The leaves of C. japonica were provided from Jeollanamdo Wando Arboretum, in Jeonnam, Korea. C. japonica leaves were collected on Joyag island, Korea (126°56′50.07″E longitude and 34°22′31.27″N latitude). A voucher specimen (MNUCSS-CJ-01) was deposited at Mokpo National University (Muan, Korea). The leaves were separated for use in the present study. The air-dried and powdered C. japonica leaf (10 g) was extracted twice with ethanol (100 ml) at room temperature for 3 days. The resultant ethanol solution was evaporated, dried and stored at −50°C. The sample was used for in vitro and in vivo experiments, and for the analysis of the active constituents.

Male ICR mice (4 weeks old) were purchased from Orient Bio Co. (Sungnam, Korea). The mice were retained in a clean room at a temperature of 20–23°C with 12-h light (07:00–19:00) and dark (19:00–07:00) cycles, and a relative humidity of 50±5%. The mice were housed in ventilated mice cages (Tecniplast, West Chester, PA, USA) under filtered and pathogen-free air, with food (Agribrands Purina Korea, Inc., Seoul, Korea) and water available ad libitum. All animal experiments were carried out according to the guidelines of the Animal Investigation Committee of Jeonnam Bioindustry Foundation (Naju, Korea) (approval no. JINR1517).

The analytical methods for the analysis based on GC-MS have been previously reported (16). The analysis of scanned organic compounds was performed using an Agilent 7890 Gas Chromatograph System, coupled to a quadrupole Agilent 5975C electron ionization (EI) (70 eV) mass spectrometric detector (Agilent Technologies, Inc., Palo Alto, CA, USA). An Agilent HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) was utilized for the identification of organic compounds. Briefly GC-MS was tuned using perfluorotribuyl-amine (PFTBA) by mass fragments of 69.0, 219.0, 502.0 m/z under EI conditions. The GC oven was heated using the following program: isothermal at 65°C for 10 min and 10 min⁻¹ to 300°C with He as the carrier gas. The transfer line was heated at 300°C, and the mass spectrometer was then operated in scan mode (50–550 amu). All mass spectra were compared with the data system library (NIST 2008). The summarized operation parameters for the GC are shown in Table I.

Constituent profiling of ECJL was performed with HPLC. All HPLC analyses were performed using the Alliance 2695 HPLC System (Waters Corp., Millford, MA, USA) equipped with a photodiode array detector. The Agilent ZORBAX Extend-C18 (5 µm, 150 mm l. × 5 mm i.d.) analytical column was used with a mobile phase consisting of solvent A (acetonitrile) and solvent B (water containing 0.2% phosphoric acid). Gradient elution (from 10/90 to 100/0, v/v) was performed at a flow rate of 1.0 ml/min (Table II). Column temperature was maintained at 25°C. The detection wavelength was set at 270 nm for rutin. The solvent was filtered through a 0.22-µm filter and degassed. The sample injection volume was 10 µl.

The determination of the antioxidant activity of ECJL was performed by the DPPH radical scavenging method. DPPH radicals have an absorption maximum of 517 nm, which disappears with reduction by an antioxidant compound. ECJL solution (1 ml) containing 1–20 mg of ECJL was added to a 0.4 mM DPPH ECJL solution (1 ml). The solution was mixed and allowed to react at room temperature in the dark for 10 min. The absorbance at 517 nm was measured using a microplate reader (PerkinElmer, Inc., Waltham, MA, USA). The radical scavenging activity was calculated as a percentage using the following equation: DPPH radical scavenging activity (%) = [1 − (A_sample/A_blank)] ×100. The DPPH free radical scavenging activities of the samples were compared in terms of their IC₅₀ (µg/ml) values, as previously described (17).

The reducing power of ECJL was determined according to a modified reducing power assay method. The sample (0.1 ml) was added to 0.2 M sodium phosphate buffer (0.5 ml) and 1% potassium ferricyanide (0.5 ml), and this mixture was incubated at 50°C for 20 min. Following incubation, 10% trichloroacetic acid solution (0.5 ml) was added to the reaction mixture, and it was centrifuged at 12,000 rpm for 10 min. The supernatant was mixed with distilled water (0.5 ml) and a 0.1% iron (III) chloride solution (0.1 ml), and the absorbance at 700 nm of the resulting solution was measured. The reducing powers of the samples were expressed as vitamin C equivalents, as previously described (17).

