A High-Performance Liquid Chromatography with Photodiode Array Detection Method for Simultaneous Determination of Three Compounds Isolated from Wikstroemia ganpi: Assessment of the Effects on Cytochrome P450-Mediated Metabolism In Vitro and In Vivo.

In natural products, the content and quality of the marker components differ depending on the part, production area, collection period, and extraction method; therefore, a standardized analysis method is required to obtain consistent results. This study developed a simultaneous analysis method for three marker components (7-methoxylutolin-5-O-glucoseide, pilloin 5-O-β-d-glucopyranoside, rutarensin) isolated and purified from Wikstroemia ganpi (W. ganpi). Simultaneous analysis was performed using high-performance liquid chromatography with photodiode array detection (HPLC-PDA) method that was validated according to the International Council for Harmonisation (ICH) guidelines. The developed analytical method exhibited linearity (r(2) > 0.999), detection limits (0.72-3.34 μg/mL), and quantification limits (2.19-10.22 μg/mL). The relative standard deviation (RSD) value of intra- and inter-day precisions was less than 1.68%, and analyte recoveries (93.42-117.55%; RSD < 1.86%) were validated according to the analytical procedures, and all parameters were within the allowable range. Quantitative analysis of the three marker components from W. ganpi MeOH extract (WGM) showed 7-methoxylutolin-5-O-glucoseide with the highest content (51.81 mg/g). The inhibitory effects of WGM on cytochrome P450 (CYP) substrate drugs were further investigated. The in vitro study revealed that WGM inhibited the CYP3A-mediated metabolism of buspirone and that 7-methoxylutolin-5-O-glucoseide and pilloin 5-O-β-d-glucopyranoside inhibited the metabolism of buspirone with IC(50) values of 2.73 and 18.7 μM, respectively. However, a single oral dose of WGM did not have significant effects on the pharmacokinetics of buspirone in rats, suggesting that WGM cannot function as an inhibitor of CYP3A-mediated metabolism in vivo.