Natural Xanthine Oxidase Inhibitor 5- O-Caffeoylshikimic Acid Ameliorates Kidney Injury Caused by Hyperuricemia in Mice

Abstract

Xanthine oxidase (XOD) inhibition has long been considered an effective anti-hyperuricemia strategy. To identify effective natural XOD inhibitors with little side effects, we performed a XOD inhibitory assay-coupled isolation of compounds from Smilacis Glabrae Rhizoma (SGR), a traditional Chinese medicine frequently prescribed as anti-hyperuricemia agent for centuries. Through the in vitro XOD inhibitory assay, we obtained a novel XOD inhibitor, 5-O-caffeoylshikimic acid (#1, 5OCSA) with IC₅₀ of 13.96 μM, as well as two known XOD inhibitors, quercetin (#3) and astilbin (#6). Meanwhile, we performed in silico molecular docking and found 5OCSA could interact with the active sites of XOD (PDB ID: 3NVY) with a binding energy of -8.6 kcal/mol, suggesting 5OCSA inhibits XOD by binding with its active site. To evaluate the in vivo effects on XOD, we generated a hyperuricemia mice model by intraperitoneal injection of potassium oxonate (300 mg/kg) and oral gavage of hypoxanthine (500 mg/kg) for 7 days. 5OCSA could inhibit both hepatic and serum XOD in vivo, together with an improvement of histological and multiple serological parameters in kidney injury and HUA. Collectively, our results suggested that 5OCSA may be developed into a safe and effective XOD inhibitor based on in vitro, in silico and in vivo evidence.

Keywords: 5-O-caffeoylshikimic acid; Smilacis Glabrae Rhizoma; XOD inhibitor; hyperuricemia; potassium oxonate.

Figure 1

Bio-assay guided fractionation and isolation from SGR. Schematic isolation procedures of fractions and compounds from SGR were described in (a). Structures of the chemicals (#1–6) were shown in (b). Inhibition rates of 10 fractions (c) and 6 compounds (d) on XOD were evaluated and plotted by Graphpad. Values were presented as mean ± SEM of five replicates. SGR: Smilacis Glabrae Rhizoma; CEE: Crude Ethanol Extracts; EAF: EtOAc Fractions; BUF: n-BuOH Fractions; CMF: CHCl₃-MeOH Fractions.

Figure 2

Interaction between 5OCSA and XOD in vitro and in silico. (a) IC₅₀ of 5OCSA and AP on XOD. Plots of v (∆OD₂₉₅/min)/XOD concentration with different concentrations of 5OCSA (b). Predicted binding mode of 5OCSA docked into XOD. 3D and 2D docking patterns of 5OCSA (c) and quercetin (d) into 3NVY has been generated by Discovery Studio 4.5. 5OCSA: 5-O-caffeoylshikimic acid; XOD: xanthine oxidase; AP: allopurinol.

Figure 3

Effects of different doses of 5OCSA on BWs and organ coefficients in HUA mice. (a) Changes of BWs in different groups (n = 8 in each group). Coefficients of liver (b) and kidney (c) were calculated and plotted via GraphPad. ** p < 0.01, compared with saline group; ∆ p < 0.05; ∆∆ p < 0.01, compared with HUA group. 5OCSA-L: low dose of 5-O-caffeoylshikimic acid; 5OCSA-M: medium dose of 5OCSA; 5OCSA-H: high dose of 5OCSA; BWs: bodyweights; HUA: hyperuricemia.

Figure 4

Effects of 5OCSA on sUA, XOD and markers of kidney damage in HUA. (a) Serum level of uric acid (μmol/L). (b) Serum XOD activity (U/g Protein). (c) Hepatic XOD activity (U/g Protein). (d) Serum creatinine (μmol/L). (e) Serum BUN (mmol/L). ** p < 0.01, *** p < 0.005, compared with saline group; ∆ p < 0.05; ∆∆ p < 0.01, ∆∆∆ p < 0.005, compared with HUA group. sUA: serum uric acid; XOD: xanthine oxidase; BUN: blood urea nitrogen.

Figure 5

Protective effects of 5OCSA on kidney histomorphometry change caused by PO/HX in mice. The right kidney of mice was fixed, embedded in paraffin, sectioned, and stained by H&E. Arrows indicate the enlarged vacuoles in renal tubular epithelial cells. 100×: 100-fold magnification; 400×: 400-fold magnification; 5OCSA: 5-O-caffeoylshikimic acid; 5 OCSA-L: low dose of 5OCSA; 5OCSA-M: medium dose of 5OCSA; 5OCSA-H: high dose of 5OCSA.

Figure 6

Effects of 5OCSA on kidney inflammation cytokines in HUA mice. (a) TNF-α (pg/mg Protein); (b) IL-1β (pg/mg Protein); (c) IL-6 (pg/mg Protein); (d) IL-18 (pg/mg Protein). Data are represented as mean ± SD (n = 8). ** p < 0.01, *** p < 0.005, compared with saline group; ∆ p < 0.05; ∆∆ p < 0.01, ∆∆∆ p < 0.005, compared with HUA group. TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; IL-6: interleukin-6; IL-18: interleukin-18.