The Hypouricemic Effect of Balanophora laxiflora Extracts and Derived Phytochemicals in Hyperuricemic Mice

The objective of this study is to evaluate the lowering of uric acid using Balanophora laxiflora extracts and derived phytochemicals on potassium-oxonate-(PO-) induced hyperuricemia in mice. The results revealed that ethyl acetate (EtOAc) fraction of B. laxiflora extracts exhibited strong xanthine-oxidase-(XOD-) inhibitory activity. In addition, among the 10 subfractions (EA1–10) derived from EtOAc fraction, subfraction 8 (EA8) exhibited the best XOD-inhibitory activity. Four specific phytochemicals, 1-O-(E)-caffeoyl-β-D-glucopyranose (1), 1-O-(E)-p-coumaroyl-β-D-glucopyranose (2), 1,3-di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (3), and 1-O-(E)-caffeoyl-4,6-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (4), were further isolated and identified from this subfraction. Compounds 3 and 4 exhibited the strongest XOD-inhibitory activity compared with other compounds, and both hydrolyzable tannins were determined to be noncompetitive inhibitors according to the Lineweaver-Burk plot. On the other hand, the in vivo hypouricemic effect in hyperuricemic mice was consistent with XOD-inhibitory activity, indicating that B. laxiflora extracts and derived phytochemicals could be potential candidates as new hypouricemic agents.


Introduction
Xanthine oxidase (XOD) is an important enzyme in humans that plays a crucial role in mediating purine metabolism. The major function of XOD is to convert hypoxanthine to xanthine and xanthine to uric acid [1,2]. Many studies have demonstrated that excessive amounts of uric acid in the human body lead to the formation of urate crystals, which can cause diseases, such as inflammation, gouty arthritis, and tophaceous gout [3][4][5][6]. An epidemiological study has shown a high prevalence of gout in male and geriatric groups [7]. XOD inhibitors are generally used for the treatment of gout and hyperuricemia, which can hinder enzyme reactions involved in uric acid synthesis, reduce the formation of uric acid and relieve symptoms of the aforementioned diseases [8,9].
Recently, scientists have attempted to find new and safe XOD inhibitors from a wide variety of traditional herbal plants [10][11][12]. Balanophora laxiflora Hemsl. (Balanophoraceae), which is traditionally used as a medicinal plant to cure cough, metrorrhagia, and hemorrhoids, is widely distributed in woodlands from mid to low altitudes in Taiwan. In our previous study, we determined that the extract of B. laxiflora exhibited good antioxidant activity [13]. Furthermore, the Balanophora species contain an abundant amount of hydrolyzable tannins and phenolic compounds [13][14][15][16][17][18]. Many studies have shown that some of these phenolic compounds have strong XOD-inhibitory activity and that the therapeutic effects of these compounds may be due to their antioxidant activity and enzyme inhibitory ability [2,3,8,10]. However, there are no prior reports on lowering uric acid using B. laxiflora extracts. Thus, in this study, the lowering of uric acid by B. laxiflora extract and its derived phytochemicals was evaluated in an in vitro XOD-inhibitory assay and in vivo potassium-oxonate-(PO-) induced acute hyperuricemic mouse model, and these results provide scientific evidence of hypouricemic efficacy for the first time.   Then, all mice were sacrificed, and their serum uric acid levels were determined using a commercial kit from Randox Laboratories (U.K.).

Statistical Analyses.
The results were expressed as the mean ± SD (n = 3) or mean ± SEM (n = 6 or 7). The significant difference was calculated by Scheffe's test; P values <0.05 were considered to be significant.

Results and Discussion
3.1. The XOD-Inhibitory Activity of B. laxiflora Crude Extract and Derived Soluble Fractions. The XOD-inhibitory activity of crude extract and its derived soluble fractions was dosedependent in Figure 1. In the presence of test samples at a concentration of 10 μg/mL, the XOD-inhibitory activity decreased in the following order: EtOAc fraction (41.9%) > crude extract (31.7%) > BuOH fraction (25.4%) > water fraction (9.8%). The EtOAc fraction exhibited the strongest XOD-inhibitory activity. In addition, the IC 50 values (the concentration required to inhibit uric acid formation by 50%) of crude extract, EtOAc fraction, BuOH fraction, and water fraction were 28.2, 14.2, 81.7, and >100 μg/mL, respectively. Havlik et al. [2] reported that the leaf of Thuja occidentalis and fruit of Prunus domestica were historically used to treat gout in Europe. Furthermore, their 80% ethanolic extracts induced 43.5 and 3.4% XOD inhibition at a concentration of 200 μg/mL. Additionally, the results reported by González et al. [11] showed that, at a concentration of 50 μg/mL, the XOD-inhibitory activity of ethanolic extracts of Crossopetalum lotabum leaves, Crossopetalum tonduzii branches, and the whole plant from Hyptis suaveolens was 31, 42, and 52%, respectively. Kong et al. [23] reported that the XOD-inhibitory activity of water extracts from Cinnamomum cassia and Morus alba barks was 31 and 14%, respectively, at a concentration of 50 μg/mL. Arimboor et al. [24] also reported that the methanolic extract from Semecarpus anacardium, an Indian traditional medicine used for the treatment of gout, rheumatoid arthritis, and inflammatory diseases, had potent XOD-inhibitory activity with an IC 50 value of 253 μg/mL. Comparisons of the aforementioned results indicated that there are abundant XOD-inhibitory phytochemicals present in the extracts of B. laxiflora, especially in the EtOAc fraction.

