萸连汤的抗炎活性“制剂质量标志物”研究

Research of preparation quality markers of Yulian Tang with anti-inflammatory activity

该研究以LPS诱导的RAW264.7细胞炎症模型为载体,观察萸连汤抗炎活性“制剂质量标志物”的体外抗炎作用。以LPS刺激RAW264.7细胞制备体外炎症模型,随机分为对照组,LPS组(50 ng·mL^(-1)),萸连汤中18种化合物低(0.1μmol·L^(-1)+LPS 50 ng·mL^(-1))、中(1μmol·L^(-1)+LPS 50 ng·mL^(-1))、高(10μmol·L^(-1)+LPS 50 ng·mL^(-1))剂量组,CCK-8法检测萸连汤中18种化合物干预RAW264.7细胞24 h的活力;ELISA法检测RAW264.7细胞上清中炎症因子TNF-α和IL-6的含量。结果显示,随着浓度的升高对TNF-α抑制率升高的化合物有小檗碱、黄连碱、木兰花碱、表小檗碱、非洲防己碱、木香烃内酯;随着浓度的升高对TNF-α抑制率降低的化合物有柠檬苦素、去氢吴茱萸碱、绿原酸、新绿原酸、格兰地新碱、吴茱萸碱、吴茱萸次碱、黄柏碱;浓度变化对TNF-α抑制率影响不大的化合物有巴马汀、药根碱、去氢木香烃内酯及隐绿原酸。随着浓度的升高对IL-6抑制率升高的化合物有去氢吴茱萸次碱、绿原酸、新绿原酸、吴茱萸碱、吴茱萸次碱、木香烃内酯、黄柏碱、隐绿原酸;随浓度变化对IL-6抑制率基本不变的有小檗碱、黄连碱、木兰花碱、表小檗碱、柠檬苦素、非洲防己碱、格兰地新碱、去氢木香烃内酯;对IL-6抑制率出现最大浓度的化合物有巴马汀、药根碱。结合萸连汤的定性、定量“制剂质量标志物”及量效实验结果,确定其抗炎活性“制剂质量标志物”15种,分别为小檗碱、黄连碱、巴马汀、木兰花碱、表小檗碱、柠檬苦素、非洲防己碱、药根碱、新绿原酸、绿原酸、格兰地新碱、吴茱萸碱、吴茱萸次碱、去氢木香烃内酯、木香烃内酯。综上所述,研究提供了一种快速筛选萸连汤的抗炎活性“制剂质量标志物”的策略,为萸连汤另外3种活性“制剂质量标志物”的确定明确了路径。

The lipopolysaccharide(LPS)-indused RAW264.7 cells inflammation model was used as a carrier to investigated the effects of the preparation quality markers of Yulian Tang with anti-inflammatory activity in vitro. RAW264.7 cells were treated with LPS(50 ng·mL^(-1)) or/and different concentrations(low dose 0.1 μmol·L^(-1);medium dose 1 μmol·L^(-1);high dose 10 μmol·L^(-1)) of 18 chemical components in Yulian Tang for 24 h. Then the activity of RAW264.7 cell was detected using Cell Counting Kit-8(CCK-8) and the concentrations of inflammatory factors TNF-α and IL-6 in the supernatant of RAW264.7 cell were detected by ELISA assay. As the concentrations of chemical components in Yulian Tang increased, berberine, coptisine, magnoflorine, epiberberine, columbamine and costunolide had stronger inhibitory effects on TNF-α, whereas limonin, dehydroevodiamine, chlorogenic acid, neochlorogenic acid, groenlandicine, evodiamine, rutaecarpine and phellodendrine showed weakened inhibitory effects on TNF-α. The concentrations of palmatine, jatrorrhizine, dehydrocostus lactone and cryptochlorogenic acid had no significant effect on their inhibitory effect on TNF-α. Furthermore, dehydrorutaecarpine, chlorogenic acid, neochlorogenic acid, evodiamine, rutaecarpine, costunolide, phellodendrine and cryptochlorogenic acid showed stronger inhibitory effect on IL-6 as their concentrations increased;berberine, coptisine, magnoflorine, epiberberine, limonin, columbamine, groenlandicine and dehydrocostus lactone had no changes in their inhibitory effects on IL-6 as the concentrations increased. Palmatine and jatrorrhizine had the best inhibitory effect on IL-6. Combining the previous analysis of qualitative and quantitative preparation quality markers of Yulian Tang with the above result of dose-response relationship, we finally identified 15 preparation quality markers of Yulian Tang with anti-inflammatory activity, namely berberine, coptisine, palmatine, magnoflorine, epiberberine, limonin, columbamine, jatrorrhizine, neochlorogenic acid, chlorogenic acid, groenlandicine, evodiamine, rutaecarpine, dehydrocostus lactone and costunolide. In conclusion, our study provides a quick strategy for screening the qualitative preparation quality markers of Yulian Tang with anti-inflammatory activity. Moreover, it also provides an explicit route for the determination of preparation quality markers of Yulian Tang with other activities.