Hypouricemic and Anti-oxidant Effects of Chrysanthemum indicum L. and Cornus o cinalis In Vitro and In Vivo Models


 Background: 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 & Zucc (Co) on hyperuricemia, both individually and in combination (FSU-CC), Methods: We used hypoxanthine-treated human liver cancer (HepG2) cells and primary mouse renal proximal tubule cells for in vitro model, and potassium oxonate-induced hyperuricemic mice for in vivo model.Results: We found that treatment of 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 and organic anion transporter 3 proteins and a decrease in the expression levels of glucose transporter 9 and urate transporter 1 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.Conclusions: 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.

kidneys and about 90% of the ltered load is usually reabsorbed in the nephrons [9]. Uric acid reabsorption occurs in the renal proximal tubules by transporters, such as glucose transporter 9 (GLUT9), urate anion transporter 1 (URAT1), and organic anion transporter 4 (OAT4), while uric acid excretion is facilitated by transporters, such as organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3). Maintaining the function of these uric acid transporters is important to prevent hyperuricemia [10,11].
In this study, we investigated the effects of Chrysanthemum indicum L. and Cornus o cinalis Siebold & Zucc on hyperuricemia in in vitro and in vivo models. C. indicum L., called Indian chrysanthemum, a member of the Compositae family and C. o cinalis Siebold & Zucc, a member of the Cornaceae family, have been reported to exhibit anti-in ammatory and anticancer effects and possess antioxidant properties [12][13][14]. The owers of C. indicum L. and fruits of C. o cinalis Siebold & Zucc are widely known for their health bene ts as traditional tea with accepted for use as food in China and Korea [15,16]. We compared the effects of treatment of extracts from C. indicum L. and C. o cinalis Siebold & Zucc, both individually and in combination, on hypoxanthine-treated human liver cancer cells, primary mouse renal proximal tubule cells, and potassium oxonate-induced hyperuricemic mice to develop agents for the prevention of hyperuricemia.

Materials And Methods
Extract preparation and HPLC Flowers of C. indicum L. were extracted using water for 8 h at 90°C. The extract was ltered with Whatman paper No. 6 and concentrated in a rotary evaporator under reduced pressure. The concentrate was lyophilized (Ci) and stored at -20°C until further use. Fruits of C. o cinalis Siebold & Zucc were extracted using water for 8 h at 90°C. The extract was ltered with Whatman paper No. 6 and concentrated in a rotary evaporator under reduced pressure. The extract was dried using hot air with dextrin (50%) (Co) and stored at -20°C until further use. Ci and Co were mixed in a ratio of 1:2 (FSH-CC) and stored at -20°C until further use. And then, we analyzed luteolin and loganin of Ci and Co, and FSH-CC by high-performance liquid chromatography (HPLC) using Agilent 1260 in nity II HPLC system (Santa Clara, CA, USA).
To obtain the primary mouse renal proximal tubule cells, kidney was isolated from Balb/c mice (22-25 g, 6 weeks, male). The kidney was minced using Hank's balanced salt solution (HBSS) containing trypsin, with the addition of 1 mg/mL deoxyribonuclease (DNAse) and 2 mg/mL collagenase Type I (Sigma-Aldrich, USA). After 30 min, the solution was passed through an 80-mesh and 1709-mesh sieve (Fisher Scienti c, Pittsburgh, PA) to remove the cell debris and glomeruli. Proximal tubule cells remained on the sieve lter and were collected by washing the sieve lter with HBSS. The proximal tubule cell suspension was centrifuged for 10 min at 1000 revolutions per minute (rpm) at 4°C and the cell pellet was collected.
HepG2 cells and primary mouse renal proximal tubule cells were cultured with Ci, Co, and FSH-CC for 24 h and treated with 4 mM hypoxanthine. After 2 h, assays were performed to measure the activity of xanthine oxidase, mRNA expression of xanthine dehydrogenase, and the expression levels of OAT1, OAT3, GLUT9, and URAT1 proteins.

