Korean Red Ginseng Ameliorates the Level of Serum Uric Acid via Downregulating URAT1 and Upregulating OAT1 and OAT3

Abstract

Hyperuricemia is caused by an imbalance of uric acid and is associated with many diseases. Although gout which is one of hyperuricemia-related diseases is curable with anti-hyperuricemic drugs some medications have side effects, such as hypersensitivity in patients with circulatory system disorders, flare reoccurrences, and increased cardiac risk. This study consisted of test tube (xanthine oxidase’s inhibition) and animal study. Animal study using with ICR mice was composed of control, potassium oxonate-induced hyperuricemia, allopurinol, and 3 Korean red ginseng water extract (KRGWE) treatment groups (62.5; 125, and 500 mg/kg). We orally administered KRGWE once a day for 7 d to induce hyperuricemia and injected PO 2 h before the final KRGWE administration. We measured serum uric acid, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen, and creatinine and analyzed the genes such as organic anion transport (OAT)-1, OAT-3, and urate transport (URAT)-1. KRGWE dose-dependently controlled xanthine oxidase activity in the serum and completely inhibited serum uric acid. KRGWE affected both uric acid excretion-related and uric acid reabsorption-related gene expression. KRGWE stimulated uric acid excretion-related gene expressions, such as OAT-1 and OAT-3, but inhibited uric acid reabsorption-related gene expression, such as URAT-1. KRGWE improved liver and kidney functioning. KRGWE improved liver/kidney functioning and is promising anti-hyperuricemic agent which can control serum uric acid via downregulating URAT1 and upregulating OAT1 and OAT3.

INTRODUCTION

Hyperuricemia is indicated by serum uric acid levels greater than 6.8 mg/dL.¹⁾ It is associated with cardiovascular disease, renal disorders, diabetes, obesity, gout, and piercing crystals composed of a high concentration of urine/serum uric acid that stimulate gout.^2–5) The level of uric acid in the body is controlled by several factors, such as the consumption of protein-rich foods and urate and excretion,⁶⁾ but regulatory malfunctions induce hyperuricemia. Several genes are related to hyperuricemia, such as urate transport (URAT)-1, glucose transporter type-9 (GLUT-9), organic anion transport (OAT)-4, BCRP, NPT-1, SLC2A-9, and ABCG-2.^7–10) Worldwide, although the population of hyperuricemic patients is approximately 21%, approximately 25% of hospitalized patients have hyperuricemia.¹⁾

Gout among hyperuricemic decreases the QOL due to a burning, gnawing, stabbing, and throbbing in the joint and periarticular sites.¹¹⁾ Gout can induce morphological changes at uric acid crystal gathering sites.⁵⁾

The clinical symptoms of hyperuricemia include gout, nephrolithiasis, and uric acid renal disease.^12,13) There are 3 types of hyperuricemia mediations: uric acid synthesis inhibitors (xanthine oxidase inhibitor), uric acid excretion stimulators (URAT-1 inhibitor), and uric acid hydrolysis regulators (uricase stimulator).¹⁴⁾ However, some medications have adverse effects, such as hypersensitivity in patients with cardiovascular disease or renal failure,¹⁵⁾ flare reoccurrences,¹⁶⁾ and increased cardiac disease risk.¹⁷⁾

Recently, the need for safe and effective new drugs for controlling hyperuricemia has increased the development of anti-hyperuricemia agents from natural products.^18–23) Korean red ginseng (Panax ginseng Meyer) is used as a folk remedy²⁴⁾ for its biological and pharmaceutical effects, such as enhanced cardiocirculatory²⁵⁾ and immune function²⁶⁾ and curative effects on hyperresponsiveness,²⁷⁾ inflammation,²⁸⁾ neoplasm,²⁹⁾ and metabolomic disorders.³⁰⁾ In this study, we investigated the anti-hyperuricemic effects and therapeutic mechanism of Korean red ginseng water extract.

