The Epimedium Effective Component Analysis And Its Metabolism In Vivo Studies

Epimedii Folium, the dried leaf of Epimedium brevicornum Maxim., Epimedium sagittatum Maxim., Epimedium pubescens Maxim., Epimedium wushanense T.S. Ying and Epimedium koreanum Nakai. is a well known traditional Chinese medicine. It is widely used as a tonic, aphrodisiac and antirheumatic in China for over 2000 years. Modern pharmacological study indicated that Epimedii Folium was able to increase the Cardio vascular and cerebral vascular blood flow, promote haematogenesis, immunity function and metabolism of bones, and it also had efficacy of anti-aging and anti-tumor, etc. The main constituents of Epimedii Folium have been elucidated to be flavonoids, such as Icariin and baohuoside I, which are considered to be the major active components of Epimedii Folium and to be used as the marker compounds for quality control of Epimedii Folium and its preparations.In this paper, a simple, sensitive and selective high-performance liquid chromatographic method coupled with ultraviolet detection and Fourier transform-ion cyclotron resonance mass spectrometry (HPLC-UV/FTICRMS) was developed to identify the flavonoids in Epimedii Folium from 7 different sources. The MS" data were obtained by parent mass list-triggered data-dependent multiple-stage and the accurate FTICRMS m/z value at a resolving power of 50,000 was collected with an accuracy within 2 ppm. The high accurate molecular mass combined with multiple-stage mass spectrometric data (MSn), enabled us rapidly to predict molecular formula and structures. As a result,68 flavonoids were identified in Epimedii Folium. In addition, by comparing the peak areas of the 68 compounds, the major flavonoids were selected as markers.Then, a sensitive and specific high performance liquid chromatographic method connected with mass spectrometry (HPLC-MS) has been developed and validated for the determination of the 7 main flavonoids, namely, epimedin A, epimedin B, epimedin C, icariin, sagittatoside B,2"-O-rhamnosyl icariside II, and baohuoside I in Epimedii Folium from 35 different species or habitats. In addition, the principal component analysis (PCA) and clustering analysis method were used to analysis of the quantitative data. A comprehensive evaluation function was obtained by the the principal component analysis (PCA). The results show that this function not only reflected the flavonoids absolute contents changed, but also reflected that relative contents changed, and is the promising quality evaluation method. Clustering analysis shows that Epimedii Folium from different species or habitats can be divided into low, medium and high three groups. Classification results and the comprehensive evaluation function results are consistent. Our results showed that it is reasonable to use multiple flavonoids as marker components to control the quality of Epimedii Folium and to evaluate the quality by the appropriate statistical processing method.A HPLC-FTICRMS method was developed to identify the active compounds and their metabolites after oral administration of the herbal extract of Epimedium koreanum Nakai to rats, using parent mass list-triggered data-dependent multiple-stage accurate mass analysis at a resolving power of 100,000 in the external calibration mode. Nine flavonoids were identified in rats. The chemical formulae with unsaturation numbers calculated from accurate m/z values of precursor and product ions were used to assign the structures of metabolites and the chemical sites of metabolism. The mass accuracies obtained for all full-scan MS and MS" spectra were within 3 ppm (< 1 ppm in most cases). The majority of the metabolites identified have been previously reported, but three compounds were first noted in rats. By contrasting the analytical results obtained from the herbal extract with those obtained from biological specimens, the metabolic pathways of Epimedii Folium flavonoids in rats were proposed.On the basis of quality control and metabolism of Epimedii Folium flavonoids, an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to detect the metabolic fingerprint in rats after oral administration of two kinds of Epimedii Folium with different characteristic chromatographic fingerprints. Results further proved that 7 main flavonoids of Epimedii Folium were widely distributed in tissues and organs, which is evidence for its wide pharmacological effects. In additon, Epimedii Folium with different characteristic chromatographic fingerprints also showed obviously different metabolic fingerprints.Finally, a sensitive and specific UHPLC-MS/MS method has been developed and validated for determination of the 7 flavonoids in dog plasma. The established method is simple, rapid, sensitive and reliable. This method has been successfully applied to the pharmacokinetic study after the oral administration of Epimedii Folium and Er-Xian Decoction extracts (EXD) to dogs respectively. The pharmacokinetic parameters indicated that 5 major active flavonoids were absorbed better from Epimedii Folium extract than EXD extract. These results suggested that the absorption of the active flavonoids from Epimedii Folium might be affected by other herbs in the EXD. It also helped to reveal the mechanism of traditional Chinese prescription.