Box-Behnken设计-响应面法结合G1-熵权法优化黄芪六一汤黄酮组分的提取纯化工艺

Optimization of extraction and purification process for flavonoids from Huangqi Liuyi Decoction based on Box-Behnken design-response surface methodology combined with G1-entropy method

目的 研究黄芪六一汤(Huangqi Liuyi Decoction,HLD,黄芪-甘草6∶1组成)黄酮组分的提取和聚酰胺树脂富集纯化工艺。方法 以毛蕊异黄酮葡萄糖苷、芹糖甘草苷、甘草苷、芹糖异甘草苷、芒柄花苷、甘草素、毛蕊异黄酮、异甘草素、芒柄花素9个成分为评价指标,以G1-熵权法确定各指标的组合权重,进行综合评分;采用Box-Behnken设计-响应面法(Box-Behnken design-response surface methodology,BBD-RSM)对HLD黄酮组分提取的关键影响因素乙醇体积分数、乙醇用量、提取时间、提取次数进行优化;采用单因素实验考察上样药液pH值、上样药液质量浓度、上样药液体积流量、上样量、水洗脱用量、洗脱剂乙醇体积分数、洗脱剂体积流量、洗脱剂用量等因素对黄酮组分聚酰胺富集纯化工艺的影响,采用Plackett-Burman设计(Plackett-Burman design,PBD)结合BBD-RSM筛选显著影响因素并对其进行工艺优化,比较聚酰胺树脂纯化前后各指标的含量。结果 最优提取工艺为69%乙醇回流提取2次,每次10倍量乙醇提取65 min;最优纯化工艺为pH值4.8、质量浓度0.20 g/mL的上样药液以0.8 mL/min体积流量按树脂-生药1∶1的质量比上样,4个柱体积(BV)纯水以1.5 mL/min的体积流量洗脱除杂,8 BV体积分数69%乙醇以1.5 mL/min体积流量洗脱,所得黄酮组分提取物中9个指标成分含量约是纯化前的14倍。结论 优选的HLD黄酮组分的提取和富集纯化工艺稳定、可行,可为进一步成药性研究和组分中药研制奠定基础。

Objective To study the extraction of flavonoids from Huangqi Liuyi Decoction(HLD,黄芪六一汤,Astragali RadixGlycyrrhizae Radix et Rhizoma 6:1) and the enrichment and purification process of polyamide resin.Methods Nine components of calycosin 7-O-β-D-glucopyranoside,liquiritin apioside,liquiritin,isoliquiritin apioside,ononin,liquiritigenin,calycosin,isoliquiritigenin and formononetin were used as evaluation indexes,and the combined weight of each index was determined by G1-entropy method,and the comprehensive score was made.Box-Behnken design-response surface methodology(BBD-RSM) was used to optimize the key factors affecting the extraction of flavonoids from HLD,such as ethanol volume fraction,ethanol dosage,extraction time and extraction times.The effects of pH value,mass concentration,volume flow rate,volume fraction of water,volume flow rate and amount of eluent on the enrichment and purification process of flavonoid component polyamide were investigated by single factor experiment.Plackett-Burman design(PBD) combined with BBD-RSM was used to screen the significant influencing factors,optimize the process,and compare the contents of each index before and after purification of polyamide resin.Results The optimal extraction process was that 69% ethanol was refluxed twice,and 10 times of ethanol was extracted for 65 min each time.The optimal purification process is as follows:the pH value is 4.8,the mass concentration is 0.20 g/mL,the volume flow rate is 0.8 mL/min,the resin-crude drug ratio is 1:1,the volume flow rate of four BV pure water is 1.5 mL/min,and the volume flow rate of eight BV 69% ethanol is 1.5m L/min.The content of nine index component in the obtained flavonoids extract is about 14 times higher than that before purification.Conclusion The optimized extraction,enrichment and purification process of flavonoids from HLD is stable and feasible,which can lay a foundation for further research on medicinal properties and development of component-based traditional Chinese medicine.