Studies On The Metabolism Of Icariin In Vivo And In Vitro And Determination Of Relevant Activities Of Its Metabolites

Herba Epimedii from many species of genus Epimedium (family Berberidaceae) has been widely used as a tonic, aphrodisiac, and antirheumatic in China. Its effective ingredient-icariin has been used as a quality control standard of Herba Epimedii. Experimental evidence has demonstrated diverse biological effects of icariin such as amelioration of cardio- and cerebro-vascular system, improvement of immunity, and modulation of endocrine, et al. And it has been reported that formulated medicines of Herba Epimedii have phytoestrogen like activities. The therapeutical effects of Herba Epimedii probably come from the phytoestogen like activities. And the determination of phytoestrogen like activities of icariin, the indicative constituent, is important for elucidation of material base of its therapeutical effects.It was reported that orally administered flavonoids usually exhibit estrogenic activities after metabolism in vivo. Herba Epimedii is often taken orally. It is necessary to study whether orally administered icariin could be transformed to possible metabolites such as icaritin and desmethylicaritin et al. and to explore the structure-activity relationship. A method of CZE (capillary zone electrophoresis) for pharmacokinetics of icariin was established and combination of CZE, HPLC and LC-MS was constructed to detect metabolites of icariin in vivo and by intestinal bacteria in vitro; main metabolites were prepared by chemical modification toinvestigate the structure-activity relationship and to provide foundation for the study of biological activities and mechanism of Herba Epimedii preparations.1. Chemical modification and purification of icaritin and desmethylicaritin from icariinIcaritin was prepared via hydrolysis by cellulase and extraction by ethyl acetate. Desmethylicaritin was prepared via oxidation by boron tribromide solvent and extraction by ethyl acetate. Then the prepared compounds were purified by preparative-HPLC and analyzed by HPLC and EI-MS.Based on the data integrated by the software the purity of icaritin reached 98.9%. The EI mass spectrum displayed a molecular ion peak at m/z 368 corresponding to that of icaritin.Five pseudomolecular ions ([M-H]") correlated with the demethylation of icaritin were observed by HPLC/ESI-MS. Only the compound of peak 5, which display a pseudomolecular ions ([M-H]) of m/z 352.6, corresponded to the molecule of desmethylicaritin. A preparative-HPLC was used for isolating desmethylicaritin, and the eluent of the same retention time of peak 5 was pooled and evaporated to dryness. Detection by HPLC analysis, the purity of desmethylicaritin was found to be 98.6% for integration by software. The EI mass spectrum of purified desmethylicaritin displayed a molecular ion peak at m/z 354 corresponding to that of desmethylicaritin.2. Determination of icariin in biological samples of rat by capillary zone electrophoresisAnalysis of icariin and its metabolites in serum, urine and feces was conducted in the following manner:An untreated fused-silica capillary tube (effective length 43.6cm 50m I.D.) was used for separation. Sodium tetraborate (30 mmol/L), monobasic sodium phosphate (50 mmol/L)- acetonitrile (50:50, v/v)(pH 10.0) was chosen as running buffer. The typical analytical conditions were: voltage, 12.0 kV; injection, 10 psi sec; capillary and carousel temperature were 25 and 10, respectively; the detectionwave length was 270nm.Standard stock solution of icariin (1mg/ml) and internal standard of carbamazepine (Img/ml) were prepared with methanol. Working standards from the concentrated stock solutions were prepared, IS added (used as final concentration of 40mg/l) and then diluted with 0.1ml rat serum, urine or feces to yield concentrations of 2.5, 10, 50, 100, 150 mg/1. 0.1ml serum sample with 0.2ml acetonitrile or 0.1ml urine and sieved fecal slurry with 0.4ml ethyl acetate containing the IS were mixed up vigorously and centrifuged. Then, its supernatant or organic layer was poured into another sample tube. The supernatant or organic layer was...