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目的:建立HPLC体外筛选黄嘌呤氧化酶抑制剂的方法。方法:采用HPLC法测定酶促反应体系中底物黄嘌呤在反应前后含量的变化,以此考察待测样品对黄嘌呤氧化酶的抑制作用。色谱条件:采用Agilent SB-C18色谱柱(4.6 mm“250mm,5#m),以0.02 mol·L-1磷酸二氢钾溶液(含甲醇1%)为流动相,流速1.0 mL·min-1,柱温为室温,检测波长254 nm,进样量20#L。结果:在该色谱条件下,酶促反应体系中的黄嘌呤与其他成分分离度良好。利用该方法对14种中药进行了筛选,发现使君子、苏木、虎杖和牡丹皮四味中药的70%乙醇提取物对黄嘌呤氧化酶抑制作用较强,其IC50分别为10.04、8.02、2.06和1.36 mg·mL-1。针对活性较好的苏木,对从其分离得到的24个化合物进行活性筛选,发现化合物7,3’,4’-三羟基-3-苄基-2氢-苯并吡喃、3-去氧苏木酮B、苏木查尔酮和原苏木素A具有一定的活性。结论:该方法简单可行,准确性较高,可用于黄嘌呤氧化酶抑制剂的快速筛选。
Objective: To establish a HPLC method for screening xanthine oxidase inhibitors in vitro. Methods: The content of xanthine in the enzymatic reaction system was determined by HPLC before and after reaction. The inhibitory effect of xanthine oxidase on the samples was investigated. Chromatographic conditions: An Agilent SB-C18 column (4.6 mm ”250 mm, 5 # m) with 0.02 mol·L-1 potassium dihydrogen phosphate solution containing 1% methanol as mobile phase at a flow rate of 1.0 mL · min- 1, the column temperature was at room temperature, the detection wavelength was 254 nm, and the injection volume was 20 # L.Results: Under the chromatographic conditions, the xanthine in the enzymatic reaction system was well separated from other components. The results showed that 70% ethanol extracts of Chinese herbal medicines such as Gentleman, Hematoxylin, cuspidatum and Cortex Moutan had stronger inhibitory effects on xanthine oxidase with IC50 of 10.04, 8.02, 2.06 and 1.36 mg · mL -1, respectively. For the more active hematoxylin, the 24 compounds isolated from them were screened for activity and found that the compound 7,3 ’, 4’-trihydroxy-3-benzyl-2-hydro-benzopyran, 3- Oxygenated hematoxylin B, hematoxylin and pro-hematoxylin A. CONCLUSION: The method is simple, feasible and accurate and can be used for the rapid screening of xanthine oxidase inhibitors.