茶芎HPLC指纹图谱的建立及差异性成分的含量测定

Establishment of HPLC fingerprint and content determination of differential components in Ligusticum sinense

目的建立不同产地茶芎的指纹图谱,筛选差异性成分并测定其含量。方法以Z-藁本内酯为参照,采用《中药色谱指纹图谱相似度评价系统(2012版)》建立12批茶芎药材的高效液相色谱(HPLC)指纹图谱,指认共有峰并进行相似度评价;进行聚类分析(CA)、主成分分析(PCA)和正交偏最小二乘-判别分析(OPLS-DA),以变量重要性投影(VIP)>1为标准筛选影响药材质量的差异性成分;采用同一HPLC法测定上述差异性成分的含量。结果12批茶芎药材指纹图谱共有17个共有峰,相似度为0.989~1.000;共指认了7个共有峰,分别为绿原酸(1号峰)、阿魏酸(2号峰)、洋川芎内酯Ⅰ(7号峰)、阿魏酸松柏酯(9号峰)、E-藁本内酯(13号峰)、洋川芎内酯A(14号峰)、Z-藁本内酯(17号峰)。CA结果显示,12批茶芎药材可分为3类,S1~S5(武宁茶芎)聚为一类,S6~S8(瑞昌茶芎)聚为一类,S9~S12(德安茶芎)聚为一类;2、13、14、17号峰(分别对应阿魏酸、E-藁本内酯、洋川芎内酯A、Z-藁本内酯)的VIP均大于1。S1~S5、S6~S8、S9~S12样品中,阿魏酸的含量分别为0.488~0.533、0.603~0.658、0.415~0.433 mg/g,洋川芎内酯A分别为1.184~1.295、1.450~1.588、1.307~1.377 mg/g,E-藁本内酯分别为0.118~0.125、0.130~0.135、0.223~0.229 mg/g,Z-藁本内酯分别为7.200~7.681、8.076~8.643、4.508~4.996 mg/g,两两比较差异均有统计学意义(P<0.05)。结论所建指纹图谱操作简便、准确,结合多元统计分析可用于评价不同产地茶芎的整体质量。阿魏酸、洋川芎内酯A、Z-藁本内酯、E-藁本内酯可能是影响不同产地茶芎药材质量的差异性成分,且前3种成分含量均以瑞昌茶芎最高,E-藁本内酯含量以德安茶芎最高。

OBJECTIVE To establish the fingerprints of Ligusticum sinense from different habitats,screen differential components and determine their contents.METHODS Using Z-ligustilide as reference,HPLC fingerprints of 12 batches of L.sinense were established by using Similarity Evaluation System of Chromatographic Fingerprints of TCM(2012 edition);common peaks were identified and their similarities were evaluated.Cluster analysis(CA),principal component analysis(PCA)and orthogonal partial least squares-discriminant analysis(OPLS-DA)were performed to screen differential components with variable importance in the projection(VIP)>1 as standard;meanwhile,the contents of above differential components were determined by the same HPLC method.RESULTS There were 17 common peaks in the fingerprints of 12 batches of L.sinense,and their similarities ranged 0.989-1.000.A total of 9 common peaks were identified,i.e.chlorogenic acid(peak 1),ferulic acid(peak 2),senkyunolideⅠ(peak 7),coniferyl ferulate(peak 9),E-ligustilide(peak 13),senkyunolide A(peak 14),Z-ligustilide(peak 17).CA results showed that 12 batches of L.sinense were divided into 3 categories,S1-S5(Wuning)were clustered into one category,S6-S8(Ruichang)were clustered into one category,S9-S12(De’an)were clustered into one category;the VIP values of peaks 2,13,14 and 17(corresponding to ferulic acid,E-ligustilide,senkyunolide A,and Z-ligustilide respectively)were all greater than 1,respectively.In S1-S5,S6-S8 and S9-S12 samples,the contents of ferulic acid were 0.488-0.533,0.603-0.658 and 0.415-0.433 mg/g,respectively;senkyunolide A were 1.184-1.295,1.450-1.588 and 1.307-1.377 mg/g,respectively;E-ligustilide were 0.118-0.125,0.130-0.135 and 0.223-0.229 mg/g,respectively;Z-ligustilide were 7.200-7.681,8.076-8.643 and 4.508-4.996 mg/g,respectively;the differences between two groups were statistically significant(P<0.05).CONCLUSIONS Established fingerprint is simple and accurate,and can be used for overall quality evaluation of L.sinense from different habitats by combining with multivariate statistical analysis.Ferulic acid,senkyunolide A,Z-ligustilide and E-ligustilide may be the differential components that affect the quality of L.sinense from different habitats,the contents of the first 3 components in L.sinense from Ruichang are the highest,and the content of E-ligustilide in samples from De’an is the highest.