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

Abstract

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 (r² > 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 IC₅₀ 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.

Keywords: 7-methoxylutolin-5-O-glucoseide; HPLC-PDA; Wikstroemia ganpi; herb–drug interaction; pharmacokinetics; pilloin 5-O-β-d-glucopyranoside; rutarensin; validation.

Figure 1

Chemical structures of the three compounds isolated from WGM.

Figure 2

HPLC chromatogram (λ = 340 nm) of WGM (a) and reference mixture (b): 7-methoxyluteolin-5-O-glucoside (1), pilloin 5-O-β-d-glucopyranoside (2), and rutarensin (3).

Figure 2

HPLC chromatogram (λ = 340 nm) of WGM (a) and reference mixture (b): 7-methoxyluteolin-5-O-glucoside (1), pilloin 5-O-β-d-glucopyranoside (2), and rutarensin (3).

Figure 3

PDA spectra of WGM (a) and reference solutions (b): 7-Methoxylutolin-5-O-glucoside (1), pilloin 5-O-β-d-glucopyranoside (2), and rutarensin (3).

Figure 4

Effects of WGM on the disappearance rate of model cytochrome P450 substrate in rat liver S9 fraction (a). Dose–response curves for the inhibitory effect of 7-methoxylutolin-5-O-glucoside (b) pilloin 5-O-β-d-glucopyranoside (c), and rutarensin (d) on the disappearance of BUS in rat liver S9 fraction (n = 5). The asterisk represents a value significantly different from that of the other groups (p < 0.05).

Figure 4

Effects of WGM on the disappearance rate of model cytochrome P450 substrate in rat liver S9 fraction (a). Dose–response curves for the inhibitory effect of 7-methoxylutolin-5-O-glucoside (b) pilloin 5-O-β-d-glucopyranoside (c), and rutarensin (d) on the disappearance of BUS in rat liver S9 fraction (n = 5). The asterisk represents a value significantly different from that of the other groups (p < 0.05).

Figure 5

The mean plasma concentration-time curve of BUS after oral administration of 30 mg/kg in rats (green circles) or following co-administration with 1 g/kg WGM (red circles). Symbols show means and error bars represent SD (n = 6).