MATERIALS AND METHODS

Korean Red Ginseng Water Extract (KRGWE)

In our previous study, we reported that KRGWE (Korea Ginseng Corporation, Daejeon, Korea) was extracted from P. ginseng roots and analyzed Rb1 (7.44 mg/g), Rb2 (2.50 mg/g), Rc (3.04 mg/g), Rd (0.91 mg/g), Re (1.86 mg/g), Rf (1.24 mg/g), Rg1 (1.79 mg/g), Rg2s (1.24 mg/g), Rg3s (1.39 mg/g), and Rh1 (1.01 mg/g).²⁷⁾ Experimental research and field studies on cultivated plant (Korean red ginseng), including the collection of plant material, were complied with relevant institutional, national, and international guidelines and legislation.

Xanthine Oxidase Inhibition Assay

To determine the xanthine oxidase inhibitory effect of KRGWE, we performed a test tube assay with 0.13 mL of 100 mM phosphate buffer (Biosolution, Seoul, Korea), 0.01 mL xanthin oxidase (0.2 U/mL, Sigma-Aldrich, MO, U.S.A.), 0.1 mL of 2 mM xanthine (Sigma-Aldrich), and 0–1000 µg/mL KRGWE. We shook the mixture at 37 °C for 15 min and added 1 M HCl to halt the reaction. We measured the absorbance value at 295 nm.

Animal Experiments

We purchased 30 male ICR mice from Samtako Korea (Osan, South Korea). The study had a control (CON, tap water treated), a gout group induced with 250 mg/kg potassium oxonate (PO, Tokyo Chemical Industry, Tokyo, Japan), a positive control group with 10 mg/kg allopurinol treatment (ALP, Sigma-Aldrich) on 250 mg/kg PO, and 3 KRGWE treated groups (62.5; 125, and 500 mg/kg) on 250 mg/kg PO. Each group contained 5 animals. Figure 1 depicts the animal schedule. We orally administered tap water; 10 mg/kg ALP (Sigma-Aldrich), and KRGWE (62.5; 125, and 500 mg/kg) in each group once per day for 7 d. We induced gout by intraperitoneal injection of 250 mg/kg PO (Tokyo chemical industry) at 166 h after the start of the experiment and euthanized the mice by intraperitoneal injection of 50 mg/kg Zoletil (Virbac, Carros, France) at 168 h.

Fig. 1. Animal Schedule

PD, pre-day for starting; D, day for studying.

Ethics Statement and Guideline

Animal care and all experimental procedures were approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chonnam National University (CNU IACUC-YB-2020-108). The study was reported in accordance with ARRIVE guidelines.

Xanthine Oxidase Activity and Uric Acid Analysis

After the Zoletil, we collected blood via intracardiac injection and euthanized the animals. We centrifuged the collected blood for 5 min at 3000 rpm (Sorvall Legend Micro 17R, Thermo Fisher Scientific, Waltham, MA, U.S.A.). We stored the separated serum at −80 °C before the experiment. We measured xanthine oxidase activity and uric acid using with Xanthin Oxidase Activity Assay Kit (Abcam, Cambridge, U.K.) and Uric Acid Assay Kit (Abcam), respectively per the manufacturer’s instructions.

Quantitative (q)-PCR Analysis

We performed a q-PCR analysis to define the change in genes related to uric acid elimination, such as OAT-1, OAT-3, and URAT-1. We collected whole renal RNA by RNeasy Mini Kit (Qiagen, Hilden, Germany), with 100 ng RNA used for the reacting template. The q-PCR cycle was composed of denaturing at 95 °C for 5 s and annealing/extending at 65 °C for 30 s for 40 cycles. We used q-Tower2.2 (Analytik Jena, GmbH, Jena, Germany) to obtain the results. The primers are listed in Table 1.

