HPLC-PDA联合Box-Behnken响应面法优选五苓散低极性部位提取工艺

Optimization of low polarity components extraction process of Wuling Powder by HPLC-PDA combined with Box-Behnken response surface methodology

目的 应用HPLC-PDA联合响应曲面法,筛选五苓散低极性部位乙醇提取最佳工艺。方法 采用多波长切换定量测定,以23-乙酰泽泻醇B、23-乙酰泽泻醇C、麦角甾醇、茯苓酸、白术内酯Ⅰ、白术内酯Ⅱ、白术内酯Ⅲ、肉桂酸、桂皮醛9种有效成分为评价指标,采用Box-Behnken响应曲面法考察提取乙醇体积分数、料液比、提取时间对提取工艺的影响,确定最佳工艺。结果 色谱柱为Agilent Proshell 120 EC-C18柱(100 mm×4.6 mm,2.7μm),体积流量为0.8 mL/min,柱温为40℃。流动相为乙腈-0.1%磷酸水溶液,梯度洗脱:0~10 min,37%~60%乙腈;10~20 min,60%乙腈;20~23 min,60%~95%乙腈;23~36 min,95%乙腈;36~37 min,95%~37%乙腈;37~42 min,37%乙腈。检测波长为280、247、220、208 nm多波长切换。最佳提取工艺为10倍量75%乙醇提取2次,每次1.5 h。结论 建立的HPLC-PDA法稳定可行,适合多种成分同时测定。响应曲面法所建模型准确,可以较好地预测9种成分含量综合评分;优选的提取工艺方法重现性良好,稳定可行,为五苓散临床应用、药效物质基础研究和新药开发提供技术参考。

Objective The optimal ethanol extraction process for the low polarity components of Wuling Powder(五苓散)was established using HPLC-PDA combined response surface method.Methods The quantitative analysis method of HPLC-PDA multi-wavelength switching was used to analyze the contents of nine active components including 23-acetate alisol B,23-acetate alisol C,ergosterol,pachymic acid,atractylenolideⅠ,atractylenolideⅡ,atractylenolideⅢ,cinnamic acid and cinnamaldehyde.Box-Behnken response surface methodology was used to investigate the effects of extraction ethanol volume fraction,solid-liquid ratio and extraction time on Wuling Powder low polarity components extraction process to determine the optimal extraction process.Results The determination was performed on an Agilent Proshell 120 EC-C18 column(100 mm×4.6 mm,2.7μm)with a volume flow rate of 0.8 mL/min and a column temperature of 40℃.The mobile phase consisted of acetonitrile-0.1%phosphoric acid aqueous solution with gradient elution:0-10 min,37%-60%acetonitrile;10-20 min,60%acetonitrile;20-23 min,60%-95%acetonitrile;23-36 min,95%acetonitrile;36-37 min,95%-37%acetonitrile;37-42 min,37%acetonitrile.The detection wavelength was multi-wavelength switched for 280,247,220,208 nm.Conclusion The established HPLC-PDA method is stable and suitable to analyze multiple components.The model established by response surface method is accurate and can be used to predict the comprehensive score of nine components well.The optimized extraction method had good reproducibility,stability and feasibility,which provided technical reference for clinical application,research of effective substances and development of new medicine of Wuling Powder.