Lund University, Department of Chemistry, Centre for Analysis and Synthesis, Lund, Sweden.
Anal Chim Acta. 2013 Jun 27;785:50-9. doi: 10.1016/j.aca.2013.04.031. Epub 2013 Apr 22.
A novel environmentally sound continuous-flow hot water extraction and enzymatic hydrolysis method for determination of quercetin in onion raw materials was successfully constructed using a stepwise optimization approach. In the first step, enzymatic hydrolysis of quercetin-3,4'-diglucoside to quercetin was optimized using a three level central composite design considering temperature (75-95°C), pH (3-6) and volume concentration of ethanol (5-15%). The enzyme used was a thermostable β-glucosidase variant (termed TnBgl1A_N221S/P342L) covalently immobilized on either of two acrylic support-materials (Eupergit(®) C 250L or monolithic cryogel). Optimal reaction conditions were irrespective of support 84°C, 5% ethanol and pH 5.5, and at these conditions, no significant loss of enzyme activity was observed during 72 h of use. In a second step, hot water extractions from chopped yellow onions, run at the optimal temperature for hydrolysis, were optimized in a two level design with respect to pH (2.6 and 5.5), ethanol concentration (0 and 5%) and flow rate (1 and 3 mL min(-1)) Obtained results showed that the total quercetin extraction yield was 1.7 times higher using a flow rate of 3 mL min(-1) (extraction time 90 min), compared to a flow rate of 1 mL min(-1) (extraction time 240 min). Presence of 5% ethanol was favorable for the extraction yield, while a further decrease in pH was not, not even for the extraction step alone. Finally, the complete continuous flow method (84°C, 5% ethanol, pH 5.5, 3 mL min(-1)) was used to extract quercetin from yellow, red and shallot onions and resulted in higher or similar yield (e.g. 8.4±0.7 μmol g(-1) fresh weight yellow onion) compared to a conventional batch extraction method using methanol as extraction solvent.
一种新颖的环保连续流动热水提取和酶解方法，用于测定洋葱原料中的槲皮素，成功地使用逐步优化方法构建。在第一步中，考虑温度（75-95°C）、pH（3-6）和乙醇体积浓度（5-15%），使用三水平中心复合设计优化了槲皮素-3,4'-二葡萄糖苷酶解为槲皮素的过程。所用的酶是一种热稳定的β-葡萄糖苷酶变体（称为 TnBgl1A_N221S/P342L），共价固定在两种丙烯酰基支持材料之一上（Eupergit(®) C 250L 或整体式 cryogel）。最佳反应条件与支持物无关，为 84°C、5%乙醇和 pH 5.5，在这些条件下，在 72 小时的使用过程中，酶活性没有明显损失。在第二步中，在水解的最佳温度下，对切碎的黄洋葱进行热水提取，在 pH（2.6 和 5.5）、乙醇浓度（0 和 5%）和流速（1 和 3 mL min(-1)）的两级设计中进行优化。得到的结果表明，与流速为 1 mL min(-1)（提取时间 240 min）相比，流速为 3 mL min(-1)（提取时间 90 min）时，总槲皮素提取产率提高了 1.7 倍。存在 5%乙醇有利于提取产率，而进一步降低 pH 甚至对单独的提取步骤也没有帮助。最后，使用完整的连续流动方法（84°C、5%乙醇、pH 5.5、3 mL min(-1)）从黄洋葱、红洋葱和葱头中提取槲皮素，得到了更高或相似的产率（例如，8.4±0.7 μmol g(-1) 鲜重黄洋葱），与使用甲醇作为提取溶剂的传统分批提取方法相比。