Table 1. The Primer Sequences

OAT-1	Forward	5′-GACAGGGTCTCATCCCTAGC-3′
	Reverse	5′-GTCCCTGACACACTGACTGA-3′
OAT-3	Forward	5′-TACAGTTGTCCGTGTCTGCT-3′
	Reverse	5′-CTTCCTCCTTCTTGCCGTTG-3′
URAT-1	Forward	5′-GATAGGTTTGGGCGCAGAAG-3′
	Reverse	5′-TCATCATGACACCTGCCACT-3′
β-Actin	Forward	5′-CCAGCCTTCCTTCTTGGGTA-3′
	Reverse	5′-CAATGCCTGGGTACATGGTG-3′

Western Blot Analysis

Kidney tissues were prepared with radio immunoprecipitation assay (RIPA)-based lysis buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific), centrifuged at 13000 rpm for 20 min, and the supernatant was collected. Protein concentration was determined using the Bradford assay, and 30 µg of protein was denatured by heating with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer at 95 °C for 5 min. After electrophoresis on the SDS-PAGE gel, the samples were transferred to polyvinylidene difluoride (PVDF) membranes, and each protein in the samples was bound to primary antibodies in 5% skim milk. The primary antibodies used were OAT-1 (26574-1-AP, Proteintech, Rosemont, IL, U.S.A.), OAT-3 (sc-293264, Santa Cruz Biotechnology, Dallas, TX, U.S.A.), URAT-1 (14937-1-AP, Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MA5-15738, Thermo Fisher Scientific). Membranes were washed with 0.1% Tween-20 Tris-buffered saline and incubated with secondary antibodies (goat anti-rabbit immunoglobulin G (IgG) (111-035-003, Jackson ImmunoResearch, West Grove, PA, U.S.A.) or goat anti-mouse IgG (115-035-003, Jackson ImmunoResearch). Bands were imaged using an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) and a Davinch-Western™ imaging system (Davinch-K, Seoul, Korea). Protein expression relative to GAPDH was analyzed using ImageJ software (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, U.S.A.).

Blood Chemistry Analysis

We used slides purchased from FUJIFILM (Tokyo, Japan) to measure the levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), and creatinine. We followed the manufacturer’s guidelines and achieved the results by DRI-CHEM NX-500i (FUJIFILM).

Statistical Analysis

The results are expressed as means ± standard deviation. We performed a one-way ANOVA and a Dunnett’s multiple comparison test followed if differences were noted. Significance was observed at p < 0.001.

RESULTS

KRGWE Dose-Dependently Increased Xanthine Oxidase Inhibition in a Test Tube Assay and Decreased Xanthine Oxidase Activity in an Animal Study

The positive control drug, allopurinol (25 µg/mL), significantly elevated xanthine oxidase inhibition to 72.78 ± 2.022% compared to the control group (0.00 ± 1.434%) (Fig. 2A). KRGWE dose-dependently increased xanthine oxidase inhibition from 26.73 ± 1.514% at 10 µg/mL to 48.62 ± 0.835% at 1000 µg/mL. In the animal study; 250 mg/kg potassium oxonate treatment (gout induction group) augmented xanthin oxidase activity compared to the control group, but KRGWE treatment dose-dependently suppressed potassium oxonate-induced xanthine oxidase activity (Fig. 2B).

Fig. 2. Korean Red Ginseng Water Extract (KRGWE) Controversially Modulated Both Xanthine Oxidase Inhibition in the Test Tube and Xanthine Oxidase Activity in the Serum in a Dose-Dependent Manner

(A) KRGWE dose-dependently increased xanthine oxidase inhibition in the test tube. N = 6. ** p < 0.001 vs. 0 µg/mL treated group; ^$$ p < 0.001 vs. 25 µg/mL allopurinol treated group; ^# p < 0.05 vs. 10 µg/mL KRGWE treated group; ^## p < 0.001 vs. 10 µg/mL KRGWE treated group. (B) KRGWE decreased xanthine oxidase activity in the serum in a dose-dependent manner. N = 5. KRGWE, Korea red ginseng water extract; ALP, allopurinol; PO, potassium oxonate. * p < 0.05 vs. CON; ^$ p < 0.05 vs. PO; ^$$ p < 0.001 vs. PO group; ^# p < 0.05 vs. ALP; ^## p < 0.001 vs. ALP.

KRGWE Dose-Dependently Suppressed Uric Acid Serum Levels

Potassium oxonate increased the level of serum uric acid compared to the control group, but 10 mg/kg allopurinol treatment significantly decreased the level of uric acid resulting from potassium oxonate treatment (Fig. 3). KRGWE suppressed the level of uric acid in the serum in a dose-dependent manner, even though the serum uric acid level in the 62.5 mg/kg KRGWE treatment group was statistically lower than that in the PO group.

