熵权法结合星点设计-效应面法优化祛湿清肺方提取工艺及体外抗炎活性评价

目的:优化祛湿清肺方提取工艺,并对祛湿清肺方提取液进行体外抗炎活性评价。方法:以绿原酸、虎杖苷、黄芩苷、汉黄芩苷、黄芩素、芦荟大黄素、汉黄芩素、大黄素成分含量及得膏率为指标,加水量及提取时间为考察因素,采用熵权法结合星点设计-效应面法对祛湿清肺方提取工艺进行优化。以脂多糖(LPS)诱导大鼠腹腔巨噬细胞(RAW_264.7)为炎症模型,酶联免疫吸附法测定白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、一氧化氮(NO)的含量,蛋白质印迹法检测磷酸化NF-κB抑制蛋白激酶(phosphorylated inhibitor of NF-κB kinase,p-IKK)、磷酸化NF-κB p65(phosphorylated NF-κB p65,p-NF-κB p65)、NF-κB抑制蛋白α(inhibitor of NF-κBα,IκBα)蛋白表达水平变化,评价祛湿清肺方提取液抗炎活性。结果:星点设计-效应面法优化所得的最佳提取工艺为加水量13倍,提取2次,每次提取时间105 min。祛湿清肺方提取液能降低IL-6、IL-1β、TNF-α、NO的含量,抑制p-IKK、p-NF-κB p65蛋白表达,促进IκBα蛋白表达,具有较好的体外抗炎活性。结论:熵权法结合星点设计-效应面法优选的祛湿清肺方提取工艺稳定可行,所得提取液具有较好的抗炎活性,为祛湿清肺方开发和现代化研究提供参考奠定基础。

Optimization of extraction process for Qushi Qingfei Recipe by combination of central composite design-response surface methodology with entropy weight method and its anti-inflammatory activity

OBJECTIVE To optimize the extraction process for Qushi Qingfei Recipe and to evaluate its anti-inflammatory activity.METHODS The contents of chlorogenic acid,polygonin,baicalin,wogonoside,baicalein,aloeemodin,wogonin and emodin and the extraction amount were determined as indexes,and the amount of water added,extraction time were used as evaluation indicators.Entropy weight method with combination of central composite design-response surface methodology was used to optimize the optimum water extraction process parameters of Qushi Qingfei Recipe.The inflammation model was established by using LPS induced RAW_264.7 cells in vitro,enzyme linked immunosorbent assay method was applied to detect the contents of interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and nitric oxide(NO),the expression of phosphorylated inhibitor of NF-κB kinase(p-IKK),phosphorylated NF-κB p65(p-NF-κB p65),inhibitor of NF-κBα(IκBα)was detected by Western blot,to evaluate the anti-inflammatory activity.RESULTS The optimal technology extraction conditions were:13-folds of water,extraction of 2 times,105 min per time.Qushi Qingfei extracting solution could decrease the level of IL-6,IL-1β,TNF-α and NO,inhibit the expression of p-IKK,p-NF-κB p65 and promote the expression of IκBα protein,and had great anti-inflammatory activity.CONCLUSION The preferred process has high extraction rate.Under this condition,Qushi Qingfei Recipe extracting solution was proved to have great anti-inflammatory activity.It provides reference for the development and modernization of Qushi Qingfei Recipe formula and lays a foundation.