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

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

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(RAW264.7)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。

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 RAW264.7cells 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.