Animals
The Institutional Animal Care and Use Committee of Kyung Hee University approved the protocol (KHGASP-20-410) for the use of animals in this study. The animals were cared for in accordance with the "Guidelines for Animal Experiments" established by the university.
Six-week-old male C57 black 6 (C57BL6) mice were purchased from SaeRon Bio (Uiwang, Korea) and housed in cages under automatically controlled temperature (22 ± 2°C), humidity (about 50%), and lighting (12:12-h light-dark cycle) conditions. The mice in the control group with normal diet were fed a commercial pelleted chow (AIN-93G rodent puri ed diet, Orient Bio, Korea) and water ad libitum. All the mice were randomly divided into eight groups of eight mice per group as follows: normal control (NC), control (C; hyperuricemia-induced mice), positive control (PC; hyperuricemia-induced mice with oral supplementation of allopurinol, xanthine oxidase inhibitor, 10 mg/kg body weight (b.w)), Ci 300 (hyperuricemia-induced mice with oral supplementation of Ci, 300 mg/kg b.w), Co 300 (hyperuricemiainduced mice with oral supplementation of Co, 300 mg/kg b.w), FSH-CC 150 (hyperuricemia-induced mice with oral supplementation of FSH-CC, 150 mg/kg b.w), FSH-CC 300 (hyperuricemia-induced mice with oral supplementation of FSH-CC, 300 mg/kg b.w), and FSH-CC 600 (hyperuricemia-induced mice with oral supplementation of FSH-CC, 600 mg/kg b.w). The extracted samples were orally administered for 21 d. To induce hyperuricemia, an intraperitoneal injection of 200 mg/kg b.w. potassium oxonate (Sigma-Aldrich Co, MO, USA) was given. After 2 h, the mice urine was collected and the mice were anesthetized with iso urane.

Levels of uric acid and creatinine in the urine and serum
Blood was centrifuged at 3000 rpm for 10 min and the serum was separated. The levels of uric acid in the urine and serum were determined using the uric acid assay kits (BioVision Inc., CA, USA), while the levels of creatinine in the urine and serum were determined using the creatinine assay kit (BioVision Inc., CA, USA).

Activity of xanthine oxidase
The activity of xanthine oxidase was determined from the HepG2 cells in the culture medium and serum from mice using the Xanthine Oxidase Activity Assay Kit (Sigma-Aldrich Co, MO, USA).

Antioxidant enzyme activity in the liver
The liver tissues were lysed using the CelLytic™ MT lysis reagent (Sigma) and the antioxidant enzyme activity was measured using the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) assay kits (Biomax Inc., Seoul). Malondialdehyde (MDA), a lipid peroxidation marker, was measured using MDA assay kits (BioVision Inc., Mountain View, CA, USA).

Statistical analysis
All data are presented as mean ± standard deviation (SD). The data were statistically evaluated using Duncan's multiple range tests after one-way analysis of variance (ANOVA) using SPSS statistical procedures (SPSS PASW Statistic v.23.0, SPSS Inc., Chicago, IL, USA). When the data were subjected to prior investigations before analysis, parametric assumptions including homoscedasticity and normality of observations were satis ed. Differences were considered to be statistically signi cant at p < 0.05 level.