Fig. 3. Korean Red Ginseng Water Extract (KRGWE) Dose-Dependently Suppressed the Serum Level of Uric Acid

N = 5. KRGWE, Korean red ginseng water extract; ALP, allopurinol; PO, potassium oxonate. * p < 0.05 vs. CON; ^$ p < 0.05 vs. PO; ^$$ p < 0.001 vs. PO group; ^# p < 0.05 vs. ALP.

KRGWE Increased Uric Acid Secretion and Inhibited Uric Acid Reabsorption in the Renal Tubule

Uric acid elimination is related to OATs and URATs. OAT-1 and OAT-3 are in the proximal renal tubule and play a role in uric acid secretion. URAT-1 is in the proximal and distal renal tubules and plays a role in uric acid reabsorption.^31,32) Potassium oxonate treatment suppressed OAT-1 and OAT-3 protein and gene expression, which were decreased to 50% compared to the levels in CON (Figs. 4A–C, E, F). However, OAT-1 level was completely recovered by ALP and KRGWE in a dose-dependent manner (Figs. 4A, B, E). ALP could not completely restitute KRGWE’s dose-dependent recovery of the OAT-3 protein and gene, but almost restored levels in both the 125 and 500 mg/kg treatment groups (Figs. 4A, C, F). The protein and gene levels of URAT-1 in PO increased almost 50% compared to that in CON, but KRGWE dose-dependently suppressed and inhibited levels in CON in the 500 mg/kg treatment (Figs. 4A, D, G).

Fig. 4. Korean Red Ginseng Water Extract (KRGWE) Dose-Dependently Modulated Uric Acid Excretion-Related Gene Expressions Including Organic Anion Transporter (OAT)-1 and OAT-3 in the Renal Proximal Tubule and Uric Acid Reabsorption-Related Gene Expression Including Urate Transporter (URAT)-1 in the Renal Proximal and Distal Tubules

(A) KRGWE increased the expression levels of OAT-1 and OAT-3 and decreased the expression levels of URAT-1. (B) The relative intensity results of the OAT-1 protein expression levels to GAPDH. (C) The relative intensity results of the OAT-3 protein expression levels to GAPDH. (D) The relative intensity results of the URAT-1 protein expression levels such as to GAPDH. (E, F) KRGWE increased the RNA expression of the uric acid excretion-related gene such as OAT-1 and OAT-3, which were decreased by potassium oxonate. (G) KRGWE decreased the RNA expression level of the uric acid reabsorption gene URAT-1, which was increased by potassium oxonate. N = 5. KRGWE, Korea red ginseng water extract; ALP, allopurinol; PO, potassium oxonate; OAT, organic anion transporter; URAT, urate transporter. * p < 0.05 vs. CON; ^$ p < 0.05 vs. PO; ^# p < 0.05 vs. ALP.

KRGWE Enhanced Both Hepatic and Renal Function

GOT and GPT are important biomarkers of liver function, as their levels are altered in liver cirrhosis and hepatoma.³³⁾ As BUN and creatinine levels are related to glomerular filtration, they are major markers of renal function.³⁴⁾ Potassium oxonate increased GOT and GPT levels compared to the CON group, but KRGWE dose-dependently suppressed them (Figs. 5A, B). In particular, the level of GOT in the 500 mg/kg KRGWE treatment group was similar to the CON group. The BUN and creatinine levels were increased by potassium oxinate compared to the CON group. KRGWE not only dose-dependently decreased BUN levels but also suppressed creatinine levels similar to the control group (Figs. 5C, D).

Fig. 5. Korean Red Ginseng Water Extract (KRGWE) Improved Hepatic and Renal Function

KRGWE suppressed (A) glutamic oxaloacetic transaminase (GOT) and (B) glutamic pyruvic transaminase in liver levels (GPT) and decreased the (C) blood urea nitrogen (BUN) and (D) creatine levels. N = 5. KRGWE, Korea red ginseng water extract; ALP, allopurinol; PO, potassium oxonate. * p < 0.05 vs. CON; ^$ p < 0.05 vs. PO; ^$$ p < 0.001 vs. PO group; ^# p < 0.05 vs. ALP; ^## p < 0.001 vs. ALP.

