天山雪莲悬浮培养细胞总黄酮提取纯化工艺研究

Extraction and purification of total flavonoids in suspension cultured cells of Saussurea involucrata

【目的】分析天山雪莲悬浮培养细胞生长过程中生物量和总黄酮含量的变化,优化总黄酮的提取纯化方法。【方法】采用L9(34)正交设计优化天山雪莲悬浮培养细胞中总黄酮的超声提取工艺,比较聚酰胺层析法、碱提酸沉法和聚酰胺酸碱法对总黄酮含量、纯度及回收率的影响。【结果】在3～15d,随着培养时间的增加,天山雪莲悬浮培养细胞的生物量和总黄酮含量明显增加,在培养15d时均较高,分别为21.9g/L和66.5mg/g,表明天山雪莲悬浮培养细胞最适收获时间为培养15d;总黄酮提取最佳工艺为:70℃超声条件下,采用体积分数70%的乙醇连续提取3次,可以得到66.5mg/g总黄酮;总黄酮纯化的最佳方法为:聚酰胺层析法和碱提酸沉法联用,即聚酰胺酸碱法,获得的总黄酮纯度和回收率均较高,分别为6.86g/kg和47.6%。【结论】获得了天山雪莲悬浮培养细胞总黄酮提取和纯化的最佳方法,为天山雪莲药制品的工业化生产提供了参考。

This study focused on the variations of biomass and flavonoids contents during cell growth of suspension cultured Saussurea involucrata cells,and the optimization of total flavonoids extraction and purification technology.【Method】L9（34） orthogonal design was used to optimize the ultrasonic extraction process of total flavonoids from the suspension cell,and three extraction methods,polyamide column chromatography,alkali extraction and acid precipitation,and combination of polyamide column chromatography and alkali extraction and acid precipitation,were compared.【Result】As the increase of culturing time,the biomass and total flavonoids contents of S.involucratain suspension cultured cells were 21.9g/L and 66.5mg/g at the 15th day,respectively,indicating the best harvest time was the 15th day.The best extraction conditions of total flavonoids were：ethanol solutions（volume fraction 70%）,and three times ultrasound treatment at 70℃ with a production rate of 66.5mg/g total flavonoids.Combination of polyamide column chromatography and alkali extraction and acid precipitation was the best purification process with the highest purity and recovery of refined flavonoids of 6.86g/kg and 47.6%,respectively.【Conclusion】This study obtained the best process to extraction and purification of total flavonoids from suspension cultured S.involucratacells.