Characterization of the Anticoagulative Constituents of Angelicae Sinensis Radix and Their Metabolites in Rats by HPLC-DAD-ESI-IT-TOF-MSn.

Abstract

Angelicae Sinensis Radix is commonly used in traditional Chinese medicine. Pharmacological studies show that Angelicae Sinensis Radix has clear anticoagulant activity. Therefore, in this study, the anticoagulant activity of crude Angelicae Sinensis Radix extracts was investigated by measuring the thrombin times of the extracts. The results revealed that the petroleum ether-soluble fraction of Angelicae Sinensis Radix exhibited significant anticoagulant activity in vitro, and 26 compounds were characterized by high-performance liquid chromatography with diode array detection combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry. In addition, 5 prototype constituents, 24 in vivo metabolites in rat urine and 7 prototype constituents, and 9 in vitro metabolites in the rat hepatic S9 incubation system of the petroleum ether-soluble fraction were tentatively identified. All metabolites were found from Angelicae Sinensis Radix for the first time. Among them, 13 (three ferulic acid-related constituents, six senkyunolide D-related constituents, and four senkyunolide F-related constituents) were identified as new metabolites (new compounds). This study is the first to qualitatively characterize the chemical constituents of the potent anticoagulative extract of Angelicae Sinensis Radix and to explore its metabolism. The result is a notable improvement in the discovery of Angelicae Sinensis Radix metabolites, and it provides the chemical basis for the effective forms and pharmacodynamic substances (prototypes, metabolites, or both) of the anticoagulant activity of Angelicae Sinensis Radix.