目的 运用生物效价测定方法评价大黄Rhei Radix et Rhizoma中10个蒽醌衍生物拮抗血小板聚集作用的强弱差异，筛选可用于酒大黄饮片质量评控的指标性成分。方法 采用血小板聚集仪测定体外二磷酸腺苷（ADP）诱导的血小板聚集率，计算10个蒽醌衍生物（芦荟大黄素-8-O-β-D-葡萄糖苷、大黄酸-8-O-β-D-葡萄糖苷、大黄酚-8-O-β-D-葡萄糖苷、大黄素-8-O-β-D-葡萄糖苷、大黄素甲醚-8-O-β-D-葡萄糖苷、芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚）不同浓度下的血小板聚集抑制率，采用质反应生物效价软件计算生物效价；采用分子对接软件计算得到大黄酸、大黄酚-8-O-β-D-葡萄糖苷与P2Y₁₂蛋白受体在最佳结合时的估计抑制常数（K_i），以此验证生物效价测定结果的准确性。结果 活血效价测定结果显示，大黄酸、大黄素的活血效价显著高于芦荟大黄素、大黄酚、大黄素甲醚的活血效价；大黄酸、大黄素的活血效价分别是阿司匹林活血效价的5.02、5.15倍，表明大黄酸、大黄素拮抗ADP诱导的血小板聚集作用较强。芦荟大黄素、大黄酚、大黄素甲醚的活血效价与阿司匹林的活血效价相当。芦荟大黄素-8-O-β-D-葡萄糖苷、大黄酸-8-O-β-D-葡萄糖苷、大黄素-8-O-β-D-葡萄糖苷、大黄酚-8-O-β-D-葡萄糖苷、大黄素甲醚-8-O-β-D-葡萄糖苷的活血效价分别是阿司匹林活血效价的4.13、4.46、9.31、5.46、7.80倍，表明5个结合型蒽醌糖苷拮抗ADP诱导的血小板聚集作用较强。分子对接结果显示，大黄酸、大黄酚-8-O-β-D-葡萄糖苷对P2Y₁₂蛋白有较高的亲和力，二者的K_i分别为5.73、2.51μmol/L，表明大黄酸、大黄酚-8-O-β-D-葡萄糖苷在较低的浓度水平即可对P2Y₁₂蛋白受体产生作用，且大黄酚-8-O-β-D-葡萄糖苷的活性强于大黄酸的活性，这与实际测得二者的抗血小板聚集作用强度相符合。结论 大黄药材中10个蒽醌衍生物的活血生物效价存在较大差异，筛选得到大黄酸、大黄素可作为酒大黄炮制过程品质评价的监控指标。

Objective To evaluate the ability of the antiplatelet aggregation of ten anthraquinone derivatives in Rhei Radix et Rhizoma by using bioassay method, and to screen for the indicators that can be used to control the quality of rhubarb wine processing product. Methods Platelet aggregation instrument was used to determine the platelet aggregation rate induced by ADP in vitro and calculate the antiplatelet aggregation rates of 10 anthraquinone derivatives (aloe-emodin, rhein, emodin, chrysophanol, physcion, aloe-emodin-8-O-β-D-glucoside, rhein-8-O-β-D-glucoside, emodin-8-O-β-D-glucoside, chrysophanol-8-O-β-D-glucoside, and physcion-8-O-β-D-glucoside) at different concentrations. The biopotency was calculated by bioavailability software. In order to verify the accuracy of the bioassay results, the estimated inhibition constant of rhein and chrysophanol-8-O-β-D-glucoside in P2Y₁₂ protein receptor were determined by molecular docking software. Results The bioassay results showed that the antiplatelet potencies of rhein and emodin were significantly higher than those of aloe-emodin, chrysophanol and physcion. Compared with the antiplatelet biopotency of aspirin, the antiplatelet potencies of rhein and emodin were 5.02 and 5.15 times higher than that of aspirin, which indicated that rhein and emodin have strong inhibitory effect on ADP-induced platelet aggregation. However, the antiplatelet potencies of aloe-emodin, chrysophanol and physcion were equivalent of that of aspirin. The antiplatelet potencies of aloe-emodin-8-O-β-D-glucoside, rhein-8-O-β-D-glucoside, emodin-8-O-β-D-glucoside, chrysophanol-8-O-β-D-glucoside, and physcion-8-O-β-D-glucoside were higher 4.13, 4.46, 9.31, 5.46, and 7.80 times than that of aspirin, respectively, which indicated that the five anthraquinone glucosides had a strong ability to antagonize ADP-induced platelet aggregation. The results of molecular docking showed that P2Y₁₂ protein had different selectivity to 10 anthraquinone derivatives, especially for rhein, chrysophanol-8-O-β-D-glucoside, and the estimated K_i value were 5.73 and 2.51 μmol/L, respectively, indicating that rhein, chrysophanol-8-O-β-D-glucoside produced a strong inhibitory effect on P2Y₁₂ protein at a lower concentration level. Moreover, the activity of chrysophanol-8-O-β-D-glucoside was stronger than rhein, which was consistent with their measured intensity of antiplatelet aggregation. Conclusion All results showed that there were some differences among antiplatelet potencies of 10 anthraquinone derivatives, and finally the screening of rhein, emodin can be used as evaluation indicators to control the quality of rhubarb wine processing product.

国家自然科学基金项目资助（81403126）；国家科技基础性工作专项（2015FY111500-080）；成都市科技局项目（2016-HM01-00312-SF）；成都中医药大学重点实验室开放基金（2016302）