加速溶剂萃取-高效液相色谱法同时测定全叶千里光中3种成分的含量

目的：建立加速溶剂萃取-高效液相色谱法同时测定全叶千里光药材中绿原酸、芦丁和金丝桃苷3种活性成分的含量。方法：运用正交设计试验优化加速溶剂萃取法（ASE法）的提取工艺，提取样品中的有效成分，比较ASE法、回流提取法和超声辅助提取法的提取效果；采用C₁₈色谱柱，以乙腈-0.3%磷酸水溶液为流动相，梯度洗脱，流速为1.0 mL·min^-1，柱温为25 ℃，检测波长为350 nm，以高效液相色谱进行分析。结果：正交试验设计优化ASE法，优化得到的最佳萃取条件为：以体积分数60%甲醇为提取溶剂，在4.8 MPa和65 ℃下，静态提取2次，每次17 min，提取液经旋转蒸发浓缩近干，以甲醇溶解并定容。经条件优化后ASE萃取得到的全叶千里光药材中3种主要活性成分的提取效果均高于传统的回流提取法和超声提取法；全叶千里光中绿原酸、芦丁和金丝桃苷3种被测成分的质量浓度分别在1.5~100.0、0.5~80.0和0.5~80.0 mg·L^-1范围内与其峰面积呈良好的线性关系，线性相关系数均大于0.999 0，检出限为0.08~0.15 mg·L^-1，样品的加标平均回收率为93.5%~102.0%，RSD为0.73%~3.47%（n=3）。变种全叶千里光和原变种麻叶千里光中绿原酸、芦丁和金丝桃苷的平均含量分别为1.65和0.77、0.09和0.10、0.68和0.45 mg·g^-1。结论：该方法可用于全叶千里光药材中3种主要化学成分的含量测定，其结果可为全叶千里光药材质量控制、合理用药及进一步研究开发提供参考。

Objective: To establish an accelerated solvent extraction(ASE)-high performance liquid chromatography(HPLC)method for simultaneous determination of three components,namely,chlorogenic acid,rutin and hyperin in Senecio cannabifolius Less.var integrilifolius(Koidz.)Kitam.Methods: The primary ASE parameters,including extraction temperature,pressure,static time and extraction solvent,were optimized by an orthogonal test,based on the recoveries of chlorogenic acid,rutin and hyperin.The extraction result of the ASE was then compared with reflux extraction and ultrasonic-aided extraction.Active ingredients were extracted by accelerated solvent extraction from S.cannabifolius var.integrifolius.The chromatographic analyses were carried out on a C₁₈ column by gradient elution with acetonitrile and 0.3% phosphoric acid aqueous solution as mobile phase. The flow rate was 1.0 mL·min^-1,the column temperature was 25 ℃,and the detection wavelength was set at 350 nm.Qualitative and quantitative analysis were adopted by retention time of components and external standard method.Results: The conditions of ASE were optimized using 60% methanol as the extraction solvent,and spiked sample was extracted by ASE at 4.8 MPa and 65 ℃ for 2 times,17 min for each time.The extract was concentrated by evaporation and dissolved in methanol.Our results showed that the recoveries of three main activity components extracted by the ASE were significantly higher than that by reflux extraction and ultraphonic extraction.The mass concentration of chlorogenic acid,rutin and hyperin exhibited a good linear range of 1.5-100.0 mg·L^-1, 0.5-80.0 mg·L^-1 and 0.5-80.0 mg·L^-1,respectively,with the correlation coefficients over 0.999 0.The limits of detection(LODs)for 3 components were between 0.08-0.15 mg·L^-1.The mean recoveries and relative standard deviations were 93.5%-102.0% and 0.73%-3.47%(n=3),respectively.The content of chlorogenic acid,rutin and hyperin were 1.65 and 0.77,0.09 and 0.10,0.68 and 0.45 mg·g^-1 for variant Senecio cannabifolius Less.var.integrilifolius(Koidz.)Kitam.and Senecio cannabifolius Less.,respectively.Conclusion: The method can be applied to the determination of chlorogenic acid,rutin and hyperin in S.cannabifolius var.integrilifolius.The determination result can be used as a reference for the rational medication,quality control,and further study of S.cannabifolius var.integrilifolius.