HPLC/LC-MS guided phytochemical and in vitro screening of Astragalus membranaceus (Fabaceae), and prediction of possible interactions with CYP2B6

Abstract

The dried roots of Astragalus membranaceus (Fabaceae) are used in African traditional medicine for the treatment of a variety of diseases. The objective of this study was to assess the potential of methanol, ethanol, aqueous and ethyl acetate extracts from the roots of A.membranaceus to alter the activity of cytochrome P450 (CYP) 2B6 using human liver microsomes (HLMs). Furthermore, the phytoconstituents possibly involved in this effect were analysed using biochemical tests, HPLC and LC–MS. Reference standards for flavonoids, alkaloids, coumarins, glycosides and phenols were used. In the HLM screening, the ethanol and ethyl acetate extracts reduced CYP2B6 activity to less than 50% (IC50 of 53.37 μg/ml and 29.7 μg/ml); IC50 of the positive control ticlopidine was14.47 μg/ml. The extracts did not show time-dependant inhibition of CYP2B6 (TDI IC50 > 100 μg/ml). The biochemical qualitative analysis confirmed the presence of flavonoids, glycosides, alkaloids, terpenoids, coumarins and phenols. The chromatographic analyses of the extracts using reference standards showed peaks with retention times matching gallic acid (HPLC) and m/z transition peaks of lanatoside C (LC–MS) and were quantified at ± 0.2 mg/ml equivalents of gallic acid/ lanatoside C. MS full scans of the extracts were consistent with the presence of isoflavonoids calycosin (m/z 283) and formononetin (m/z 267) in the negative scans, and astragalosides I, II and IV (m/z 869, 827 and 785) in the positive scans. It is likely that some of these compounds are responsible for the observed CYP2B6 inhibition. The putative gastrointestinal tract (GIT) concentration for both extracts was calculated as 8000 μg/ml and concentrations in the hepatic circulation were estimated as 945.6 μg/ml for the ethanolic extract, and 390.4 μg/ml for the ethyl acetate extract.In conclusion, the root extracts of A.membranaceus have been shown to inhibit CYP2B6, with IC50 values below the estimated hepatic circulation concentrations. This herb therefore may cause adverse drug interactions with CYPs, when co-administered with medications metabolized by CYP2B6.