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.
In the present study, we studied the inhibitory effects of chelidonine and rutaecarpin on porcine pancreatic a-amylase （PPA） catalyzed hydrolysis using 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside （Gal-G2-α-CNP）. We, for the first time, provided kinetic report and detailed inhibitory effects of both compounds on PPA. Lineweaver-Burk plot revealed that the inhibition was a mixed-noncompetitive type, and only one molecule of inhibitor bound to the enzyme or to the enzyme-substrate complex. Kinetic constants calculated from secondary plots were in millimole range. Dissociation constants of enzyme-inhibitor complex （KEI） were 0.9 mM and 3.5 mM, respectively. Moreover, dissociation constants of enzyme-inhibitor-substrate complex （KESI） were 0.04 mM and 0.31 mM, respectively. These data indicated that the inhibition was more inclined to competitive to Gal-G2-α-CNP hydrolysis. Further molecular docking study manifested that hydrogen bonding formed between acarbose and aspartic acid （Asp300）, histidine （His305） and glycine （Gly3-6）, while hydrogen bonding was observed between chelidonine and glutamic acid （Glu233）, lysine （Lys200） and His305. In addition, rutaecarpine had only one hydrogen bond with Lys200. Our data indicated that chelidonine and rutaecarpine were two promising drug candidates, and chelidonine possessed stronger inhibitory effect compared with rutaecarpine.
