DKB114, A Mixture of Chrysanthemum Indicum Linne Flower and Cinnamomum Cassia (L.) J. Presl Bark Extracts, Improves Hyperuricemia through Inhibition of Xanthine Oxidase Activity and Increasing Urine Excretion

Chrysanthemum indicum Linne flower (CF) and Cinnamomum cassia (L.) J. Presl bark (CB) extracts have been used as the main ingredients in several prescriptions to treat the hyperuricemia and gout in traditional medicine. In the present study, we investigated the antihyperuricemic effects of DKB114, a CF, and CB mixture, and the underlying mechanisms in vitro and in vivo. DKB114 markedly reduced serum uric acid levels in normal rats and rats with PO-induced hyperuricemia, while increasing renal uric acid excretion. Furthermore, it inhibited the activity of xanthine oxidase (XOD) in vitro and in the liver in addition to reducing hepatic uric acid production. DKB114 decreased cellular uric acid uptake in oocytes and HEK293 cells expressing human urate transporter (hURAT)1 and decreased the protein expression levels of urate transporters, URAT1, and glucose transporter, GLUT9, associated with the reabsorption of uric acid in the kidney. DKB114 exerts antihyperuricemic effects and uricosuric effects, which are accompanied, partially, by a reduction in the production of uric acid and promotion of uric acid excretion via the inhibition of XOD activity and reabsorption of uric acid. Therefore, it may have potential as a treatment for hyperuricemia and gout.


Introduction
Hyperuricemia is characterized by elevated blood uric acid levels [1], which cause accumulation of urate crystals in joints and the kidney, leading to gout and gouty arthritis [2,3]. The prevalence of hyperuricemia and gout is increasing worldwide, and uric acid is a risk factor of fatty liver, insulin resistance, hypertension, and cardiovascular diseases [4]. Therefore, there is a growing interest in hyperuricemia and uric acid regulation. Hyperuricemia is caused by increased production or impaired uric acid excretion or a combination of these two mechanisms [5,6]. Uric acid is produced by the activities of xanthine oxidase (XOD), a key enzyme that converts hypoxanthine and xanthine, which is then converted to uric acid in purine metabolism [7]. The elimination of uric acid occurs via complex urate transporters that regulate its reabsorption and secretion, such as apical urate/anion exchanger, URAT1, and basolateral glucose transporter 9 (GLUT9) in the kidney [8]. Accordingly, reducing uric acid production and increasing uric acid excretion may be useful therapeutic approach for hyperuricemia treatment. Currently, XOD inhibitors such as indicum flower and C. cassia bark mixture, DKB114 was selected and prepared by mixing previously prepared C. indicum flower and C. cassia bark extracts at a weight ratio of 1:2 based on our previous report [22].

Animals
Sprague-Dawley rats (male, age: 7 weeks) were purchased from Orient Bio (Seongnam, Korea) and maintained in a room under the following conditions: A temperature of 22 ± 1 • C, humidity of 50 ± 10%, and a 12-h light/dark cycle. The rats were allowed free access to diet (AIN-76A, Research Diet, New Brunswick, NJ, USA) and water. The experimental method was approved by the Committee on Animal Care of Korea Institute of Oriental Medicine, and all experiments were performed in accordance with the committee guidelines (Approval No.17-067)

Collection of Serum, Urine, and Tissues
Urine was collected using metabolic cages at 1, 2, 3, and 4.5 h after PO injection and sample administration on the first day. Collected urine samples were and centrifuged (3000× g, 10 min, 4 • C) to remove particulate contaminants. The supernatants were stored at −80 • C until analysis. Blood was collected via cardiac puncture under anesthesia 2 h after PO injection and DKB114 treatment on the second day. Serum was obtained via centrifugation (3000× g, 10 min, 4 • C), and the separated serum was stored at −80 • C until analysis. After blood collection, liver, and kindey tissues were dissected immediately, rinsed, weighed, frozen in liquid nitrogen, and stored at −80 • C until analysis.

