Anti-hyperuricemic effect of isorhamnetin in cultured hepatocytes and model mice: structure-activity relationships of methylquercetins as inhibitors of uric acid production

Abstract

Hyperuricemia is an important risk factor for gout. Isorhamnetin (3'-O-methylquercetin) is an O-methylated flavonol, which occurs in onion, almond and sea buckthorn. It is also one of the metabolites of quercetin in mammals. In the present study, we investigated anti-hyperuricemic effect of isorhamnetin adopting both cultured hepatocytes and mice with hyperuricemia induced by purine bodies. In cultured hepatocytes, isorhamnetin as well as quercetin significantly and dose-dependently inhibited uric acid (UA) production. We also examined the inhibitory effects on UA production of other mono-methylquercetins, i.e., tamarixetin, 3-O-methylquercetin, azaleatin, and rhamnetin in addition to isorhamnetin for studying their structure-activity relationships. From the results obtained, hydroxyl groups at C-3, C-5, and especially C-7, but not C-3' and C-4' of quercetin are demonstrated to play a critical role in suppressing UA production in the AML12 hepatocytes. Oral administration of isorhamnetin significantly reduced plasma and hepatic UA levels in the hyperuricemic model mice. Isorhamnetin also decreased hepatic xanthine oxidase (XO) activity without changes in XO protein expression, indicating that anti-hyperuricemic effect of isorhamnetin could be, at least partly, attributable to suppression of UA production by directly inhibiting XO activity in the liver. These findings demonstrate that isorhamnetin has a potent anti-hyperuricemic effect and may be a potential candidate for prevention and remediation of hyperuricemia.

Keywords: AML12 hepatocyte; Hyperuricemia; Isorhamnetin; Uric acid.

Fig. 1

Chemical structures of quercetin and mono-methylated quercetin isorhamnetin, tamarixetin, 3-O-methylquercetin, azaleatin and rhamnetin (a). Effects of quercetin and methylquercetin on cell viability in AML12 cells (b). The cells were incubated in BSS with or without various concentrations of quercetin and methylquercetin (0, 3, 10, 30 µM) for 2 h. WST-8 reagent was added 1 h before measurement of OD 450 nm. Data are expressed as a percentage of the vehicle control (DMSO). Each value represents mean ± SEM for six wells

Fig. 2

Effects of quercetin and mono-methylated quercetin on UA production in AML12 cells. AML hepatocytes were treated with 0, 3, 10 and 30 µM querctin (a), isorhamnetin (b), tamrixetin (c), 3-O-methylquerctin (d), azaleatin (e) and rhamnetin (f) for 2 h in BSS containing guanosine + inosine (100 µM each). Each value represents mean ± SEM for six wells (duplicate measurement per well). Values not sharing a common letter are significantly different at P < 0.05 (Tukey’s test)

Fig. 3

Comparison of effects of quercetin and mono-methylquercetin at the same concentration on UA production in AML12 hepatocytes. AML cells were treated with querctin, isorhamnetin, tamrixetin, 3-O-mrthylquerctin, azaleatin and rhamnetin at 3 (a), 10 (b) and 30 (c) µM for 2 h in BSS containing guanosine + inosine (100 µM each). DMSO alone was used as control. Each value represents mean ± SEM for six wells (duplicate measurement per well). Values not sharing a common letter are significantly different at P < 0.05 (Tukey’s test)

Fig. 4

Effects of quercetin and isorhamnetin on plasma UA levels in hyperuricemic model mice. The mice were perorally administered with quercetin (a) or isorhamnetin (b) at the different doses indicated. The mice were then intraperitoneally injected with both IMP and GMP (300 mg/kg body weight) to induce hyperuricemia. Normal control and model control groups were treated with 0.5% CMC-Na instead of test samples. Normal group was injected with PBS (–) instead of nucleotides. Each value represents mean ± SEM for 8–12 mice (duplicate measurement per mouse). For statistical significance, *P < 0.05 and **P < 0.01 when the treated groups were compared with the model control group (Dunnett’s test)

Fig. 5

Effects of quercetin and isorahmnetin on hepatic uric acid levels and xanthine oxidase (XO) activity in hyperuricemic mice. a and b Indicate liver uric acid levels in the mice treated with quercetin and isorhamentin, respectively. c and d Show liver XO activity in the mice treated with quercetin and isorhamentin, respectively. Each value represents mean ± SEM for 8–12 mice (duplicate measurement per mouse). For statistical significance, *P < 0.05 and **P < 0.01 when the treated groups were compared with the model control group (Dunnett’s test)

Fig. 6

Effects of quercetin and isorhamnetin on liver xanthine oxidase (XO) protein expression in hyperuricemic mice by Western blot analysis. a and b Indicate liver XO protein levels in the mice treated with quercetin and isorhamentin, respectively. Each value represents mean ± SEM for 4 mice