Simultaneous screening, identification, quantitation, and activity evaluation of six acetylcholinesterase (AChE) inhibitors in Coptidis Rhizoma by online UPLC-DAD coupled with AChE biochemical detection.

Acetylcholinesterase (AChE) inhibition is a common treatment in the early stages of dementia and neurodegenerative diseases, including Alzheimer's disease (AD). Coptidis Rhizoma (CR), a traditional Chinese medicine (TCM), contains numerous isoquinoline alkaloids that substantially inhibit AChE played neuroprotective effects in the treatment of cognitive diseases. We established a method using an ultra-high-performance liquid chromatography-diode array detector coupled with AChE biochemical detection (UPLC-DAD-AChEBCD) to screen and identify AChE inhibitors (AChEIs), measure AChEIs content and activity, and evaluate the quality of CR derived from different plant species and growth year. The chromatographic fingerprint and AChEIs activity profiles of CR were simultaneously obtained by UPLC-DAD-AChEBCD, and six alkaloids including groenlandicine, coptisine, epiberberine, jatrorrhizine, berberine, and palmatine, were identified by UPLC-electrospray ionization-tandem mass spectrometry. Data analysis based on AChEIs content and total activity of 12 batches of CR indicated differences among different species and growth year. Therefore, the online method could be used to rapidly identify AChEIs in complex matrixes and screen potential agents for neurodegenerative prevention and treatment, as well as provide information for the identification and quantitation of active markers directly associated with herbal medicine quality.

UHPLC With On-Line Coupled Biochemical Detection for High Throughput Screening of Acetylcholinesterase Inhibitors in Coptidis Rhizoma and Cortex Phellodendri.

We developed a new on-line method of ultra-performance liquid chromatography coupled with biochemical detection (UHPLC-BCD) to screen acetylcholinesterase (AChE) inhibitors in complex matrixes. Chromatography separation was performed using an Xtimate UHPLC C18 column (100 mm × 2.1 mm, 1.8 µm) and a gradient elution with methanol-0.1% formic acid at a flow rate of 0.08 mL/min. The BCD was based on a colorimetric method using Ellman's reagent, and the detection wavelength was at 405 nm. Galanthamine was used as a positive reference to validate the methodology. The detection and quantitation limits of the UHPLC-BCD method were 0.018 and 0.060 µg, respectively. A functional equation was generated in terms of the negative peak area (X) and galanthamine concentration (Y, µg/mL). The regression equation was Y = 0.0028X2 + 0.4574X + 50.7776, R2 = 0.9993. UHPLC-fourier-transform mass spectrometry detection results revealed that five alkaloids showed obvious AChE inhibitory activities including coptisin, epiberberine, jatrorrhizine, berberine and palmatine. The relative AChE inhibitory activities of jatrorrhizine, berberine and palmatine in the Coptidis Rhizoma sample were equal to that of 257.0, 2355 and 283.9 µg/mL of galanthamine, respectively. This work demonstrated that the UHPLC-BCD method was convenient and feasible, and could be widely used for the screening and activity evaluation of the bioactive components in the complex extracts.

Multiple on-line HPLC coupled with biochemical detection methods to evaluate bioactive compounds in Danshen injection.

On-line high performance liquid chromatography (HPLC) coupled with three biochemical detection (BCD) methods was applied to evaluate bioactive components in Danshen injection. On-line HPLC-photo-diode array-fluorescence detection based on the fluorogenic substrate 7-acetoxy-1-methyl quinolinium iodide, was built to search acetylcholinesterase (AChE) inhibitors in Danshen injection. On-line HPLC coupled with the scavenging assay of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals was developed to screen antioxidants. The three active profiles were obviously different. Radical scavenging profiles revealed seven strong peaks in the chromatographic fingerprint possessing obvious free radical inhibition effects, while some minor peaks exhibited stronger AChE inhibition activities. The main radical scavengers and AChE inhibitors were identified by HPLC-MS. Several unknown ingredients showing strong AChE inhibition activities needed further identification except protocatechuic aldehydrate, salvianolic acid H or I and lithospermic acid. The on-line multiple on-line HPLC-BCD methods will provide powerful tools in the field of pharmacognosy for fast-track identification of interesting and/or novel bioactive compounds.