Ethanol Extract of Cudrania tricuspidata Leaf Ameliorates Hyperuricemia in Mice via Inhibition of Hepatic and Serum Xanthine Oxidase Activity

Abstract

Cudrania tricuspidata Bureau (Moraceae) (CT) is a dietary and medicinal plant distributed widely in Northeast Asia. There have been no studies on the effect of CT and/or its active constituents on in vivo xanthine oxidase (XO) activity, hyperuricemia, and gout. The aim of this study was to investigate XO inhibitory and antihyperuricemic effects of the ethanol extract of CT leaf (CTLE) and its active constituents in vitro and in vivo. Gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) analyses were used to determine a chemical profile of CTLE. XO inhibitory and antihyperuricemic effects of CTLE given orally (30 and 100 mg/kg per day for 1 week) were examined in potassium oxonate-induced hyperuricemic ICR mice. CTLE exhibited XO inhibitory activity in vitro with an IC₅₀ of 368.2 μg/mL, significantly reduced serum uric acid levels by approximately 2-fold (7.9 nM in normal mice; 3.8 nM in 30 mg/kg CTLE; 3.9 nM in 100 mg/kg CTLE), and significantly alleviated hyperuricemia by reducing hepatic (by 39.1 and 41.8% in 30 and 100 mg/kg, respectively) and serum XO activity (by 30.7 and 50.1% in 30 and 100 mg/kg, respectively) in hyperuricemic mice. Moreover, several XO inhibitory and/or antihyperuricemic phytochemicals, such as stigmasterol, β-sitosterol, vitamin E, rutin, and kaempferol, were identified from CTLE. Compared with rutin, kaempferol showed markedly higher XO inhibitory activity in vitro. Our present results demonstrate that CTLE may offer a promising alternative to allopurinol for the treatment of hyperuricemia and gout.

Figure 1

In vitro xanthine oxidase (XO) inhibitory activity of CTLE at concentrations between 0 and 2000 μg/mL. The rectangular bars and their error bars represent the means and standard deviations, respectively (n = 5). The asterisks indicate values that are significantly different from those of the control (0 μg/mL) group (p < 0.05).

Figure 2

Serum uric acid levels after the oral administration of saline in normal mice (NOR) and after the oral administration of saline (HU), allopurinol at a dose of 10 mg/kg (HU+ALP), or CTLE at doses of 30 mg/kg (HU+CTLE30) and 100 mg/kg (HU+CTLE100) for 7 days prior to the induction of hyperuricemia in mice. The rectangular bars and their error bars represent the means and standard deviations, respectively (n = 5). The asterisks and pound sign indicate values that are significantly different from those of the HU and NOR group, respectively (p < 0.05).

Figure 3

Relative activity of hepatic (a) and serum (b) xanthine oxidase (XO) after the oral administration of saline in normal mice (NOR) and after the oral administration of saline (HU), allopurinol at a dose of 10 mg/kg (HU+ALP), or CTLE at doses of 30 mg/kg (HU+CTLE30) and 100 mg/kg (HU+CTLE100) in hyperuricemic mice for 7 days. The rectangular bars and their error bars represent the means and standard deviations, respectively (n = 5). The asterisks indicate values that are significantly different from those of the HU group (p < 0.05).

Figure 4

Representative GC-MS chromatogram of CTLE.

Figure 5

Representative chromatographic profiles showing the bioactive constituents of CTLE. (a) Elution profile of CTLE using prep-LC; (b) elution profile of purified rutin (RUT) using prep-LC; (c) elution profile of purified kaempferol (KAE) using prep-LC; (d) HPLC chromatogram of CTLE.

Figure 6

Xanthine oxidase (XO) inhibitory activity of allopurinol (ALP) and bioactive phytochemicals identified from CTLE (kaempferol, KAE; rutin, RUT). The bullet symbols and their error bars represent the means and standard deviations, respectively (n = 5). ALP was tested at a concentration of 136 μg/mL, and KAE and RUT were tested at concentrations between 0 and 1000 μg/mL.