Abstract:

In order to obtain active ingredient sinigrin with high efficiency and high yield from seeds of Thlaspi arvense， the enzymolysis assisted-ultrasonic extraction method was used. The content of sinigrin was determined by high performance liquid chromatography （HPLC）. Based on the single factor experiment， enzymolysis time， enzymolysis temperature and enzyme addition amount were the key factors， and the yield of sinigrin was taken as the response value. The Design-Expert 11 software was applied and the Both-Behnken Design response surface methodology was used to optimize the enzymolysis assisted ultrasonic extraction process of sinigrin. The yield of sinigrin was compared with that of ultrasonic and Soxhlet extraction. The results showed that Agilent HC-C18 column （25 mm×4.6 mm×5 μm）， mobile phase A methanol， mobile phase B 0.5% phosphoric acid aqueous solution， detection wavelength 215 nm， column temperature 25 ℃， flow rate 1 mL·min^-1， sample size 10 μL， and the same degree elution for 10 min， the separation effect and peak shape were better. Using the screened alkaline protease as enzyme preparation， the optimal extraction conditions were that the enzymolysis temperature was 49 ℃， the enzyme dosage was 2.90%， the enzymolysis time was 18 min， the yield of sinigrin was 2.679 mg·g^-1 under optimal conditions， which was no significant difference with the theoretical value （2.687 mg·g^-1）. The yield of sinigrin was significantly higher than that of ultrasonic extraction （2.036 mg·g^-1） and Soxhlet extraction （1.702 mg·g^-1） （P<0.05）. The enzymolysis assisted-ultrasonic extraction process of sinigrin from T. arvense was simple， energy saving， eco-friendly， high efficiency and high yield， and it could be applied to the production and utilization of sinigrin from T. arvense， which laid a theoretical foundation for the in-depth development of T. arvense.

Key words: Thlaspi arvense, sinigrin, enzyme assisted-ultrasonic extraction, response surface methodology

摘要：

为从菥蓂种子中高效提取高得率的目的活性成分黑芥子苷，采用酶解辅助-超声提取法，并利用高效液相色谱测定其含量。在单因素试验的基础上，以酶添加量、酶解温度和酶解时间为关键因素，以黑芥子苷得率为响应值，应用Design-Expert V11软件，Box-Behnken Design响应面法优化菥蓂种子中黑芥子苷酶解辅助-超声提取工艺；并与单独超声、索氏提取2种提取法的黑芥子苷得率进行比较。结果表明，采用Agilent HC-C18柱（25 mm×4.6 mm×5 μm），流动相A和B分别为甲醇和0.5%磷酸水溶液，检测波长215 nm，柱温25 ℃，流速1 mL·min^-1，进样量10 μL，在等度洗脱10 min下，获得的分离效果和峰型较好。以筛选出的碱性蛋白酶为酶制剂，最佳提取工艺条件为：酶解温度49 ℃，酶用量2.90%，酶解时间18 min；在此条件下，菥蓂种子中黑芥子苷得率为2.679 mg·g^-1，经方差分析与理论值（2.687 mg·g^-1）差异不显著，但显著高于单独超声提取（2.036 mg·g^-1）和索氏提取（1.702 mg·g^-1）（P<0.05）。优化得到的菥蓂中黑芥子苷酶解辅助-超声提取工艺，方法简单、节能、环保、高效，目的活性成分得率高，可应用于菥蓂中黑芥子苷的生产实践，为菥蓂的深度开发奠定了理论基础。

关键词: 菥蓂, 黑芥子苷, 酶解辅助-超声提取, 响应面法