Hypouricemic Effects of Chrysanthemum indicum L. and Cornus officinalis on Hyperuricemia-Induced HepG2 Cells, Renal Cells, and Mice

Abstract

Hyperuricemia, abnormally excess accumulation of uric acid, is caused by an imbalance between the production and excretion of uric acid and is a major cause of gout. We compared the effects of extracts from Chrysanthemum indicum L. (Ci) and Cornus officinalis Siebold and Zucc. (Co) on hyperuricemia, both individually and in combination (FSU-CC), using hypoxanthine-treated human liver cancer (HepG2) cells, primary mouse renal proximal tubule cells, and potassium oxonate induced hyperuricemic mice. The Ci contained 7.62 mg/g luteolin and 0 mg/g loganin, Co contained 0 mg/g luteolin and 4.90 mg/g loganin, and FSH-CC contained 3.95 mg/g luteolin and 2.48 mg/g loganin. We found that treatment with Ci, Co, and FSU-CC suppressed the activity of xanthine oxidase and mRNA expression of xanthine dehydrogenase while inducing an increase in the expression levels of the organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) proteins and a decrease in the expression levels of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) proteins. Particularly, treatment and supplementation with FSU-CC showed stronger effects than those of supplementation with either Ci or Co alone. We observed that the excretion of creatinine and uric acid in the combination of Ci and Co was higher than that observed in their individual supplementations and was similar to that of the normal group. Therefore, our data suggest that a combination of Ci and Co may potentially be used for the development of effective natural anti-hyperuricemic functional foods.

Keywords: Chrysanthemum indicum L.; Cornus officinalis Siebold and Zucc.; hyperuricemia; uric acid.

Figure 1

High-performance liquid chromatography analysis of luteolin (A) and loganin (B) levels in Ci, Co, and FSH-CC.

Figure 2

Xanthine oxidase activity (A) and xanthine dehydrogenase mRNA expression (B) in hypoxanthine-treated human liver cancer (HepG2) cells, with and without Ci, Co, or FSU-CC. NC: normal control, C: 4 mM hypoxanthine, PC: 4 mM hypoxanthine + 100 μM allopurinol, Ci 150: 4 mM hypoxanthine + 150 μg/mL Chrysanthemum indicum L., Ci 300: 4 mM hypoxanthine + 300 μg/mL C. indicum L., Co 150: 4 mM hypoxanthine + 150 μg/mL Cornus officinalis Siebold and Zucc., Co 300: 4 mM hypoxanthine + 300 μg/mL C. officinalis Siebold and Zucc., FSU-CC 150: 4 mM hypoxanthine + 150 μg/mL mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2), FSU-CC 300: 4 mM hypoxanthine + 300 μg/mL mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2). Different letters indicate significant difference at p < 0.05, as determined by Duncan’s multiple range test.

Figure 3

Expression levels of proteins by Western blotting (band images (A), relative protein expression (B–E)) in the hypoxanthine-treated primary mouse renal proximal tubule cells, with and without Ci, Co, or FSU-CC. NC: normal control, C: 4 mM hypoxanthine, PC: 4 mM hypoxanthine + 100 μM allopurinol, Ci 150: 4 mM hypoxanthine + 150 μg/mL C. indicum L., Ci 300: 4 mM hypoxanthine + 300 μg/mL C. indicum L., Co 150: 4 mM hypoxanthine + 150 μg/mL C. officinalis Siebold and Zucc., Co 300: 4 mM hypoxanthine + 300 μg/mL C. officinalis Siebold and Zucc., FSU-CC 150: 4 mM hypoxanthine + 150 μg/mL mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2), FSU-CC 300: 4 mM hypoxanthine + 300 μg/mL mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2). Different letters indicate significant difference at p < 0.05, as determined by Duncan’s multiple range test.

Figure 4

Levels of creatinine in serum (A) and urine (B) and uric acid in serum (C) and urine (D) in the hyperuricemia-induced mice supplemented with and without Ci, Co, or FSU-CC. NC: normal control, C: hyperuricemia-induced mice, PC: hyperuricemia-induced mice with oral supplementation of allopurinol 10 mg/kg b.w., Ci 300: hyperuricemia-induced mice with oral supplementation of C. indicum L. 300 mg/kg b.w., Co 300: hyperuricemia-induced mice with oral supplementation of C. officinalis Siebold and Zucc. 300 mg/kg b.w., FSU-CC 150: hyperuricemia-induced mice with oral supplementation of mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2) 150 mg/kg b.w., FSU-CC 300: hyperuricemia-induced mice with oral supplementation of mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2) 300 mg/kg b.w., FSU-CC 600: hyperuricemia-induced mice with oral supplementation of mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2) 600 mg/kg b.w. Different letters indicate significant difference at p < 0.05, as determined by Duncan’s multiple range test.

Figure 5

Xanthine oxidase activity (A); xanthine dehydrogenase mRNA expression (B); malondialdehyde (MDA) levels (C); and activities of superoxide dismutase (SOD) (D), catalase (CAT) (E), and glutathione peroxidase (GPx) (F) in the liver from hyperuricemia-induced mice supplemented with and without Ci, Co, or FSU-CC. NC: normal control, C: hyperuricemia-induced mice, PC: hyperuricemia-induced mice with oral supplementation of allopurinol 10 mg/kg b.w., Ci 300: hyperuricemia-induced mice with oral supplementation of C. indicum L. 300 mg/kg b.w., Co 300: hyperuricemia-induced mice with oral supplementation of C. officinalis Siebold and Zucc. 300 mg/kg b.w., FSU-CC 150: hyperuricemia-induced mice with oral supplementation of mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2) 150 mg/kg b.w., FSU-CC 300: hyperuricemia-induced mice with oral supplementation of mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2) 300 mg/kg b.w., FSU-CC 600: hyperuricemia-induced mice with oral supplementation of mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2) 600 mg/kg b.w. Different letters indicate significant difference at p < 0.05, as determined by Duncan’s multiple range test.

Figure 6

Expression levels of proteins by Western blotting (band images (A), relative protein expression (B–E)) in the kidney of hyperuricemia-induced mice supplemented with and without Ci, Co, or FSU-CC. NC: normal control, C: hyperuricemia-induced mice, PC: hyperuricemia-induced mice with oral supplementation of allopurinol 10 mg/kg b.w., Ci 300: hyperuricemia-induced mice with oral supplementation of C. indicum L. 300 mg/kg b.w., Co 300: hyperuricemia-induced mice with oral supplementation of C. officinalis Siebold and Zucc. 300 mg/kg b.w., FSU-CC 150: hyperuricemia-induced mice with oral supplementation of mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2) 150 mg/kg b.w., FSU-CC 300: hyperuricemia-induced mice with oral supplementation of mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2) 300 mg/kg b.w., FSU-CC 600: hyperuricemia-induced mice with oral supplementation of mixture of C. indicum L. and C. officinalis Siebold and Zucc. (1:2) 600 mg/kg b.w. Different letters indicate significant difference at p < 0.05, as determined by Duncan’s multiple range test.

Figure 7

Schematic representation of the effect of Ci, Co, or FSU-CC on hyperuricemia. Ci, Co, or FSU-CC inhibited the production of uric acid in the liver and excretion of uric acid in the renal cells.