DISCUSSION

The prevalence of hyperuricemia varies by country. In China, the 2004 prevalence was 13.2%, while it was 13.4% in Japan in 2017; 5.1% in Korea in 2013; 15.9% in Taiwan in 2008, and 21.4% in the U.S.A. in 2008.³⁵⁾ Gout is a serious metabolic disorder caused by hyperuricemia that occurs when uric acid accumulates in the blood and tissues.¹⁾ Accumulated uric acid can form pointed crystals in the joints to induce inflammatory arthritis.⁵⁾ Gout flares decrease QOL due to severe pain.¹¹⁾ The prevalence of gout ranges from <1 to 6.8% and the incidence from 0.58–2.89 per 1000 person-years.³⁶⁾

The pathogenesis of hyperuricemia might be uric acid synthesis in the serum and accumulation in tissue. The synthesis of uric acid in the serum is related to the uric acid synthetic enzyme, xanthine oxidase,³⁷⁾ and tissue accumulation is related to synthesized uric acid excretion.¹⁾ There are two means of decreasing serum uric acid synthesis: restrictions of purine-rich foods, such as beer, blue-colored fish, soybean, and meat,^38–40) and the use of xanthine oxidase inhibitors, such as allopurinol, febuxosatat, and topiroxostat.⁴¹⁾ Especially, the reason of PO-induced acute hyperuricemia murine model is that as PO is selectively uricase inhibitor it blocks to change from uric acid to allantoin.⁴²⁾ Zhang reported that vitamin B12 and folate suppressed hyperuricemia in males and folate controlled levels in females.⁴³⁾ Some transporters in the renal tubule, such as OATs and URATs, inhibit uric acid accumulation in the tissues. OAT-1 and OAT-3 in the proximal renal tubule stimulate uric acid secretion from the body to the renal tubule,³¹⁾ but URAT-1 in the proximal and distal renal tubules induces uric acid reabsorption from the renal tubule to the body.³²⁾

As unbalance or malfunction of these transporters accelerates hyperuricemia. Anti-hyperuricemic drugs can be categorized as xanthine oxidase inhibitors, URAT-1 inhibitors, and uricase inhibitors,¹⁴⁾ but some drugs have severe adverse effects, such as hypersensitivity in patients with cardiovascular or renal failure,¹⁵⁾ flare reoccurrences,¹⁶⁾ and increased cardiac risk.¹⁷⁾ To overcome these adverse effects, many are turning to natural products with an anti-hyperuricemic effect such as Baccharis trimera (Less.) DC,¹⁸⁾ Camellia japonica,⁴⁴⁾ Corylopsis coreana Uyeki (Hamamelidaceae) flos,⁴⁵⁾ Cudrania tricuspidate,^19,46) Dendropanax morbifera H. Lev,²⁰⁾ and Quercus acuta Thunb (Fagaceae).⁴⁷⁾

KRGWE dose-dependently stimulated xanthine oxidase inhibition in a test tube (Fig. 2A) but controversially suppressed xanthine oxidase activity in an animal study (Fig. 2B). KRGWE controlled serum uric acid levels, which were increased by potassium oxonate (Fig. 3). KRGWE treatment modulated the uric acid excretion genes OAT-1 and OAT-3 and the uric acid reabsorption gene, URAT-1 (Fig. 4). KRGWE improved hepatic function by modulating GOT and GPT levels (Figs. 5A, B) and enhanced renal function by regulating BUN and creatine levels (Figs. 5C, D).

CONCLUSION

KRGWE ameliorated potassium oxonate-induced hyperuricemia by suppressing xanthine oxidase activity, modulating OAT-1, OAT-3, and URAT-1 levels in the renal tubule, and improving liver and kidney function. KRGWE is a promising anti-hyperuricemic drug.

Acknowledgments

This work was supported by the 2020 Grant from the Korean Society of Ginseng and by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MSIT) (2022R1A5A8033794).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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