响应面法优化丹参中抗无乳链球菌活性成分的提取工艺

Optimization of the Extraction Conditions of Active Components for Inhibition of Streptococcus agalactiae from Salvia miltiorrhiza Bge. Using Response Surface Methodology

在单因素提取工艺优化的基础上,选取液料比、浸润时间、提取时间和提取温度进行四因素三水平的Box-behnken实验设计,并结合响应面分析法进一步优化丹参中抗无乳链球菌(Streptococcus agalactiae,SA)活性成分提取工艺。优化所得最佳提取工艺为液料比37.8:1.0(ml:g)、浸润时间104 min、提取温度86.7℃、提取时间182 min。响应面模型预测在该提取工艺下抗SA活性成分提取得率极值为25.95%,经4次平行验证实验的实际提取得率为(25.87±0.03)%,为优化前的2.59倍,表明优化方法高效可靠。

In recent years Streptococcus agalactiae（SA） has become the most harmful pathogen to cultured tilapia in Hainan and many southern provinces of China. Salvia miltiorrhiza Bge is highly resistant to SA, but it contains complex components and which component is responsible for the resistance to SA has not been reported. Here we investigated the effects of extraction conditions on the yield of anti-SA components in S. miltiorrhiza Bge and optimized the extraction method. We first developed the agar diffusion method for the quantitative analysis of extracted anti-SA components; then we used the yields of anti-SA components from Salvia miltiorrhiza Bge as the responsive values to study the effects of extraction conditions on the yield of the anti-SA components, including the extraction solvent, extraction times, the ratio of liquid to solid, infiltration time, temperature and extraction time in single-factor experiments; finally we further optimized the ratio of liquid to solid, infiltration time, temperature and extraction time using the four-factor-three-level Box-Behnken experimental design with response surface method. The results of the agar diffusion showed that the standard equation for the determination of anti-SA components from S. miltiorrhiza Bge was Y = 4.842 X ＋ 16.707（R^2 = 0.9873）. The results of the single-factor experiments showed that the most suitable extraction solvent and times was 20% ethanol aqueous solution for 1 time. The results of the Box-Behnken experimental design with response surface method showed that the infiltration time, the extraction temperature, and the ratio of liquid to solid had extremely vital effects on the responsive values. The optimum extraction conditions were as follows： the ratio of liquid to solid was 37.8：1.0（ml：g）, the infiltration time was 104 min, the extraction temperature was 86.7℃ and the extraction time was 182 min. The predicted maximum yield of anti-SA components was 25.95%, under the RSM model-generated optimal extraction conditions. The results of four parallel validation experiments showed that the actual yield of anti-SA components was（25.87 ±0.03）%, and the relative error between predicted and actual values was 0.31%. The yield of anti-SA components was 2.59 times higher than before. These demonstrated that our optimization method was efficient and reliable. Furthermore, our study provided important clues to future research on the anti-SA components of S. miltiorrhiza Bge, and significantly contributed to the safe and efficient control of diseases caused by S. agalactiae.