Analysis of Uric Acid Levels in Serum, Urine, and Liver Tissues
To analyze hepatic uric acid levels, liver tissues were homogenized in 80 mM potassium phosphate buffer (pH 7.4) and the homogenate was centrifuged (10,000× g, 10 min, 4 • C). The resulting supernatant was used to determine the uric acid concentration. Serum, urine, and liver uric acid levels were measured by using commercial assay kits (Biovision, Milpitas, CA, USA) according to manufacturer's protocols.

In Vitro and In Vivo XOD Inhibition Assay
For the analysis of the in vitro XOD activity, the reaction mixture comprising 50 mM sodium phosphate buffer (pH 7.6), 17.9 nM xanthine sodium salt, 0.04 units of xanthine oxidase, and DKB114 at various concentrations was analyzed for decreased uric acid production at 295 nm. AP served as a positive control for XOD activity analysis. All experiments were performed in triplicate. Liver XOD activity was measured using commercial assay kits (Sigma, Saint Louis, MO, USA) according to the manufacturer's protocols. Protein concentrations were measured by bicinchoninic acid protein assays using bovine serum albumin (BSA) as the standard to normalize XOD activity. XOD activity was presented as nanomoles of uric acid formed per minute per milligram of protein.

Western Blotting Analysis of Kidney Samples
Proteins from kidney tissues were extracted using RIPA buffer (ATTO Corporation, Tokyo, Japan), and protein concentrations were determined by BCA protein assays (Thermo Fisher Scientific, Waltham, MA, USA) using BSA as a standard. Proteins were separated by 10% SDS polyacrylamide gel electrophoresis and were electroblotted onto a nitrocellulose membrane (Amersham, Piscataway, NJ, USA). The membranes were blocked with EzBlockChemi blocking solution (ATTO Corporation, Tokyo, Japan) and subsequently incubated with anti-URAT1 (MyBioSource.com, San Diego, CA, USA); organic anion transporter (OAT)1, GLUT9, OAT3, and GAPDH primary antibodies and a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunocomplexes were developed by enhanced chemiluminescence (Amersham, Buckinghamshire, UK) and exposed to ImageQuant LAS 4000 system software (GE Healthcare, Chicago, IL, USA). The images obtained were subjected to a densitometric analysis using Image J1.49 software (http://rsb.info.nih.gov/ij/download.html; National Institute of Health, National Institute of Health, Bethesda, MD, USA). The URAT1, GLUT9, OAT1, and OAT3 protein expression levels were presented relative to those in the PO group after normalization to the GAPDH protein levels.

Establishment of hURAT1-Expressing Oocytes and HEK293 Cells
To establish hURAT1-expressing oocytes, a pcDNA3.1 (+) expression vector containing the cDNA of hURAT1 (GenBank accession number AB071863) was linearized using restriction enzymes NheI and XbaI and then transcribed into complementary RNA (cRNA). cRNA was microinjected into Xenopus laevis oocytes. The hURAT1-expressing oocytes were used for a uric acid uptake experiment at 2 days after microinjection. To establish hURAT1-expressing HEK293 cells, the cDNA of hURAT1 (GenBank accession number AB071863) from the human kidney was subcloned into pcDNA 3.1 (+) (Invitrogen, Carlsbad, CA, USA) using restriction enzymes NheI and XbaI. HEK293-URAT1 cells were obtained by transient transfection of HEK293 cells with a hURAT1 expression vector by using Lipofectamine 2000 according to the manufacturer's protocols (Invitrogen, Carlsbad, CA, USA). An empty pcDNA 3.1 (+) vector was transfected into the HEK293 cells as a control. The hURAT1-expressing HEK293 cells were used for a uric acid uptake experiment at 1 day after transfection.

Statistical Analysis
Data are expressed as the mean ± SEM. Differences among the treatment groups were analyzed by one-way ANOVA, and a Dunnett's multiple comparison test was applied to identify the significance Nutrients 2018, 10, 1381 5 of 12 using Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA); and p < 0.05 was considered statistically significant.