The total phenolic content was determined by Folin-Ciocalteu assay, as previously described (18). Water solution (1 ml) containing 5 mg of ECJL or standard was mixed with 1 ml of 2% sodium carbonate solution and 1 ml of 10% Folin-Ciocalteu's phenol reagent. After 10 min, the absorbance was measured at 750 nm using a microplate reader (PerkinElmer, Inc.). The measurement was compared to calibration curve of gallic acid. The results were expressed as milligrams of gallic acid equivalents per gram of sample, as previously described (17).

The XO inhibitory activity was measured by monitoring uric acid formation in a XO system, as previously described (19). The assay system consisted of 0.6 ml phosphate buffer (100 mM; pH 7.4), 0.1 ml sample, 0.1 ml XO (0.2 U/ml) and 0.2 ml xanthine (1 mM; dissolved in 0.1 N NaOH). The reaction was initiated by adding the enzyme with or without inhibitors, and the change in absorbance of the mixture at 290 nm for 15 min was measured against a reagent blank. A 0.2 ml aliquot of 1 N HCl was used to terminate the enzymatic reaction. Allopurinol was used as a positive control.

Six groups of mice (n=5 for each group) were treated once daily for 7 days as follows: the mice in the 2 negative control groups (NOR and HU groups) received 0.3% CMC-Na aqueous solution; the mice in the positive control group (ALLO group) received allopurinol solution at a dose of 10 mg/kg; the mice in the ECJL 30, ECJL 100, and ECJL 300 groups received the ECJL solution at doses of 30, 100 and 300 mg/kg, respectively. Hyperuricemia was induced in the mice by potassium oxonate, a uricase inhibitor (20). Briefly, ECJL (30, 100 and 300 mg/kg) or allopurinol (10 mg/kg) were dissolved in 0.3% CMC-Na aqueous solution. The resultant drug solutions were orally administered once per day for 7 days. Food, except water, was withdrawn from the mice at 1.5 h prior to drug administration, and the mice were intraperitoneally injected with potassium oxonate (300 mg/kg) 1 h before the final drug administration on the 7th day in order to induce hyperuricemia. Blood samples were collected via the tail vein of the mice at 1 h after the final drug administration on the 7th day. The blood samples were allowed to clot for approximately 1 h at room temperature and then centrifuged at 10,000 × g for 15 min to obtain serum. The serum samples were stored at −80°C until use. The serum uric acid concentration was measured using standard diagnostic kits (Abcam, Cambridge, UK). Each assay was performed in triplicate.

The residual activity of XO in the mouse liver and plasma were spectrophotometrically determined by monitoring uric acid formation from xanthine (12). The mouse livers (0.5 g) were homogenized in an 1 ml aliquot of 50 mM sodium phosphate buffer (pH 7.4). The homogenates were centrifuged at 3,000 × g for 10 min at 4°C. After removing the lipid layer, the supernatant was centrifuged at 10,000 × g for 60 min at 4°C. The resultant supernatant was then used for determining XO residual activity and the total protein concentration. A 0.12 ml aliquot of xanthine solution (250 mM) was added to a test tube containing 10 µl liver homogenate and 0.54 ml potassium oxonate solution (1 mM) in 50-mM sodium phosphate buffer (pH 7.4) that were previously incubated at 35°C for 15 min. The reaction was termined after 0 and 30 min by the addition of a 0.1 ml aliquot of 0.6 M HCl. Thereafter, the test tube was centrifuged at 3,000 × g for 5 min, and the absorbance of the supernatant was measured at 295 nm using a UV/VIS spectrophotometer (Biochrom Libra S12; serial no. 116997; Biochrom Ltd., Cambridge, UK). The total protein concentration was determined spectrophotometrically by the Bradford method (21). XO activity was expressed as micromoles of uric acid formed per minute (U) per milligram protein.

A value of P<0.05 was considered to indicate a statistically significant difference and was determined using a t-test between the two means for the unpaired data or an ANOVA (post hoc test: Tukey's multiple range test) among the 3 or more means for the unpaired data. All data were expressed as the means ± standard deviation and rounded to 2 decimal places.

