Box-Behnken设计—响应面法优化地参总三萜酸纯化工艺

Purification process of total triterpenic acids from Lycopus lucidus Turcz. by Box-Behnken design-response surface methodology

  • 摘要: 【目的】建立响应面模型优化合适树脂下地参总三萜酸的纯化工艺,为研究地参总三萜酸活性提供技术参考。【方法】运用Box-Behnken设计方法,采用大孔树脂吸附纯化地参总三萜酸,以吸附率和解吸率为指标,测定5种大孔树脂(AB-8、D101、HPD600、DA201和S-8型)对地参总三萜酸的纯化效果。以上样浓度、上样流速、洗脱浓度和洗脱流速为自变量,回收率为因变量,运用响应面法优化地参总三萜酸纯化工艺参数。【结果】最佳显色条件:显色温度60℃,5%香草醛—冰乙酸0.2 mL,高氯酸0.4 mL,检测波长545 nm。AB-8型树脂对地参总三萜酸的吸附和解吸效果最佳,解吸率为88.61%,吸附率为70.54%。上样体积在40 mL,80%乙醇100 mL为最佳洗脱体积。建立的回归方程: Y=0.6887+0.0196A-0.0085B-0.0287C+0.0052D+0.0299AB-0.0107AC-0.0122AD+0.0128BC+0.0211BD+0.026A2-0.0765B2-0.0877C2-0.0932D2 (A为上样浓度,B为上样流速,C为洗脱浓度,D为洗脱流速,Y为总三萜酸回收率)。由Box-Behnken方差分析结果可得,上样浓度和洗脱浓度2个因素,以及上样浓度与上样流速的交互作用、洗脱浓度与洗脱流速的交互作用对地参总三萜酸回收率影响极显著(P<0.01),上样流速与洗脱流速的交互作用对总三萜酸回收率影响显著(P<0.05),4个因素对地参总三萜酸回收率影响顺序为洗脱浓度>上样浓度>上样流速>洗脱流速。优化后的地参总三萜酸纯化工艺:上样浓度0.66 mg/mL、上样流速2 BV/h、洗脱浓度80%、洗脱流速2 BV/h,在此条件下,总三萜酸平均回收率为67.37%。【结论】建立的回归模型可用于地参总三萜酸纯化工艺的预测; AB-8型大孔树脂对地参总三萜酸的分离纯化效果较好,适用于总三萜酸物质的纯化。

     

    Abstract: 【Objective】 A response surface model was established to optimize the purification process of total triterpenic acids from Lycopus lucidus Turcz. with suitable resin, so as to provide technical reference for further study on the activity of total triterpenic acids from L. lucidus.【Method】Using the Box-Behnken design method,the total triterpenic acids of L. lucidus was purified by macroporous resin adsorption. The purification effects of macroporous resins(AB-8 type, D101 type,HPD600 type,DA201 type and S-8 type)on total triterpenic acids of L. lucidus were determined by using the adsorption rate and desorption rate as indexes. The sample concentration, sample loading speed,eluent concentration and eluentvelocity were the independent variables,and recovery rate was the dependent variable. The purification process parameters of total triterpenic acids from L. lucidus were optimized by response surface methodology.【Result】 Optimum color development conditions were:color development temperature of 60 ℃,5% vanillin-glacial acetic acid of 0.2 mL,perchloric acid of 0.4 mL,and detection wavelength of 545 nm. The results showed that AB-8 type macroporous resin had the best adsorption and desorption effectsontotal triterpenic acids of L. lucidus,withthe desorption rate of 88.61% and the adsorption rate of 70.54%. The loading volume was 40 mL,and 80% ethanol 100 mL was the best eluent volume. A regression equation was set up: Y=0.6887+0.0196A-0.0085B-0.0287C+0.0052D+0.0299AB-0.0107AC-0.0122AD+0.0128BC+ 0.0211BD+0.026A2-0.0765B2-0.0877C2-0.0932D2 (A: sample concentration, B: sample loading speed, C: eluent concentration, D: eluent velocity, Y: recovery rate of total triterpenic acids). According to Box-Behnken variance analysis, sample concentration,eluent concentration, the interaction between sample concentration and sample loading speed, and the interaction between eluent concentration and eluent velocity had extremely significant effects on the recovery rate of total triterpenic acids from L. lucidusP<0.01),while the interaction between sample loading speed and eluent velocity had significant effects on the recovery rate of total triterpenic acids from L. lucidusP<0.05). The four single factors affected the recovery rate of total triterpenic acids of L. lucidus in the order of eluent concentration>sample concentration>sample loading speed>eluent velocity from L. lucidus. The optimized purification process for total triterpenic acids of L. lucidus was:sample concentration of 0.66 mg/mL,sample loading speed of 2 BV/h,eluent concentration of 80% and from L. lucidus of 2 BV/h. Under these conditions,the average recovery rate of total triterpenic acids was 67.37%.【Conclusion】The established regression model can be used to predict the purification process of total triterpenic acids from L. lucidus;the AB-8 type macroporous resin has a relatively good effect on the separation and purification of total triterpenic acids from L. lucidus,which is suitable for the purification of total triterpenic acids.

     

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