% ; those of Chlorogenic Acid and Coffeic Acid in batch 9’ ( stored for 1a) were respectively 0. 023% and 0. 005% . CONCLUSIONS: The method is simple, reproducible and suitable for the quality control of Fruit of Chaenomeles. The best time for harvesting batch 5 is on July 12, and the best process for this batch is solarization. The storage time has certain influence on the content of Chlorogenic Acid.
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Objective To establish a HPLC method for the determination of geniposide in Mengning Tablets and to investigate the thermal stability of geniposide. Methods The content of geniposide in Mengning Tablets was determined on a E- clipse XDB-C_(18) column with a mobile phase consisting of 0.2% triethylamine (containing 0.1% phosphate) and acetonitrile (85: 15) at a flow rate of 1.0 mL?min~(-1) and detected at a wavelength of 240 nm. The thermal stability of geniposide was investigated by hyperthermal accelerated test. Results A linearity was obtained from 0. 10 ?g to 0.52 ?g of geniposide (r=0.9997, n=5) and the recovery was 98.92%(n=5). The variation of geniposide in Mengning Tablets was in first-order reaction and the expiry date of the medicine at normal temperature was 2.83 years when geniposide content was used as criteria. Conclusion This is a fast method for the determination of the expiry date of a Chinese patent medicine and this method is useful infor the development of a new medicine.