基于离线2D-HPLC-DPPH-ESI-Q-TOF/MS联用技术的金银花抗氧化成分系统筛选研究

Rapid discovery of trace antioxidants in Lonicerae Japonicae Flos by 2D-HPLC-DPPH-ESI-TOF/MS

目的建立基于离线二维色谱-二苯基三硝基苯肼-电喷雾飞行时间质谱结合技术(2D-HPLC-DPPH-ESI-Q-TOF/MS)的金银花中抗氧化成分系统筛选方法。方法采用离线二维色谱对金银花提取物进行分段富集、分析,第一维采用semi-PREP-HPLC技术对金银花提取物进行极性分段制备和富集浓缩,第二维分别采用亲水和反相色谱技术进行分析;进一步结合DPPH在线筛选技术实现金银花复杂组分中抗氧化成分的快速发现;采用ESI-Q-TOF/MS技术进行初步鉴别,并通过部分单体化合物的体外抗氧化活性评价对筛选结果进行验证。结果该方法可以实现金银花中不同极性和不同含量抗氧化成分的快速筛选,从金银花提取物中共筛选出36种具有抗氧化活性的成分,采用高分辨质谱初步鉴定出12种,对9种成分进行活性验证,其中7种成分(异绿原酸B、异绿原酸A、1,3-二咖啡酰奎宁酸、异绿原酸C、芦丁、隐绿原酸、绿原酸)的半数抑制浓度(IC50)优于阳性对照维生素C。结论该方法高效、快速,为复杂体系天然产物中不同极性和含量抗氧化成分的发现提供了技术和平台支持。

Objective A two-dimensional liquid chromatography(2 D-HPLC-DPPH-ESI-Q-TOF/MS) method combined with on-line antioxidant screening technique was established for rapid detection of trace antioxidants in Lonicerae Japonicae Flos. Methods The 2 D-HPLC method was used for the preparation,which can achieve the enrichment and polarity grouping of trace components in Lonicerae Japonicae Flos. In the first dimension,the extract was subjected to polar grouping and enrichment by semi-PREP-HPLC technique. In the second dimension,HILIC and RP2-UPLC techniques were used for analysis. Furthermore,DPPH online screening technology was combined to achieve the rapid discovery and screening of trace antioxidant components in the complex components of Lonicerae Japonicae Flos. ESI-O-TOF/MS technique was used for preliminary identification,and the screening results were verified by the in vitro antioxidant activity evaluation of some monomeric compounds. Results The results showed that this method can rapidly enrich and screen different polar antioxidants in Lonicerae Japonicae Flos,and 36 compounds with antioxidant activity were screened in Lonicerae Japonicae Flos. Twelve compounds were identified by high resolution mass spectrometry. The activity of nine components was verified,and the IC50 value of seven compounds(isochlorogenic acid B,isochlorogenic acid A,1,3-dicaffeoylquinic acid,isochlorogenic acid C,rutin,cryptochlorogenic acid,and chlorogenic acid) was better than positive control-vitamin C. Conclusion The method is efficient and rapid,which provides technical basis and platform support for the rapid discovery of trace antioxidant components in natural products of complex systems.