黄连解毒汤自沉淀化学成分及其抗PC12细胞损伤研究

Study on chemical constituents of self-settling from Huanglian Jiedu Decoction and effects on PC12 induced by CoCl_2

目的探讨黄连解毒汤(HJD)自沉淀制备条件,测定自沉淀主要成分量及自沉淀对PC12细胞损伤模型的保护作用。方法以提取时间和投水量为影响因素,确定自沉淀的制备工艺;采用HPLC法,测定自沉淀中黄芩苷、小檗碱、栀子苷、巴马汀、黄连碱、汉黄芩苷的量;采用CCK-8法评价HJD上清液、自沉淀对CoCl_2损伤的PC12细胞模型作用。结果在10倍量水回流提取30 min的煎煮条件下,干燥后HJD自沉淀质量约占上清液质量的10%,自沉淀中81.28%的成分为酸碱性化合物,其中黄芩苷占42.12%,小檗碱占31.17%,黄连碱占5.89%,汉黄芩苷占1.50%,巴马汀占0.60%,未检测到中性成分栀子苷。上清液、自沉淀对CoCl_2损伤的PC12细胞的EC50值分别28.25、19.58μg/m L。结论自沉淀制备工艺操作简单,重复性好,通过成分分析,得出自沉淀的形成机制以酸碱络合为主的假想;与上清液相比,自沉淀表现出较好的神经保护活性,为HJD的二次开发提供参考。

Objective To optimize the preparation conditions of precipitation from Huanglian Jiedu Decoction（HJD）. The main components of precipitation content were measured and the protective effect of compound precipitation on injured PC12 cell was evaluated. Methods The preparation process of precipitation was determined by time and water supply; Contents of baicalin, berberine, geniposide, palmatine, coptis, and wogonoside were determined by HPLC. The effect of HJD was evaluated and the precipitation on neurotoxic PC12 cells were injured by Co Cl2 with CCK-8 assay. Results Under the condition（extracted by refluxing for 30 min using the amount of water which was 10 times of the quality of herbs）, the quality of dry precipitation was about 10% of mass of the supernatant, and 81% of the precipitation were organic acids and alkaloids, baicalin was 42.12%, berberine was 31.17%, coptis was 5.89%, wogonoside was 1.50%, palmatine was 0.60%, and geniposide was not detected. EC50 of supernatant and precipitation on injured PC12 was 28.25 μg/m L and 19.58 μg/m L, respectively. Conclusion The preparation process of precipitation is simple and reproducible, and this study reveals that the compound precipitation from HJD is mainly composed of acid-alkali complex. Compared with the supernatant, the compound precipitation shows better neuroprotective activity, and provides basic data for the secondary development of HJD.