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目的建立高效液相色谱法(HPLC)测定过山枫药材中扁蒴藤素含量的方法,研究扁蒴藤素对人类风湿关节炎滑膜成纤维细胞(HFLS-RA)活性和迁移能力的影响。方法采用C18色谱柱(4.6 mm×250 mm,5μm),甲醇-0.1%磷酸水为流动相进行梯度洗脱,流速1.40 m L·min-1,柱温30℃,检测波长424 nm;MTT法测定HFLS-RA的活性;划痕实验观察扁蒴藤素对HFLS-RA迁移的影响。结果扁蒴藤素在浓度为0.08~0.64g·L-1呈良好的线性关系(r=0.9995),平均回收率为101.09%,RSD=2.58%;浓度为0.50~1.0μmol·L-1的扁蒴藤素,抑制HFLS-RA活性具有剂量和时间的依赖性关系,能够明显减少血管内皮生长因子(VEGF)诱导HFLS-RA的迁移。结论所建立的扁蒴藤素含量测定方法简便、准确,可用于过山枫药材质量的控制;扁蒴藤素具有抑制HFLS-RA活性和迁移的作用。
OBJECTIVE To establish a HPLC method for the determination of pristigenin in Hypericum japonicum and to study the effect of Pristimerin on the activity and migration of human rheumatoid arthritis synovial fibroblasts (HFLS-RA) . Methods The mobile phase consisted of C18 column (4.6 mm × 250 mm, 5 μm) and gradient elution of methanol-0.1% phosphoric acid in water. The flow rate was 1.40 m L · min-1 and the column temperature was 30 ℃. The detection wavelength was 424 nm. The activity of HFLS-RA was measured. The scratch test was used to observe the effect of Pristimerin on the migration of HFLS-RA. RESULTS Pristinone showed a good linearity (r = 0.9995) at a concentration of 0.08-0.64 g · L -1, with an average recovery of 101.09% and a RSD of 2.58%. A linear range of 0.50-1.0 μmol·L -1 Pristimerin inhibits the HFLS-RA activity in a dose-and time-dependent manner, and can significantly reduce the vascular endothelial growth factor (VEGF) -induced migration of HFLS-RA. Conclusion The established determination of Pristimerin content is simple and accurate and can be used for the quality control of Prunus mongolica. Pristimerin can inhibit the activity and migration of HFLS-RA.