We investigated whether P. cuspidatum's metabolites exhibited BNA inhibition. Consistent with our hypothesis, we found several inhibitors from the methanol extract of this plant, and then fully characterized their inhibitory mechanisms.
Activity guided separation of methanol extract led to isolation of individual constituents, and subsequently their structures were elucidated by spectroscopic analysis. Detailed kinetic behaviors of BNA inhibitors were explored by showing the changes of Km and Vmax, the ratios of KI/KIS and Kik/Kiv, and fluorescence quenching effect.
This study attempted to isolate the responsible metabolites and elucidate the BNA inhibitory mechanism. The principal BNA inhibitory compounds (2–6) were identified as emodin (2), physcion-8-O-β-D-glucopyranoside (3), emodin-8-O-β-D-glucopyranoside (4), emodin-1-O-β-D-glucopyranoside (5), and 2-methoxy-6-acetyl-7-methyljuglone (6). Unexpectedly, anthraquinone glucosides (3–5) were much more potent than their corresponding aglycones (1 and 2). For example, emodin (2) had an IC50=5.4 μM, whereas its glucosides (4 and 5) had IC50=0.85 μM and 0.43 μM respectively. A similar trend was observed with physcion (1, IC50>200 μM) and its glucoside (3, IC50=6.2 μM). The anthraquinone (2) was mixed type I inhibitor, whereas its glucosides (4 and 5) were noncompetitive. In addition, the fluorescence quenching study showed that the affinity constants (KSV) of inhibitors increased in proportion to their inhibitory potencies. Furthermore, we quantified the major and minor metabolites through UPLC-PDA-Q-TOF/MS, and revealed that the most potent inhibitors were the major constituents. This result contributes to our understanding of P. cuspidatum utility as functional food stuff and widely used herbal medicine.