Effects of DKB114 on Serum and Urinary Uric Acid Levels in Normal Rats and Rats with PO-Induced Hyperuricemia
As shown in Figure 1A, 150 and 200 mg/kg DKB114 significantly decreased serum uric acid levels by 16.8% and 20.4%, respectively (both, p < 0.05) and 10 mg/kg AP also decreased serum uric acid levels by 32.9% (p < 0.005) compared to those in the NC group. Serum uric acid levels were significantly higher in the PO group than in the NC group (p < 0.005). On the other hand, DKB114 at doses of 100, 150, and 200 mg/kg significantly reduced serum uric acid levels by 26.2%, 23.5%, and 38.3%, respectively (p < 0.005, p < 0.01, and p < 0.005, respectively), and AP decreased serum uric acid levels by 66.6% compared to those in the PO group ( Figure 1B). Furthermore, 200 mg/kg DKB114 markedly reduced serum uric acid levels by 18% (p < 0.05, p < 0.05, and p < 0.01, respectively) and 50 mg/kg Ben, the positive control, decreased serum uric acid levels by 18% compared to those in the PO group in the experiment for uric acid excretion-promoting effect ( Figure 1C). At 4.5 h after PO injection, urinary uric acid levels decreased in the PO group compared to those in the NC group (p < 0.001). In contrast, 200 mg/kg DKB114 significantly increased urinary uric acid levels by 1.8-fold (p < 0.05) and 2.1-fold (p < 0.005) at 3 and 4.5 h, respectively, after PO injection. Ben (50 mg/kg) also markedly increased urinary uric acid levels by 2.4-fold (p < 0.005), 2.8-fold (p < 0.005), and 2.9-fold (p < 0.005), respectively, at 2, 3, and 4.5 h after PO injection ( Figure 1D).

Statistical Analysis
Data are expressed as the mean ± SEM. Differences among the treatment groups were analyzed by one-way ANOVA, and a Dunnett's multiple comparison test was applied to identify the significance using Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA); and p < 0.05 was considered statistically significant.

Effects of DKB114 on XOD Inhibition Activity in Vitro/Vivo and Liver Uric Acid Levels
The effects of the DKB114 on in vitro and in vivo XOD inhibition activity are shown in Figure 2. In the in vitro XOD inhibition assay, 31.25, 62.5, 125, 250, and 500 µg/mL DKB114 inhibited XOD activity by 17.6%, 32.1%, 58.8%, 72.0%, and 131.8%, respectively, and the IC 50 values for DKB114 were 104.4 µg/mL (Figure 2A). The in vivo liver XOD activity in the PO group increased compared to that in the NC group (p < 0.05). However, the 200 mg/kg DKB114 and 10 mg/kg AP groups showed markedly reduced XOD activity and inhibited it by 66.7% and 95.6%, respectively (p < 0.01 and p < 0.005, respectively, compared to that in the PO group ( Figure 2B). On the other hand, 200 mg/kg DKB114 was shown to remarkably lower liver uric acid levels compared to those in the PO group (p < 0.05). AP (10 mg/kg) also significantly decreased liver uric acid levels (p < 0.05) ( Figure 2C).

Effects of DKB114 on XOD Inhibition Activity in Vitro/Vivo and Liver Uric Acid Levels
The effects of the DKB114 on in vitro and in vivo XOD inhibition activity are shown in Figure 2. In the in vitro XOD inhibition assay, 31.25, 62.5, 125, 250, and 500 μg/mL DKB114 inhibited XOD activity by 17.6%, 32.1%, 58.8%, 72.0%, and 131.8%, respectively, and the IC50 values for DKB114 were 104.4 μg/mL (Figure 2A). The in vivo liver XOD activity in the PO group increased compared to that in the NC group (p < 0.05). However, the 200 mg/kg DKB114 and 10 mg/kg AP groups showed markedly reduced XOD activity and inhibited it by 66.7% and 95.6%, respectively (p < 0.01 and p < 0.005, respectively, compared to that in the PO group ( Figure 2B). On the other hand, 200 mg/kg DKB114 was shown to remarkably lower liver uric acid levels compared to those in the PO group (p < 0.05). AP (10 mg/kg) also significantly decreased liver uric acid levels (p < 0.05) ( Figure 2C).  4) and representative of three independent experiments in vitro XOD activity and in vivo data are expressed as the mean ± SEM (n = 6). # p < 0.05 vs. the NC group; * p < 0.05, ** p < 0.01, and *** p < 0.005 vs. the PO group.

