Phloroglucinol Derivatives from Dryopteris crassirhizoma as Potent Xanthine Oxidase Inhibitors

Abstract

Dryopteris crassirhizoma rhizomes are used as a traditional medicine in Asia. The EtOAc extract of these roots has shown potent xanthine oxidase (XO) inhibitory activity. However, the main phloroglucinols in D. crassirhizoma rhizomes have not been analyzed. Thus, we investigated the major constituents responsible for this effect. Bioassay-guided purification isolated four compounds: flavaspidic acid AP (1), flavaspidic acid AB (2), flavaspidic acid PB (3), and flavaspidic acid BB (4). Among these, 1 showed the most potent inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) value of 6.3 µM, similar to that of allopurinol (IC₅₀ = 5.7 µM) and better than that of oxypurinol (IC₅₀ = 43.1 µM), which are XO inhibitors. A comparative activity screen indicated that the acetyl group at C3 and C3' is crucial for XO inhibition. For example, 1 showed nearly 4-fold higher efficacy than 4 (IC₅₀ = 20.9 µM). Representative inhibitors (1-4) in the rhizomes of D. crassirhizoma showed reversible and noncompetitive inhibition toward XO. Furthermore, the potent inhibitors were shown to be present in high quantities in the rhizomes by a UPLC-QTOF-MS analysis. Therefore, the rhizomes of D. crassirhizoma could be used to develop nutraceuticals and medicines for the treatment of gout.

Keywords: Dryopteris crassirhizoma; flavaspidic acid; gout; phloroglucinols; traditional medicine; xanthine oxidase.

Figure 1

Chemical structures of isolated compounds 1–4 from rhizomes of Dryopteris crassirhizoma.

Figure 2

(a) Inhibitory effects of compounds 1−4 on the activity of xanthine oxidase (XO) based on the oxidation of xanthine to uric acid. (b) Catalytic activity of XO as a function of enzyme concentration at different concentrations of compound 1. (c) Lineweaver–Burk plots were constructed for the inhibition of XO by compound 1. The plot is expressed as 1/velocity versus 1/xanthine (S) with or without an inhibitor in the reaction solutions. (d) Dixon plots of XO inhibition by compound 1. The graphical symbols are substrate concentrations (50 μM, ●; 100 μM, ○; 200 μM, ▼).

Figure 3

Representative chromatograms of EtOAc rhizome extract of Dryopteris crassirhizoma. (a) Diode array detector (DAD) chromatogram and (b) total ion current-base peak intensity (TIC-BPI) chromatogram.

Figure 4

Negative ion mass spectrum acquired by UPLC-ESI/MS and MS/MS analysis of phloroglucinols (a–d, 1–4, respectively) from rhizomes of Dryopteris crassirhizoma. Abbreviations: RF, residue of a fragment.