The Bioassay-Guided Isolation of the EtOAc Fraction from B. laxiflora Extracts.
Among B. laxiflora crude extracts and derived soluble fractions, the EtOAc fraction showed the best XOD-inhibitory activity. Using a bioassay-guided isolation, the constituents of the EtOAc fraction were further investigated in this study. The EtOAc soluble fraction was further divided into 10 subfractions by column chromatography. The elution solvent, collected weight, and XOD-inhibitory activity of these 10 subfractions are shown in Table 1. Of these subfractions, EA8 eluted with 100% of EtOAc and exhibited the strongest inhibitory activity (73.8%) against XOD at a concentration of 20 μg/mL. Thus, the EA8 subfraction was isolated by semipreparative HPLC with diode-array detector (DAD).  Figure 3, the XOD-inhibitory activity of phytochemicals from B. laxiflora extracts was compared with allopurinol, which is clinically used as an XOD inhibitor. Among the four phytochemicals, CHDGP (4) exhibited the best XOD-inhibitory activity (81.3%) at a concentration of 100 μM, followed by GHGDP (64.5%) with an IC 50 value of 71.5 μM. Lin et al. [26] [28]. These results reveal that hydrolyzable tannins GHDGP and CHDGP from B. laxiflora show excellent XOD-inhibitory activity. Furthermore, these tannins may be suitable for the treatment of XOD-related diseases. The Lineweaver-Burk plot was used to determine the kinetic mechanisms of inhibition by GHDGP and CHDGP. As shown in Figures 4(a) and 4(b), all straight lines in the Lineweaver-Burk plot intersect at the same point on the xaxis, which is characteristic of noncompetitive inhibition. Thus, these results indicate that the mode of XOD inhibition by both hydrolyzable tannins GHDGP and CHDGP is noncompetitive. In addition, the kinetic constants, K m and K i , were determined from the Lineweaver-Burk and Dixon plots, respectively. Accordingly, the K m for GHDGP and CHDGP were 57.6 ± 0.8 and 64.2 ± 1.4 μM, and the K i for GHDGP and CHDGP were 89.0 ± 2.0 and 70.8 ± 0.9 μM, respectively.

The Hypouricemic Effect of B. laxiflora Extracts and
Derived Phytochemicals in Hyperuricemic Mice. The hypouricemic effect of the EtOAc fraction and derived phytochemicals on PO-induced hyperuricemic mice is shown in Figure 5. In vehicle control mice, the serum uric acid level  The results represent the mean ± SEM (n = 6 or 7). Bars marked with different letters are significantly different from each other (P < 0.05). was 1.48 ± 0.13 mg/dL. In PO-induced mice (PO group), the serum uric acid level elevated to 2.31 ± 0.61 mg/dL after 3 h of PO injection. Thus, the serum uric acid level of PO group mice increased more than 1.5-fold as compared with the vehicle control. The administration of allopurinol (10 mg/kg) significantly reduced the serum uric acid level (0.40 ± 0.08 mg/dL) by 83% as compared with the PO group. At the same dosage (10 mg/kg), mice treated with the EtOAc fraction of B. laxiflora extracts (1.77 ± 0.60 mg/dL), GHDGP (1.43 ± 0.49 mg/dL), and CHDGP (1.08 ± 0.30 mg/dL) had uric acid levels that were significantly reduced by 23, 38 and 53%, respectively, relative to the PO group (P < 0.05). On the other hand, uric acid levels, between PO group and the animal treated with CFGP (1.89 ± 0.48 mg/dL) and CMGP (1.89 ± 0.57 mg/dL) at a concentration of 10 mg/kg, were not significantly different. Mo et al. [29] reported that at a dosage of 100 mg/kg quercetin, morin, myricetin, kaempferol, and icariin significantly reduced uric acid levels by 29, 31, 27, 35, and 13%, respectively. Wang et al. [30] reported that the administration of cinnamaldehyde (150 mg/kg) in hyperuricemic mice reduced 60% of the serum uric acid levels compared with the PO group. Comparisons of these results indicate that B. laxiflora extracts and their active components, GHDGP, and CHDGP, exhibit excellent hypouricemic effects. It is well known that XOD inhibitors are used to treat gout and hyperuricemia, which can hinder the enzymes involved in the synthesis of uric acid and reduce the formation of uric acid. Therefore, the hypouricemic effect of B. laxiflora extracts and derived phytochemicals may be due to their XOD-inhibitory activity. In addition, the in vivo lowering effect on uric acid was consistent with XODinhibitory activity, indicating that B. laxiflora extracts and derived phytochemicals possess potent hypouricemic effects and could be potential candidates for new hypouricemic agents.

Conclusions
In this study, the XOD-inhibitory activity and the hypouricemic effect of B. laxiflora extracts and derived phytochemicals were addressed for the first time. Four specific phytochemicals were isolated and identified from the EtOAc fraction of B. laxiflora extracts. The hydrolyzable tannins 1,3-di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-βd-glucopyranose (GHDGP) and 1-O-(E)-caffeoyl-4,6-(S)-hexahydroxydiphenoyl-β-d-glucopyranose (CHDGP) exhibited potent XOD-inhibitory activity via noncompetitive inhibition. Furthermore, the hypouricemic effect of B. laxiflora extracts and derived phytochemicals was determined using a PO-induced hyperuricemic mouse model. Our results also revealed that the hydrolyzable tannins GHDGP and CHDGP significantly reduced serum uric acid levels. Accordingly, our results suggest that the bioactive phytochemicals from B. laxiflora extracts may represent a new type of hypouricemic agent and provide a potent hypouricemic effect for clinical use.

Author's Contribution
S.-T. Ho and Y.-T. Tung contributed equally to this work.