超高效液相色谱-多元统计分析法评价蜂胶提取物质量

Evaluation of propolis extract quality by ultra performance liquid chromatography-multivariate statistical analysis

目的建立超高效液相色谱法(ultra performance liquid chromatography,UPLC)快速测定蜂胶提取物中的14种化学成分,结合多元统计分析方法对不同厂家的蜂胶提取物质量进行综合评价。方法收集来自不同厂家的17批蜂胶提取物样品,采用UPLC采集色谱图,甲醇-0.2%磷酸水溶液为流动相,梯度洗脱,同时测定咖啡酸、p-香豆酸、阿魏酸、异阿魏酸、3,4-二甲氧基肉桂酸、咖啡酸苯乙酯、阿替匹林C、槲皮素、山奈素、芹菜素、异鼠李素、乔松素、白杨素、高良姜素的含量,运用统计学软件进行主成分分析(principal component analysis,PCA)、聚类分析(clustering analysis,CA)、偏最小二乘-判别分析(partial least squares-discriminant analysis,PLS-DA),筛选分析质量差异标志物。通过熵权法计算各指标权重,将结果应用于优劣解距离法(technique for order preference by similarity to ideal solution,TOPSIS)和秩和比法(rank sum ratio,RSR)构建综合评价模型,评价不同批次的蜂胶提取物质量优劣。结果14个指标成分在各自的浓度范围内线性关系良好(r≥0.9992),平均加样回收率是96.37%~102.21%,相对标准偏差小于2%。化学计量学结果表明17批样品聚为4类,同一个厂家的样品聚为一类,不同厂家的样品存在明显差异,3,4-二甲氧基肉桂酸、异阿魏酸、槲皮素、高良姜素、阿替匹林C、咖啡酸苯乙酯可能是影响厂家质量差异的潜在标志物。通过熵权-TOPSIS、熵权-RSR以及两者相结合的方式构建的综合质量评价模型,对不同批次蜂胶提取物的质量优劣排序结果较为一致。结论基于UPLC的多指标测定方法准确便捷,结合PCA、CA、PLS-DA和TOPSIS-RSR建立的评价模式能够有效分析不同厂家的差异性,为蜂胶提取物的整体质量评价提供参考。

Objective To establish a method for the rapid determination of 14 kinds of chemical components in propolis extracts by ultra performance liquid chromatography(UPLC),and to comprehensively evaluate the quality of propolis extracts from different manufacturers by multivariate statistical analysis.Methods The 17 batches of propolis extract samples from different manufacturers were collected and chromatographed by UPLC with methanol-0.2%phosphoric acid aqueous solution as mobile phase and gradient elution,the content of caffeic acid,P-coumaric acid,ferulic acid,isoferulic acid,3,4-dimethoxycinnamic acid,phenethyl caffeic acid,atipilin C,quercetin,kaempin,apigenin,isorhamnetin,josinetin,albumin and galangin were determined.Principal component analysis(PCA),cluster analysis(CA),partial least squares-discriminant analysis(PLS-DA)were used to screen and analyze the quality difference markers by statistical software,meanwhile,the weights of each index were calculated by entropy weight method,and the results were applied to the superiority and inferiority solution distance method(TOPSIS)and rank sum ratio method(RSR)to construct a comprehensive evaluation model to evaluate the quality of different,the results were used to evaluate the quality of different batches of propolis extracts.Results The linear relationships of the 14 components were good(r≥0.9992)in their respective concentration ranges.The average recoveries were 96.37%–102.21%,and the relative standard deviations were less than 2%.The chemometric results showed that the 17 batches of samples were clustered into 4 categories,samples from the same manufacturer were clustered into one category,and there were significant differences between samples from different manufacturers.3,4-dimethoxycinnamic acid,isoferulic acid,quercetin,galangin,atipyrine C,and caffeic acid phenethyl ester might be potential markers affecting the quality differences of manufacturers.The comprehensive quality evaluation models constructed by entropy-TOPSIS,entropy-RSR and a combination of both showed more consistent results in ranking the quality of different batches of propolis extracts.Conclusion The UPLC-based multi-indicator determination method is accurate and convenient,and the evaluation model established by combining PCA,CA,PLS-DA and TOPSIS-RSR can effectively analyze the variability of different manufacturers and provide a reference for the overall quality evaluation of propolis extracts.