酸水解提取HPLC法测定不同来源莲须中槲皮素和山柰酚的含量

目的: 研究莲须的酸水解提取条件及其水解苷元槲皮素和山柰酚的HPLC含量测定方法,并分析不同来源莲须中上述成分的含量差异。方法: 莲须样品经体积分数为25%的盐酸-甲醇溶液(1:4)水解提取后,采用高效液相色谱法测定其水解液中槲皮素和山柰酚的含量。色谱柱为Thermo Scientific Syncronis C₁₈ 柱(250 mm×4.6 mm,5 μm),流动相为甲醇-0.012 5%磷酸溶液(60:40),等度洗脱,流速1.0 mL · min^-1,检测波长370 nm,柱温30 ℃。结果: 方法学考察结果表明,槲皮素和山柰酚进样量分别在0.013 99~0.279 8 μg(r=0.999 9)和0.028 70~0.574 2 μg(r=0.999 9)范围内与其对应的峰面积线性关系良好;它们的低、中、高3个加入水平的平均回收率(n=3)分别为102.4%(RSD=1.9%)和100.1%(RSD=1.7%)、101.2%(RSD=0.87%)和96.0%(RSD=0.18%)、101.8%(RSD=1.7%)和95.5%(RSD=1.6%)。15批不同来源的莲须样品中槲皮素和山柰酚含量差异较大,分别为0.07~0.76 mg · g^-1 和0.24~2.40 mg · g^-1。其中1号样品(福建福州1)中槲皮素和山柰酚的含量最高,而14号样品(福建永春)中槲皮素和山柰酚的含量最低。结论: 酸水解提取HPLC法可用于莲须药材中槲皮素和山柰酚含量的测定;15批不同来源莲须的含量测定结果为进一步修订莲须的质量标准提供了实验依据。

Objective: To establish a novel HPLC method with acid hydrolytic extraction for simultaneous determination of quercetin and kaempferol, and compare their contents in Nelumbins Stamen from different origins. Methods: The sample was hydrolyzed and extracted with methanol-chlorhydric acid(volume fraction of 25%)mixed solution(4:1)at 85 ℃ for 1.5 h.Quantification was performed by HPLC at 30 ℃ using Thermo Scientific Syncronis C₁₈ column with the mobile phase of methanol-0.012 5% phosphoric acid solution(60:40)for isocratic elution at the flow rate of 1.0 mL · min^-1, with the detection wavelength of 370 nm and the column temperature of 30 ℃. Results: The linear ranges of quercetin and kaempferol were 0.013 99-0.279 8 μg(r=0.999 9)and 0.028 70-0.574 2 μg(r=0.999 9), respectively.The average recoveries of quercetin and kaempferol at three different concentrations(n=3)were 102.4%(RSD=1.9%)and 100.1%(RSD=1.7%), 101.2%(RSD=0.87%)and 96.0%(RSD=0.18%), 101.8%(RSD=1.7%)and 95.5%(RSD=1.6%), respectively.The contents were 0.07-0.76 mg · g^-1 for quercetin and 0.24-2.40 mg · g^-1 for kaempferol in the fifteen samples collected from different origins, in which the No.1 sample(Fujian Fuzhou 1)had the highest content of quercetin and kaempferol, while the No.14 sample(Fujian Yongchun)had the lowest content of these two components. Conclusion: The novel HPLC method with acid hydrolytic extraction can be used for determination of quercetin and kaempferol in Nelumbins Stamen.And the content results for the 15 batches of samples provide an experimental basis for quality standard revision of Nelumbins Stamen in the future.