Results
Luteolin and loganin of Ci and Co, and FSH-CC The HPLC analysis of the Ci and Co, and FSH-CC revealed three peaks matching those of the commercial standards luteolin (Fig. 1A) and loganin (Fig. 1B), The Ci contained 7.62 mg/g luteolin, Co contained 4.90 mg/g loganin, and FSH-CC contained 3.95 mg/g luteolin and 2.48 mg/g loganin.
The combination of Ci and Co suppressed the xanthine oxidase activity and xanthine dehydrogenase mRNA expression in liver cells more than their individual treatments We found that hypoxanthine treatment (C) increased the activity of xanthine oxidase and mRNA expression of xanthine dehydrogenase as compared with that in the normal control (NC). However, Ci and Co treatment revealed a signi cant decrease in the activity of xanthine oxidase and mRNA expression of xanthine dehydrogenase as compared with that in the control group. In addition, the combination of Ci and Co (FSU-CC) suppressed xanthine oxidase activity and xanthine dehydrogenase mRNA expression more than the individual treatment of either Ci or Co alone. Moreover, FSU-CC 300 treatment resulted in the most signi cant reduction of the xanthine oxidase activity and xanthine dehydrogenase mRNA expression among all the hypoxanthine-treated HepG2 cells (p < 0.05) (Fig. 2).
The combination of Ci and Co increased the expression levels of OAT1 and OAT3 and suppressed the expression levels of GLUT9 and URAT1 in renal proximal tubule cells more than their individual treatments We con rmed the uric acid excretion transporters, OAT1 and OAT3, as well as the uric acid reabsorption transporters, GLUT9 and URAT1, in the primary mouse renal proximal tubule cells. Hypoxanthine treatment in these cells induced a decrease in the expression levels of OAT1 and OAT3 compared with that in the normal control group, while Ci and Co treatment groups increased the expression levels of OAT1 and OAT3 compared with that in the hypoxanthine control group. Moreover, the combination of Ci and Co (FSU-CC) increased the expression levels of OAT1 and OAT3 more than the individual treatment of either Ci or Co alone (p < 0.05) (Fig. 3B,C).
Compared to the normal control group, hypoxanthine treatment induced an increase in the expression levels of GLUT9 and URAT1 in the primary mouse renal proximal tubule cells, while Ci and Co treatment groups signi cantly decreased the expression levels of GLUT9 and URAT1 compared with that in the hypoxanthine treatment control group. Moreover, the combination of Ci and Co (FSU-CC) decreased expression levels of GLUT9 and URAT1 more than the individual treatment of either Ci or Co alone (p < 0.05) (Fig. 3D,E).
The combination of Ci and Co increased the excretion of creatinine and uric acid in hyperuricemiainduced mice more than their individual treatments We investigated the effects of Ci and Co supplementation on hyperuricemia-induced mice and found that Ci and Co supplementation did not affect the change in the levels of serum ALT, AST, triglycerides, total cholesterol, HDL cholesterol, and LDL cholesterol in hyperuricemia-induced mice ( Table 1). We measured the levels of creatinine and uric acid in the serum and urine in hyperuricemia-induced mice to con rm whether Ci and Co supplementation affects the excretion of creatinine and uric acid in them. Compared to the normal control, hyperuricemia-induced mice showed a signi cant increase in the levels of creatinine and uric acid in the serum and a signi cant decrease in the levels of creatinine and uric acid in the urine. Ci 300 and Co 300 supplementation groups signi cantly decreased the levels of creatinine and uric acid in the serum and increased the levels of creatinine and uric acid in the urine as compared with that in the control group. FSU-CC 300 decreased the levels of creatinine and uric acid in the serum and increased the levels of creatinine and uric acid in the urine than the individual supplementation of either Ci 300 or Co 300 alone (p < 0.05) (Fig. 4).  The combination of Ci and Co increased the xanthine oxidase activity and xanthine dehydrogenase mRNA expression and inhibited the oxidative stress in hyperuricemia-induced mice more than their individual treatments We measured the xanthine oxidase activity, xanthine dehydrogenase mRNA expression, and oxidative stress in the liver of hyperuricemia-induced mice. The xanthine oxidase activity and xanthine dehydrogenase mRNA expression in the liver were signi cantly increased in the hyperuricemia-induced mice group compared with that in the normal control group. However, Ci 300 and Co 300 supplementation suppressed the xanthine oxidase activity and xanthine dehydrogenase mRNA expression in the liver of hyperuricemia-induced mice. FSU-CC 300 supplementation signi cantly decreased the xanthine oxidase activity and xanthine dehydrogenase mRNA expression in the liver of hyperuricemia-induced mice more than the individual supplementation of either Ci 300 or Co 300 alone (p < 0.05) (Fig. 5A,B).
We found that hyperuricemia-induced mice caused an increase in the MDA levels and a decrease in the activities of antioxidant enzymes, including SOD, CAT, and GPx compared with that in the normal control group. Ci 300, Co 300, and FSU-CC supplementation induced a decrease in the MDA levels and an increase in the antioxidant enzyme activities compared with that in the control group (p < 0.05) (Fig. 5C-F).
The combination of Ci and Co increased the expression levels of OAT1 and OAT3 and suppressed the expression levels of GLUT9 and URAT1 in hyperuricemia-induced mice than their individual treatments Hyperuricemia-induced mice showed a decrease in the expression levels of OAT1 and OAT3 in the kidney compared with that in the normal control group. Ci 300 and Co 300 supplementation increased the expression levels of OAT1 and OAT3 in the kidney of hyperuricemia-induced mice compared with that in the control group. In addition, FSU-CC 300 supplementation signi cantly increased the expression levels of OAT1 and OAT3 in the kidney more than the individual supplementation of either Ci 300 or Co 300 alone (p < 0.05) (Fig. 6B,C).
Hyperuricemia-induced mice induced an increase in the expression levels of GLUT9 and URAT1 in the kidney as compared to the normal control. Ci 300 and Co 300 supplementation signi cantly decreased the expression levels of GLUT9 and URAT1 in the kidney compared with that in the control group. Moreover, FSU-CC 300 decreased the expression levels of GLUT9 and URAT1 in the kidney of hyperuricemia-induced mice more than the individual supplementation of either Ci 300 or Co 300 alone (p < 0.05) (Fig. 6D,E).

