Liu Jin-Liang, Li Long-Yun, He Guang-Hua
Institute of Material Medical Planting, Chongqing Academy of Chinese Materia Medica (Chongqing Engineering Research Center for Fine Variety Breeding Techniques of Chinese Materia Medica, Chongqing Key Laboratory of Chinese Medicine Resources), Chongqing Sub-Center of National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Science, Chongqing 400065, China.
College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China.
Molecules. 2016 Mar 2;21(3):296. doi: 10.3390/molecules21030296.
Microwave-assisted extraction was applied to extract rutin; quercetin; genistein; kaempferol; and isorhamnetin from Flos Sophorae Immaturus. Six independent variables; namely; solvent type; particle size; extraction frequency; liquid-to-solid ratio; microwave power; and extraction time were examined. Response surface methodology using a central composite design was employed to optimize experimental conditions (liquid-to-solid ratio; microwave power; and extraction time) based on the results of single factor tests to extract the five major components in Flos Sophorae Immaturus. Experimental data were fitted to a second-order polynomial equation using multiple regression analysis. Data were also analyzed using appropriate statistical methods. Optimal extraction conditions were as follows: extraction solvent; 100% methanol; particle size; 100 mesh; extraction frequency; 1; liquid-to-solid ratio; 50:1; microwave power; 287 W; and extraction time; 80 s. A rapid and sensitive ultra-high performance liquid chromatography method coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (EIS-Q-TOF MS/MS) was developed and validated for the simultaneous determination of rutin; quercetin; genistein; kaempferol; and isorhamnetin in Flos Sophorae Immaturus. Chromatographic separation was accomplished on a Kinetex C18 column (100 mm × 2.1 mm; 2.6 μm) at 40 °C within 5 min. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile (71:29; v/v). Isocratic elution was carried out at a flow rate of 0.35 mL/min. The constituents of Flos Sophorae Immaturus were simultaneously identified by EIS-Q-TOF MS/MS in multiple reaction monitoring mode. During quantitative analysis; all of the calibration curves showed good linear relationships (R² > 0.999) within the tested ranges; and mean recoveries ranged from 96.0216% to 101.0601%. The precision determined through intra- and inter-day studies showed an RSD% of <2.833%. These results demonstrate that the developed method is accurate and effective and could be readily utilized for the comprehensive quality control of Flos Sophorae Immaturus.
采用微波辅助萃取法从槐米中提取芦丁、槲皮素、染料木素、山柰酚和异鼠李素。考察了六个独立变量，即溶剂类型、粒度、萃取频率、液固比、微波功率和萃取时间。基于单因素试验结果，采用中心复合设计的响应面法优化实验条件（液固比、微波功率和萃取时间），以提取槐米中的五种主要成分。使用多元回归分析将实验数据拟合为二阶多项式方程。还使用适当的统计方法对数据进行了分析。最佳萃取条件如下：萃取溶剂为100%甲醇，粒度为100目，萃取频率为1，液固比为50:1，微波功率为287 W，萃取时间为80 s。建立了一种快速灵敏的超高效液相色谱法，结合电喷雾电离四极杆飞行时间串联质谱（EIS-Q-TOF MS/MS），用于同时测定槐米中的芦丁、槲皮素、染料木素、山柰酚和异鼠李素。在40℃下，于5分钟内在Kinetex C18柱（100 mm×2.1 mm，2.6μm）上完成色谱分离。流动相由0.1%甲酸水溶液和乙腈（71:29，v/v）组成。以0.35 mL/min的流速进行等度洗脱。通过EIS-Q-TOF MS/MS在多反应监测模式下同时鉴定槐米的成分。在定量分析中，所有校准曲线在测试范围内均显示出良好的线性关系（R²>0.999），平均回收率在96.0216%至101.0601%之间。通过日内和日间研究确定的精密度显示相对标准偏差（RSD%）<2.83%。这些结果表明，所建立的方法准确有效，可方便地用于槐米的全面质量控制。