The antioxidant potential of ECJL was determined by DPPH and reducing power assays. Plants have been reported to be useful sources of phytochemicals, such as phenolics, and various health benefits, such as antioxidant activity have been suggested (22). In addition, the various antioxidant activities of natural resources significantly correlate with their major compound contents, such as polyphenols. Therefore, in this study, we determined the total phenolic contents of the extract obtained from ECJL.

The DPPH scavenging effect is a widely used method to evaluate the free radical scavenging ability of plant extracts. Table III shows the measured DPPH radical scavenging activity. A low IC₅₀ value indicates a strong antioxidant activity in ECJL. The IC₅₀ value of the extract was 38.53±0.72 µg/ml.

Fe³⁺ was transformed into Fe²⁺ in the presence of extracts to measure the reductive capability. At 50 µg/ml of the extract, the value, which is expressed in vitamin C equivalents, of the reducing power of vitamin C was 13.34±1.7 µg/50 µg ECJL.

Total phenol compounds, as determined by the Folin-Ciocalteu method, as previously described (17), are reported as gallic acid equivalents by reference to a standard curve (r²=0.999). Table III shows the total phenolic content of the extract. The amount of the phenolic compounds in the extract was 46.6±1.6 mg/g eq. gallic acid.

Fig. 1 shows the XO inhibitory activity of ECJL. XO inhibitory activity was expressed as the suppression rate of uric acid production. Allopurinol (as a positive control) at a concentration of 30 µg/ml significantly inhibited XO activity by 64.6±3.4%. Notably, the dose-dependent XO inhibitory activity of ECJL significantly increased. ECJL at a concentration of 2 mg/ml inhibited XO activity by 41.3±5.5%.

Fig. 2 shows the effects of ECJL on the serum uric acid levels in mice with potassium oxonate-induced hyperuricemia. The concentrations were significantly higher in the mice with hyperuricemia compared to the normal mice, indicating that the mouse model of hyperuricemia was successfully established at 1 h after the intraperitoneal injection of potassium oxonate, a urate oxidase inhibitor, which is consistent with the findings of a previous study (20). The serum uric acid concentrations in the normal mice were comparable with those in the hyperuricemic mice that were administered allopurinol or the 3 different doses of ECJL (30, 100 and 300 mg/kg over a period of 7 days.

In our in vivo model, the level of serum uric acid was effectively increased by potassium oxonate at a dose of 300 mg/kg via intraperitoneal injection (14.19±1.62 µM). The serum levels of uric acid in the allopurinol-treated group and ECJL treatment groups (30, 100, 300 mg/kg) were 6.65±0.74, 12.89±1.13, 8.89±1.17 and 7.23±1.63 µM, respectively. Allopurinol at a dose of 10 mg/kg suppressed the serum uric acid levels by 53% and ECJL at a dose of 300 mg significantly suppressed the uric acid levels by 49.1%, similar to allopurinol.

Figs. 3 and 4 show the effects of ECJL on the hepatic and serum XO activity in the mice with potassium oxonate-induced hyperuricemia. Pre-treatment with allopurinol for 1 week (oral administration) and ECJL at the doses of 100 and 300 mg/kg significantly reduced hepatic XO activity by 66.1, 55.2 and 64.7%, respectively (Fig. 3). Similarly, the serum XO activity in the mice pre-treated orally with allopurinol and ECJL at the doses of 100 and 300 mg/kg was reduced by 65, 45.2 and 59.3%, respectively.

However, there were no significant differences in hepatic and plasma XO activity between the normal and hyperuricemic control groups. Taken together, our findings demonstrated that ECJL at the dose of 300 mg/kg exerted XO inhibitory activity similar to that of allopurinol at a dose of 10 mg/kg.

In the present study, we identified active compounds related to XO inhibitory activity from C. japonica leaf using GC-MS and liquid chromatography analysis. We identified one XO inhibitor, namely rutin (5.87%) using liquid chromatography. We identified 8 active compounds, which were vitamin E (25.35%) n-eicosane (10.2%), neophytadiene (0.91%), all trans-squalene (3.32%), n-octacosane (2.65%), 6,9-pentadecadien-1-ol (1.51%), α-linolenic acid (1.41%), and n-hexadecanoic acid (0.61%) using GC-MS.

Figs. 5 and 6 show typical GC-MS and HPLC chromatograms, respectively, which show the phytochemical constituents. After clarifying the active substances, we expected that the potent XO inhibitory activity of ECJL was due to the synergism of antioxidant and XO inhibitory substances.