在线HPLC-DAD-MS^n-DNA-AO-FLD系统的建立及紫草中DNA结合活性化合物的研究（英文）

On-line HPLC-DAD coupled with ESI-IT-TOF-MS and fluorescence detection to identify DNA-binding compounds from Lithospermum erythrorhizon using acridine orange as the fluorescence probe

本文首次以吖啶橙为荧光探针建立了在线HPLC--DAD--MSn-D-NA--AO--FLD检测系统并应用于天然药物的活性成分研究中,该系统可以同时给出所测天然药物的指纹图谱、各色谱峰的紫外光谱、总离子流图,多级高分辨MS数据以及各化合物与DNA结合的活性图谱,能够对天然药物进行高效分离、结构鉴定和活性检测以及构效、量效关系和作用机理研究。本文采用该系统对紫草正己烷提取物进行了快速分离、结构鉴定和活性研究,证明5个萘醌类化合物具有明显的DNA结合活性?构效和量效关系研究表明,紫草萘醌化合物的母核能嵌插在DNA碱基对中,阻断DNA的复制,侧链上的羟基酰化能够促进与DNA结合,对活性有较大的影响。本文为天然药物中发现抗肿瘤药物提供了简便、快速、精密度高、稳定性好的研究方法。

We have developed an on-line detection method using acridine orange as the fluorescence probe and applied this method to rapidly identify active compounds in herbal medicines. This on-line method was equipped with a high-performance liquid chromatography tandem diode array detector, electrospray ionization-ion-trap time-of-flight mass spectrometry and DNA- acridine orange fluorescence detection （HPLC-DAD-MSn-DNA-AO-FLD）. A large amount of information could be simultaneously obtained during one run, which included HPLC fingerprint, ultraviolet spectra, total ion chromatograms, MSn data of high-resolution mass spectrometry and activity profile of each compound binding with DNA. The method also provided information on structureactivity relationships and mechanism of interaction. We used this on-line method to identify five DNA-binding activity components from Lithospermum erythrorhizon sample for the first time. The result showed that the parent nucleus of shikonin derivatives could bind with DNA. The structure-activity relationship showed that the parent nucleus of shikonin derivatives plays a major role in DNA binding, not the carboxyl group on the side chain. This simple, rapid, high precision and good stability on-line method should be useful for compound separation, structural identification and screening of DNA-binding compounds in herbal medicines.