Effects of DKB114 on the Expression of Uric Acid Transporters in Rats with PO-Induced Hyperuricemia
URAT1 and GLUT9 protein expression levels were higher in the PO group than in the NC group (p < 0.05), but the expression levels were significantly suppressed in the 200 mg/kg DKB114 group and the 50 mg/kg Ben group compared to that in the PO group (all, p < 0.05) ( Figure 4A-C). OAT1 protein expression levels did not alter among the groups ( Figure 3A,D). OAT3 protein expression levels were lower in the PO group than in the NC group (p < 0.05), but did not alter among the PO, DKB114, and Ben groups ( Figure 4A,E).

Effects of DKB114 on the Expression of Uric Acid Transporters in Rats with PO-Induced Hyperuricemia
URAT1 and GLUT9 protein expression levels were higher in the PO group than in the NC group (p < 0.05), but the expression levels were significantly suppressed in the 200 mg/kg DKB114 group and the 50 mg/kg Ben group compared to that in the PO group (all, p < 0.05) ( Figure 4A-C). OAT1 protein expression levels did not alter among the groups ( Figure 3A,D). OAT3 protein expression levels were lower in the PO group than in the NC group (p < 0.05), but did not alter among the PO, DKB114, and Ben groups ( Figure 4A,E).

Effects of DKB114 on Mitochondrial Toxicity in HepG2 Cells
It is known that Ben has hepatic toxicity, and mitochondrial toxicity has been proposed as the underlying mechanism of hepatic toxicity. To assess the mitochondrial toxicity of DKB114 treatment, we determined the MMP in HepG2 cells. As shown in Figure 5A, DKB114 treatment did not change the MMP. Additionally, DKB114 did not affect cell viability ( Figure 5B).

Effects of DKB114 on Mitochondrial Toxicity in HepG2 Cells
It is known that Ben has hepatic toxicity, and mitochondrial toxicity has been proposed as the underlying mechanism of hepatic toxicity. To assess the mitochondrial toxicity of DKB114 treatment, we determined the MMP in HepG2 cells. As shown in Figure 5A, DKB114 treatment did not change the MMP. Additionally, DKB114 did not affect cell viability ( Figure 5B).

Effects of DKB114 on Mitochondrial Toxicity in HepG2 Cells
It is known that Ben has hepatic toxicity, and mitochondrial toxicity has been proposed as the underlying mechanism of hepatic toxicity. To assess the mitochondrial toxicity of DKB114 treatment, we determined the MMP in HepG2 cells. As shown in Figure 5A, DKB114 treatment did not change the MMP. Additionally, DKB114 did not affect cell viability ( Figure 5B).

