响应面法优化大孔树脂纯化仿刺参中皂苷工艺研究

Optimization of Purification Process of Saponins from Sea Cucumber Apostichopus japonicus with Macroporous Resin by Response Surface Methodology

为优化大孔树脂纯化皂苷Holotoxin A_(1)(HA_(1))的最佳工艺条件,选择常用于纯化海参皂苷的4种不同结构的大孔树脂(AB-8、HP-20、D101和NKA-9),以皂苷HA_(1)为标准品,采用高效液相色谱法测定HA_(1)的含量,通过比较其静态吸附-解吸能力和静态吸附曲线,筛选合适树脂后,以粗皂苷质量浓度、乙醇体积分数和醇洗体积为因素,在单因素试验的基础上,采用Box-Behnken响应面试验确定最佳动态纯化工艺条件。试验结果显示,AB-8树脂对HA_(1)纯化效果最好,最佳纯化工艺为:粗皂苷质量浓度25mg/mL、流速1mL/min、上样体积25mL、水洗体积2倍柱体积、乙醇体积分数73%、醇洗体积7倍柱体积,此条件下,HA_(1)回收率为91.40%,纯度由0.16%提高至0.58%。本试验得到的纯化工艺适用于仿刺参中皂苷HA_(1)的纯化。

In order to optimize the optimum process conditions for purifying saponin Holotoxin A_(1)(HA_(1))by macroporous resin,four different structural macroporous resins(AB-8,HP-20,D101 and NKA-9)commonly used for purification of saponins in sea cucumber Apostichopus japonicus were selected.The content of HA_(1)was determined by high performance liquid chromatography(HPLC)using sea cucumber saponin HA_(1)as a standard.By comparing their static adsorption-desorption capacity and static adsorption curves,the suitable resin type was selected.The crude saponin mass concentration,ethanol volume fraction and ethanol washing volume were used as factors to determine the optimal dynamic purification process conditions based on the single factor experiments and Box-Behnken response surface experiment.The results showed that AB-8 resin had the best purification effect on HA_(1),and the optimal purification process was as follows:crude saponin mass concentration of 25 mg/mL,flow rate 1 mL/min,sample loading volume 25 mL,washing volume 2 times column volume(BV),ethanol volume fraction 73%,and ethanol washing volume 7 BV.Under these conditions,the recovery rate of HA_(1)was 91.40%,and the purity was increased from 0.16%to 0.58%.The purification process obtained in this study is suitable for the purification of saponin HA_(1)in Apostichopus japonicus.