Safety of Botanical Dietary Supplements – Licorice: in vitro Investigation of Drug-Botanical Interactions

The root of licorice (Glycyrrhiza sp.) is popular worldwide for use in botanical dietary supplements. There are three commonly used species, namely Glycyrrhiza glabra L., Glycyrrhiza uralensis Fish. ex DC and Glycyrrhiza inflata Batalin. We are investigating the possibility of drug-botanical interactions involving licorice extracts and whether different licorice species show different interactions against specific cytochrome P450 (CYP) drug metabolizing enzymes. These three licorice species are difficult to distinguish by root morphological examination alone and are often marketed without differentiation. An ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify 14 components (liquiritin, isoliquiritin, liquiritin apioside, isoliquiritin apioside, licuraside, liquiritigenin, isoliquiritigenin, glycyrrhizin, glycyrrhetinic acid, glabridin, glycycoumarin, licoricidin, licochalcone A and p-hydroxybenzylmalonic acid) found in licorice. Using this method, three botanically authenticated licorice species from different sources (six for G. glabra, five for G. uralensis, one for G. inflata) could be differentiated, and five commercial supplements were investigated. The understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (CYP) enzymes is the most common cause of drug-botanical interactions. There are multiple CYP isoforms found to be important in metabolizing drugs. To be cost-effective and time-saving, a rapid and comprehensive mass spectrometry–based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4, this cocktail simultaneously assesses at least as many CYP enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using UHPLC-MS/MS. The method was validated using known inhibitors of each CYP enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (G. glabra) as an example. Three authenticated licorice extracts showed different inhibitory profiles using this cocktail assay. G. uralensis strongly inhibited CYP2B6 (both reversibly and irreversibly), and G. inflata had potent inhibition against CYP2C8, CYP2C9, CYP2C19 and CYP3A4 (midazolam). 14 licorice constituents were also screened for potential inhibition activities. Licochalcone A, abundant only in G. inflata, was one of the major contributors to the G. inflata inhibition effects and was found to be a mechanism-based inhibitor for CYP3A4 (midazolam).