Discussion
In our previous study, we found that DKB114, a combination of CF and CB extracts, exerted antihyperuricemic effects in rat models of PO-induced hyperuricemia. Based on our previous research, we further study the understanding of the mechanism underlying the antihyperuricemic effects. In this study, we found that DKB114 significantly decreased serum uric acid levels in normal rats and rats with PO-induced hyperuricemia, consistent with that of our previous study and other studies [18][19][20][21]. Furthermore, DKB114 increased uric acid levels in urine, indicating that DKB114 has the ability to increase uric acid excretion and that it might be a potent uricosuric agent, which may explain its antihyperuricemic effects.
Generally, hyperuricemia in humans can be divided into two categories based on pathophysiology, uric acid overproduction, and underexcretion. Uric acid production is mainly regulated by XOD, a key enzyme that converts hypoxanthine and xanthine to uric acid [23,24]. Thus, inhibition of XOD activity should be a target to control hyperuricemia. In our study, DKB114 inhibited the in vitro XOD and hepatic XOD activity in rats with PO-induced hyperuricemia, which supports the findings of previous studies [18][19][20][21]. Furthermore, DKB114 decreased liver uric levels in hyperuricemic models, indicating that inhibition of XOD activity by DKB114 might be attributable to the reduction in uric acid production. Thus, it seems likely that the beneficial antihyperuricemic effects of DKB114 may be attributable, at least in part, to its inhibitory effects on XOD activity and reduction of hepatic uric acid production.
Genome-wide association studies have demonstrated that approximately 90% of hyperuricemia patients show underexcretion of uric acid [25]. Excretion of uric acid mainly occurs in the kidney and intestine. Handling of uric acid in the kidney is achieved through a complex interplay between the reabsorption and secretion of uric acid. Multiple transporters are involved in uric acid transport in the kidney. Among the transporters, URAT1 and GLUT9 are localized on apical and basolateral membranes of renal proximal tubule cells, respectively, and they mediate uric acid reabsorption through mediates the reabsorption of uric acid from the proximal tubule transport of uric acid from the kidney lumen to blood [26,27]. Uricosuric agents, Ben and probenecid, effectively decrease serum uric acid levels through uric acid reabsorption [28]. OAT1 and OAT3, localized in the basolateral membrane of proximal tubules, are responsible for uric acid transport from the blood to epithelial cells [25]. Since more than 90% of the excreted uric acid is reabsorbed and about 10% is excreted in the urine, it is accepted that regulation of uric acid reabsorption plays an important role in uric acid excretion [29,30]. Therefore, URAT1 and GLUT9 are considered attractive therapeutic targets for hyperuricemia. As mentioned above, DKB114 exhibits potential as a potent uricosuric agent. In the present study, we examined the effects of DKB114 on these renal transporters in vitro and in vivo. Interestingly, in hURAT1-expressing oocytes and HEK293 cells, DKB114 inhibited uric acid uptake into the cells without causing cytotoxicity. In addition, DKB114 decreased the URAT1 and GLUT9 protein expression levels in the kidney in rats with PO-induced hyperuricemia, although OAT1 and OAT3 protein expression levels did not change. These results suggest that reduction of cellular uric acid uptake by URAT1 transporters and decrease in the expression of renal transporters can inhibit uric acid reabsorption, which may contribute to the promotion of uric acid excretion and thus alleviate hyperuricemia.
Drugs to treat hyperuricemia and gout are currently available, but treatment failure and adverse effects has been reported [31]. Uricosuric drugs such as probenecid and Ben also increased the risk for kidney damage [32]. In particular, hepatotoxicity has been a big concern associated with Ben, although the extract mechanisms underlying its hepatotoxicity remain unclear. Recently, mitochondrial toxicity and reactive metabolite formation were proposed as potential mechanisms [33,34]. In our study, DKB114 did not decrease MP or cell viability of HepG2 cells, indicating that DKB114 probably did not have the potential to cause mitochondrial toxicity and has mild side effects.
There are limitations in our study; (1) DKB114 is crude extract of two different plant sources that might contain numerous components and compounds. (2) Effects of DKB114 is direct or indirect unknown. In our previous study, HPLC showed that DKB114 contains six components: Chlorogenic acid and 3,4-dicaffeoylquinic acid from CF and coumarin, cinnamaldehyde, trans-cinnamaldehyde, and o-methoxycinnamaldehyde from CB; these components exerted XOD inhibitory activity in vitro [21]. This suggests that these components may be partially responsible for the antihyperuricemic effects of DKB114. However, we did not examine whether these components enhance the excretion of uric acid through regulation of various transporters associated with uric acid excretion. Therefore, the mechanism underlying the promotion of uric acid excretion and analysis of bioactive compounds needs to be investigated further in future.

Conclusions
The present study demonstrated that DKB114 reduced serum uric acid levels in normal rats and rats with PO-induced hyperuricemia and promoted the excretion of uric acid in urine, indicating that DKB114 has an antihyperuricemic effect and may be a potent uricosuric agent. In addition, DKB114 inhibited XOD activity and hepatic uric acid production in vitro and in vivo, as well as cellular uptake of uric acid in vitro. Furthermore, it decreased the protein expression levels of renal transporters such as URAT1 and GLUT9 in the kidney. Thus, the antihyperuricemic effects of DKB114 may be due, at least in part, to its inhibitory effects on XOD and reabsorption of uric acid. On the basis of these results, we propose that DKB114 exerts beneficial effects on hyperuricemia and may be useful in the treatment of hyperuricemia.