高效液相色谱法结合多元统计分析用于苍术药材的质量评价

Quality evaluation of Atractylodes by high-performance liquid chromatography and multivariate statistical analysis

目的:测定3种苍术药材中7个主要成分(白术内酯Ⅲ、Ⅱ、Ⅰ及(4E,6E,12E)-十四癸三烯-8,10-二炔-1,3-二乙酸酯、苍术素、β-桉叶醇、苍术酮)的含量并进行多元统计分析,以实现对苍术药材的质量评价。方法:采用CAPCELL PAK C18色谱柱(250 mm×4.6 mm,5μm),以0.1%磷酸水-甲醇为流动相梯度洗脱,流速1.0 m L·min^(-1),检测波长220 nm(0~28 min,检测白术内酯Ⅲ、白术内酯Ⅱ)、270 nm(28~40 min,检测白术内酯Ⅰ、(4E,6E,12E)-十四癸三烯-8,10-二炔-1,3-二乙酸酯)、203 nm(40~50 min,检测苍术素、β-桉叶醇)、220 nm(50~77 min,检测苍术酮),柱温25℃;茅苍术样品中β-桉叶醇含量测定以0.1%磷酸水-甲醇(20∶80)为流动相等度洗脱,检测波长203 nm。采用主成分分析法(PCA法)和正交偏最小二乘判别分析法(OPLS-DA法)对含量测定数据进行分析。结果:含量测定方法学验证结果良好。茅苍术中7个成分(白术内酯Ⅲ、Ⅱ、Ⅰ及(4E,6E,12E)-十四癸三烯-8,10-二炔-1,3-二乙酸酯、苍术素、β-桉叶醇、苍术酮)的含量范围分别为0.010~0.145、0~0.014、0.031~0.281、0.379~1.073、2.227~3.756、17.14~32.15、0.002~3.730 mg·g-1,北苍术中7个成分的含量范围分别为0.045~0.708、0.017~0.592、0.101~1.360、0.095~2.340、0.432~7.059、0.303~7.031、2.070~8.487 mg·g-1,关苍术中7个成分的含量范围分别为0.198~0.773、0.124~0.944、0.515~0.899、1.590~2.277、0.115~2.094、0.090~0.175、10.71~17.98 mg·g-1,个别样本中白术内酯Ⅲ、Ⅱ及β-桉叶醇的含量低于定量限或检测限,茅苍术样本中白术内酯II含量低于定量限,仅有1个样本可定量(0.014 mg·g^(-1))。采用PCA对含量测定结果进行分析,PCA得分散点图显示苍术药材被明显分为4组,其中组1和组4均为北苍术,组2为关苍术,组3为茅苍术,表明PCA能实现茅苍术、北苍术和关苍术的有效区分。在PCA的基础上进行OPLS-DA分析,筛选出苍术素和β-桉叶醇是区分3种苍术的关键差异成分。结论:本研究所建立的含量测定方法可以用于苍术药材中7个成分的定量分析,PCA和OPLS-DA可用于区分茅苍术、北苍术和关苍术,并揭示区分3种苍术的关键差异成分,为苍术药材质量控制和评价提供科学依据。

Objective：To establish an HPLC method for simultaneous determination of seven principal components（atractylenolide Ⅲ,atractylenolide Ⅱ,atractylenolide Ⅰ,（4 E,6 E,12 E）-tetradecatriene-8,10-diyne-1,3-diacetate,atractylodin,β-eudesmol and atractylon） in three kinds of Atractylodes and analyze the data by multivariate statistical analysis for quality evaluation of Atractylodes.Methods：HPLC was performed on a CAPCELL PAK C18（4.6 mm×250 mm,5 μm） column with gradient elution of methanol-0.1% phosphoric acid solution at a flow rate of 1.0 mL·min^-1.The column temperature was 25 ℃ and the detection wavelength was set at 220 nm from 0 to 28 min（atractylenolide Ⅲ and atractylenolide Ⅱ）,270 nm from 28 to 40 min（atractylenolide Ⅰ and（4 E,6 E,12 E）-tetradecatriene-8,10-diyne-1,3-diacetate）,203 nm from 40 to 50 min（atractylodin and β-eudesmol） and 220 nm from 50 to 77 min（atractylon）.The HPLC conditions for the determination of β-eudesmol in A.lancea samples were as follows：an isocratic mobile phase consisting of 0.1% phosphoric acid solution-methanol（20∶80） and a detection wavelength of 203 nm.Principal component analysis（PCA） and orthogonal partial least square discrimination analysis（OPLS-DA） were employed for the analysis of seven components in Atractylodes.Results：Methodology validation of this content determination method was good.The contents of seven components（atractylenolide Ⅲ,atractylenolide Ⅱ,atractylenolide Ⅰ,（4 E,6 E,12 E）-tetradecatriene-8,10-diyne-1,3-diacetate,atractylodin,β-eudesmol and atractylon） were 0.010-0.145,0-0.014,0.031-0.281,0.379-1.073,2.227-3.756,17.14-32.15,0.002-3.730 mg·g^-1 in A.lancea samples;0.045-0.708,0.017-0.592,0.101-1.360,0.095-2.340,0.432-7.059,0.303-7.031,2.070-8.487 mg·g^-1 in A.chinensis samples;0.198-0.773,0.124-0.944,0.515-0.899,1.590-2.277,0.115-2.094,0.090-0.175,10.71-17.98 mg·g^-1 in A.japonica samples.The contents of atractylenolide Ⅲ,atractylenolide Ⅱ and β-eudesmol in individual samples were below the limits of quantification（LOQs） or the limits of detection（LODs）.The contents of atractylenolide Ⅱ in A.lancea samples were below the LOQ,except for only one sample could be quantified（0.014 mg·g^-1）.The results of quantitative analysis were analyzed by PCA.According to the PCA score scatter plot,40 batches of samples were classified into group 1,group 2,group 3 and group 4 corresponding to A.chinensis,A.japonica,A.lancea and A.chinensis.And the classification result indicated that PCA could achieve the effective differentiation of three kinds of Atractylodes.OPLS-DA was carried out on the basis of PCA,screening out the cucial components which could differentiate three kinds of samples.Conclusion：The method could be used for simultaneous determination of seven components in Atractylodes.PCA and OPLS-DA could be employed to differentiate A.chinensis,A.japonica and A.lancea and reveal the crucially differential components,thus providing a reference for quality control and evaluation of Atractylodes.