Discussion
Recently, the prevalence of hyperuricemia-induced gout is increasing and the treatment for gout includes the use of non-steroidal anti-in ammatory drugs to relieve the symptoms of the illness and the use of allopurinol and xanthine oxidase inhibitors to reduce the production of uric acid [17]. However, these drugs have side effects, including gastrointestinal toxicity and bleeding, renal toxicity, and hypersensitivity reactions [18]. Therefore, alternative therapies have been explored in an attempt to treat and prevent hyperuricemia [19,20]. We compared the effects of Ci, Co, and a combination of Ci and Co (FSU-CC) on hyperuricemia-induced HepG2 cells, primary mouse renal proximal tubule cells, and potassium oxonate-induced hyperuricemic mice. The present study aimed to compare the effects of Ci, Co, and a combination and to develop agents for the prevention of hyperuricemia.
Xanthine oxidase, converted from xanthine dehydrogenase, acts as a key enzyme for the oxidation of hypoxanthine and xanthine during the production of uric acid in the liver. It is well known that the mitochondrial ROS are produced during the xanthine oxidase-mediated production of uric acid [7,8]. We found increase in the xanthine oxidase activity and xanthine dehydrogenase mRNA expression in hypoxanthine-treated HepG2 cells and the liver of potassium oxonate-induced hyperuricemic mice as compared to that in the normal cells and liver of healthy mice. In addition, the potassium oxonateinduced hyperuricemic mice caused oxidative stress in the liver by decreasing the antioxidant enzyme activities and increasing the MDA levels. However, treatment of Ci, Co, and FSU-CC suppressed the xanthine oxidase activity, xanthine dehydrogenase mRNA expression, and oxidative stress in hypoxanthine-treated HepG2 cells and the liver of potassium oxonate-induced hyperuricemic mice. We found that the combination of Ci and Co inhibited the activity of xanthine oxidase more than either of the two given separately.
The study of Nishida [21] has demonstrated the signi cant positive correlations between the excretion of creatinine and uric acid in urine. Thus, we measured the levels of creatinine and uric acid in the serum and urine to observe the excretion process of creatinine and uric acid. Supplementation of Ci, Co, and FSU-CC increased the excretion of creatinine and uric acid through urine, which was suppressed by potassium oxonate injection in mice. Moreover, the excretion of creatinine and uric acid in the combination of Ci and Co was higher than that of individual supplementation and was similar to that of the normal group. These results indicate that the combination of Ci and Co helps to treat hyperuricemia more than individual dietary supplements.
In order to elucidate the mechanisms of Ci, Co, and FSU-CC that mediate the excretion of uric acid in hyperuricemia, we observed the expression of the transporters involved in uric acid excretion in hypoxanthine-treated primary mouse renal proximal tubule cells and the kidney from potassium oxonateinduced hyperuricemic mice. Previous studies have identi ed GLUT9 and URAT1, involved in uric acid reabsorption, and OAT1 and OAT3, involved in uric acid excretion, as potential therapeutic targets for hyperuricemia [10,22,23]. We have shown in the present study that Ci, Co, and FSU-CC signi cantly increased the expression levels of OAT1 and OAT3, while decreasing the expression levels of GLUT9 and URAT1 in hypoxanthine-treated primary mouse renal proximal tubule cells and the kidney from potassium oxonate-induced hyperuricemic mice. Moreover, the combination of Ci and Co increased the expression levels of OAT1 and OAT3 and suppressed the expression levels of GLUT9 and URAT1 more than the individual treatments. Therefore, we hypothesize that FSU-CC aids in maintaining the function of these uric acid transporters to prevent hyperuricemia more than the individual treatment of either Ci or Co alone.
We showed that Ci contained luteolin, a common avonoid, and Co contained loganin, an iridoid glycoside. Matsuda et al. isolated new avanone glycosides and phenylbutanoid glycoside from the owers of C. indicum L. and found the inhibitory activity for rat lens aldose reductase [24]. Dong [25][26][27]. According to these reports and our present results, we can assume that phenolic compounds from Ci and Co can have hypouricemic effects on hyperuricemiainduced HepG2 cells, renal cells, and mice. However, further human clinical trials are needed to fully understand the effects of dietary supplementation on hyperuricemia, with both Ci and Co.

Conclusions
We compared the effects of extracts from Ci, Co, and FSU-CC on hyperuricemia using hypoxanthinetreated HepG2 cells, primary mouse renal proximal tubule cells, and potassium oxonate-induced hyperuricemic mice. We found that the FSU-CC treatment inhibited the production and excretion of uric acid more than the individual treatment of either Ci or Co alone in both in vitro and in vivo models. We con rmed that treatment with FSU-CC directly inhibited the production of uric acid in hepatocytes and increased the expression of uric acid transporters in renal cells, which are involved in the excretion process. This study provides scienti c evidence and describes the underlying mechanisms responsible for the anti-hyperuricemic effects of Ci and Co. Therefore, our data suggest that a combination of Ci and The datasets used during this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate
The Institutional Animal Care and Use Committee of Kyung Hee University approved the protocol (KHGASP-20-410) for the use of animals in this study. The animals were cared for in accordance with the "Guidelines for Animal Experiments" established by the university.

Consent for publication
Not applicable. Figure 1 High-performance liquid chromatography analysis of luteolin (A) and loganin (B) levels in Ci and Co, and FSH-CC