基于UPLC-MS/MS分析方法的枳实“肉”与“瓤”中黄酮类活性成分的比较研究

Comparative Study on the Active Components of Favonoids between Peel and Pulp of Aurantii Fructus Immaturus by UPLC-MS/MS

关键词:  枳实  UPLC-MS/MS  性状特征  黄酮  辨状论质

Key words:  Aurantii Fructus Immaturus  UPLC-MS/MS  characteristics  flavonoid  Bianzhuang Lunzhi

摘要:

目的 建立UPLC-MS/MS同时测定枳实“肉”与“瓤”中7个黄酮类活性成分含量的方法，并比较两个部位的含量差异。方法 采用Waters ACQUITY UPLC HSS T3 色谱柱 (2.1 mm×100 mm，1.8 μm)，以0.2%甲酸水溶液-乙腈为流动相进行梯度洗脱，流速0.4 mL·min-1，柱温30℃，进样量2 μL；质谱采用电喷雾离子源，正离子多反应监测模式采集数据；对24批次枳实“肉”与“瓤”含量测定结果采用聚类分析、主成分分析、正交偏最小二乘法判别分析进行综合分析。结果 7个黄酮类活性成分在各自的质量浓度范围内线性关系良好；与枳实“肉”组比较，芹菜素和山柰酚在“瓤”中的含量高，5,7,3',4'-四甲氧基黄酮、6-去甲氧基橘皮素、5,7,3',4',5'-五甲氧基黄酮、异甜橙黄酮和5-去甲基川陈皮素在“瓤”中的含量低，均具有显著性差异；枳实“肉”与“瓤”可按不同部位分为两类，5,7,3',4',5'-五甲氧基黄酮、异甜橙黄酮、6-去甲氧基橘皮素、5-去甲基川陈皮素对区分“肉”和“瓤”贡献较大，尤其是5,7,3',4',5'-五甲氧基黄酮和5-去甲基川陈皮素，可作为区分“肉”和“瓤”的主要差异性成分。结论 所建立的方法准确可靠，枳实“肉”与“瓤”中7个黄酮类活性成分的含量存在显著差异，可为枳实“肉厚瓤小”性状特征的科学内涵提供更充分的科学依据。

Abstract:

OBJECTIVE To develop an UPLC-MS/MS method for simultaneous determination of 7 flavonoids including apigenin, kaempferol, 5,7,3',4'-tetramethoxyflavone, 6-demethoxytangeretin, 5,7,3',4',5'-pentamethoxyflavone，isosinensetin and 5-demethylnobiletin, between peel and pulp of Aurantii Fructus Immaturus, and to compare the content differences in different parts of Aurantii Fructus Immaturus. METHODS The analysis was carried out on a Waters ACQUITY UPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm) at 30℃, with the mobile phase of water containing 0.2% formic acid (A) and acetonitrile (B) under gradient elution, the flow rate of 0.4 mL·min-1, and the injection volume of 2 μL. The electrospray ionization (ESI) source in the positive ion mode and multiple reaction monitoring (MRM) were used for MS quantitative analysis. The content results were analyzed by the cluster analysis (CA), principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA). RESULTS The linearity of the 7 components is good within their linear range. The results of 24 batches of samples showed that the contents of apigenin and kaempferol were higher in the pulp group, while the contents of 5,7,3',4'-tetramethoxyflavone, 6-dimethoxytangeretin, 5,7,3',4',5'-pentamethoxyflavone, isosinensetin, and 5-demethylnobiletin were lower in the pulp group than in the peelp group, and all of them were significantly different. The results of multivariate statistical analysis showed that peel and pulp could be could be divided into two categories according to different tissue parts. Among them, 5,7,3',4',5'-pentamethoxyflavone, isosinensetin, 6-dimethoxytangeretin and 5-demethylnobiletin contributed more to differentiate between peel and pulp. In particular, 5,7,3',4',5'-pentamethoxyflavone and 5-demethylnobiletin could be the main differentiating components between the peel and pulp. CONCLUSION An accurate and reliable UPLC-MS/MS quantitative method for the determination of 7 flavonoid components in the peel and pulp was established. There were significant differences in the contents of 7 flavonoids between peel and pulp, which can provide a more adequate scientific basis for the scientific connotation about the appearance characteristic of Aurantii Fructus Immaturus.