补阳还五汤质量评价及其活性成分羟基红花黄色素A对内皮细胞损伤保护作用的研究

Quality evaluation of Buyang Huanwu Decoction and the protective effect of hydroxy-safflower yellow A on endothelial cell injury

目的建立同时测定补阳还五汤中毛蕊异黄酮葡萄糖苷、阿魏酸、羟基红花黄色素A、芍药苷、苦杏仁苷5种成分的高效液相色谱(HPLC)分析方法,并研究羟基红花黄色素A对内皮细胞损伤的作用。方法采用HPLC法对补阳还五汤中的5种成分进行质量分数测定及方法学考察。细胞计数试剂(cell counting kit-8,CCK8)法检测羟基红花黄色素A不同浓度对EA.hy926细胞的细胞毒性;qRT-PCR法检测羟基红花黄色素A对TNF-a诱导的EA.hy926细胞损伤中TF、IL-6、IL-1β、MCP-1的mRNA水平。结果5种成分线性关系均良好,相关系数(R2)均在0.9950以上;平均加样回收率在93.80%~101.48%之间,RSD均小于3%,表明方法准确度良好;5种成分质量分数的RSD值在1.78%~2.59%之间。TNF-a(10 ng/mL)刺激EA.hy926细胞后,TF、IL-6、IL-1β、MCP-1的mRNA水平显著升高(P<0.05),而羟基红花黄色素A干预后呈浓度依赖性下调(P<0.05)。结论该方法专属性强、重复性好,稳定、可控,可为补阳还五汤的质量评价提供参考。其活性成分羟基红花黄色素A可改善TNF-a诱导的Ey.hA926内皮细胞损伤,为阐明补其药效物质基础提供参考。

Objective To establish A high performance liquid chromatography(HPLC)method for simultaneous determination of Calycosin-7-O-beta-D-glucoside,ferulic acid,hydroxy-safflower Yellow A,paeoniflorin and amygdalin in Buyang Huanwu Decoction,and to study the effect of hydroxy-safflowerYellow A on endothelial cell injury.Methods The content of 5 components in Buyang Huanwu Decoction was determined by HPLC and the methodology was investigated.The cytotoxicity of hydroxy-safflower yellow A at different concentrations to EA.hy926 cells was detected by CCK8 assay.The mRNA levels of TF,IL-6,IL-1βand MCP-1 in TNF-a induced EA.hy926 cells were detected by qRT-PCR.Results All the five components had good linear relationship,and the correlation coefficient(R2)was above 0.9950.The average recoveries ranged from 93.80%to 101.48%,and RSD was all lower than 3%,indicating that the method was accurate.The RSD of the five components measured by extrinsic standard method was 1.78%-2.59%.The mRNA levels of TF,IL-6,IL-1βand MCP-1 were significantly increased after TNF-a(10 ng/mL)stimulation of EA.hy926 cells(P<0.05),and hydroxy-safflower yellow A was down-regulated in a concentration-dependent manner after intervention(P<0.05).Conclusion The method has strong specificity,good repeatability,stability and controllability,which can provide reference for the quality evaluation of Buyang Huanwu Decoction.Its active ingredient hydroxy-safflower yellow A can improve the TNF-a-induced Ey.hA926 endothelial cell injury,which provides experimental reference for elucidating the material basis of Buyang Huanwu Decoction.