筋骨草药材UPLC指纹图谱及多指标成分定量研究

Study on the UPLC fingerprint and quantitative analysis of multi-index components of Ajugae Herba medicinal materials

目的立筋骨草药材超高效液相色谱(UPLC)指纹图谱及5种成分的质量分数测定方法。方法采用UPLC法,采用Agilent Eclipse Plus C_(18)色谱柱(100 mm×2.1 mm,1.8μm),以甲醇和0.1%(φ)磷酸水溶液作为流动相梯度洗脱,流速为0.25 m L/min,柱温为25℃,检测波长为207 nm。运用相似度评价、聚类分析(hierarchical cluster analy‐sis,HCA)、偏最小二乘法判别分析(orthogonal partial least squares discriminant analysis,OPLS-DA)等化学模式识别方法对指纹图谱进行综合评价。结果建立的筋骨草药材UPLC指纹图谱共标示了10个共有峰,指认了哈巴苷、乙酰哈巴苷、毛蕊花糖苷、木犀草素-7-O-二葡萄糖醛酸苷、木犀草苷5种成分。质量分数测定结果显示,15批筋骨草药材中哈巴苷、乙酰哈巴苷、毛蕊花糖苷、木犀草素-7-O-二葡萄糖醛酸苷、木犀草苷的质量分数范围分别为5.37~18.48、4.64~24.25、0.05~0.35、0.74~2.24、0.68~1.12 mg/g。多批样品指纹图谱相似度均在0.90以上;HCA将样品分为3类,通过OPLS-DA分析结合VIP值(variable importance in the projection,VIP)筛选出影响筋骨草药材质量差异的4种主成分。结论所建立的筋骨草药材指纹图谱及质量分数测定方法重复性高、稳定性好,可用于筋骨草药材的质量控制及评价。

Objective To establish the UPLC fingerprint and the determination of five components in Ajugae Herba.Methods The determination was performed by UPLC with an Agilent Eclipse Plus C_(18) column(100 mm×2.1 mm,1.8μm),a mobile phase of methanol and 0.1%aqueous phosphoric acid solution by gradient elution,a volumetric flow rate of 0.25 mL/min,a column temperature of 25℃,and a detection wavelength of 207 nm.Fingerprints were evaluated by chemical pattern recognition techniques such as similarity evaluation,orthogonal partial least squares discriminant analysis(OPLS-DA),and hierarchical cluster analysis(HCA).Results A total of 10 common peaks were matched in 15 batches of UPLC fingerprints,and five components were identified:harpagide,acetylharpagide,verbascoside luteolin-7-O-diglucuronide,and luteoloside.The content ranges of harpagide,acetylharpagide,verbascoside luteolin-7-O-diglucuronide,and luteolosidein 15 batches of Ajugae Herba were 5.37-18.48,4.64-24.25,0.05-0.35,0.74-2.24and 0.68-1.12 mg/g.The similarity of fingerprints was above 0.90.Through HCA and OPLS-DA analysis combined withvariable importance in the projection(VIP)value,Ajugae Herba from different habitats could be divided into three categories,and four principal compounds that led to differences were screened out.Conclusion The established fingerprint spectrum and quality fraction determination method for the medicinal herb of Ajugae Herba have high repeatability and good stability,which can be used for quality control and evaluation of Ajugae Herba.