基于HPLC指纹图谱结合化学计量学评价不同产地佛手药材质量

Quality Evaluation of Citri Sarcodactylis Fructus from Different Origins Based on HPLC Fingerprint and Chemometrics

目的:建立佛手的高效液相色谱法(HPLC)指纹图谱,结合化学计量学方法寻找表征不同产地佛手质量差异的标志物。方法:采用Thermo Hypersil GOLD C_(18)色谱柱(4.6 mm×250 mm,5μm),流动相0.05%磷酸水溶液-乙腈梯度洗脱,检测波长254 nm,对31批佛手样品进行分析,建立指纹图谱,采用“中药色谱指纹图谱相似度评价系统”(2012版)进行相似度评价,确认共有峰,通过对照品比对对共有峰进行指认,并结合化学计量学方法对不同产地佛手质量及其控制方法进行分析和评价,同时随机收集佛手、枳实、枳壳、青皮和陈皮5种芸香科植物的饮片各3批进行分析,对建立的佛手指纹图谱的有效性和可靠性进行评价。结果:建立了佛手样品的HPLC指纹图谱,共标定了22个共有峰,通过对照品比对指认了其中7个共有峰(6,7-二甲氧基香豆素、香叶木苷、橙皮苷、白当归素、佛手柑内酯、氧化前胡素和5,7-二甲氧基香豆素);除2批样品外,其他29批佛手样品指纹图谱与对照指纹图谱的相似度均>0.9;聚类分析和主成分分析将31批佛手基本分为三类,与3个不同产地分类一致;正交偏最小二乘法-判别分析筛选得到8个差异标志物,经对照品指认了其中4个差异性成分,分别为5,7-二甲氧基香豆素、佛手柑内酯、香叶木苷和6,7-二甲氧基香豆素;以佛手对照指纹图谱为参照图谱,对5种芸香科植物的饮片的图谱进行相似度评价,佛手的相似度在0.892~0.977,其余4种饮片的相似度均在0.215~0.517。结论:该研究建立的指纹图谱方法合理、有效、准确,结合化学计量学分析方法,表征信息更加全面,可为佛手的质量控制与品质评价提供科学依据和参考。

Objective:To establish the high performance liquid chromatography(HPLC)fingerprint of Citri Sarcodactylis Fructus,and to search for makers to characterize the quality difference of Citri Sarcodactylis Fructus from different origins coupled with chemometrics.Method:The analysis was performed on a Thermo Hypersil GOLD C_(18) column(4.6 mm×250 mm,5μm)with mobile phase consisted of acetonitrile-0.05%phosphoric acid solution for gradient elution,and the detection wavelength was set at 254 nm.A total of 31 batches of samples were analyzed to establish the HPLC fingerprint of Citri Sarcodactylis Fructus.Similarity evaluation was performed by Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2012 edition)to confirm the common peaks,which were identified by comparison of reference substances.On the basis,chemometrics methods were used to analyze and evaluate the quality of Citri Sarcodactylis Fructus from different origins.At the same time,3 batches of 5 species of decoction pieces from the genus Citrus in the family Rutaceae,including Citri Sarcodactylis Fructus,Aurantii Fructus Immaturus,Aurantii Fructus,Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium,were randomly collected for evaluating the effectiveness and reliability of the established HPLC fingerprint of Citri Sarcodactylis Fructus.Result:HPLC fingerprint of Citri Sarcodactylis Fructus was established and 22 common peaks were identified.And seven common peaks among them were identified as 6,7-dimethoxycoumarin,diosmin,hesperidin,byakangelicin,5,7-dimethoxycoumarin,bergapten and oxypeucedanin.Except for 2 batches of samples,the similarities of fingerprints between other 29 batches of samples were>0.9.The 31 batches of Citri Sarcodactylis Fructus were basically divided into 3 groups by cluster analysis and principal component analysis,which were consistent with the classification of three different producing areas.Eight differential markers were screened by orthogonal partial least squares discriminant analysis and four of them(5,7-dimethoxycoumarin,bergapten,6,7-dimethoxycoumarin and diosmin)were identified by reference substances.Similarity evaluation of 5 species of decoction pieces from genus Citrus in the family Rutaceae was carried out by taking the reference fingerprint of Citri Sarcodactylis Fructus as treference chromatogram,similarity of Citri Sarcodactylis Fructus decoction pieces was 0.892-0.977,and the similarities of the other 4 kinds of decoction pieces were 0.215-0.517.Conclusion:The established fingerprint method is reasonable,effective and accurate for quality control of Citri Sarcodactylis Fructus,the characterization information is more comprehensive combined with chemometrics.