On‐line coupling pressurised liquid extraction with two‐dimensional counter current chromatography for isolation of natural acetylcholinesterase inhibitors from Astragalus membranaceus

INTRODUCTION: Radix Astragali, the dried root of Astragalus membranaceus (Fish.) Bge. (family Fabaceae), which is known as Huangqi in China, has been proven to be an immunostimulant, diuretic, antidiabetic, analgesic, and it has also been used as a health food supplement in some Asian populations and also serves as a lead herb in many traditional Chinese medicine formulations as well as in Chinese ethnic tonifying soups. OBJECTIVE: Screening and purification of bioactive compounds from natural products is challenging work due to their complexity. We present the first report on the use of pressurised liquid extraction and on‐line two‐dimensional counter current chromatography as an efficient medium for scaled‐up extraction and separation of six bioactive compounds from Astragalus membranaceus. METHOD: We applied the established method with ultrafiltration‐liquid chromatography to screen acetylcholinesterase inhibitors, which were then evaluated and confirmed for anti‐Alzheimer activity using PC12 cell model. RESULTS: Six major compounds, namely, calycosin‐7‐O‐β‐d‐glucoside, pratensein‐7‐O‐β‐d‐glucoside, formononetin‐7‐O‐β‐d‐glucoside, calycosin, genistein, and formononetin, with acetylcholinesterase binding affinities were identified and isolated from the raw plant materials via two sets of n‐hexane/ethyl acetate/0.2% acetic acid (first‐stage counter current chromatography) and n‐hexane/ethyl acetate/methanol/water (second‐stage counter current chromatography) solvent systems: 1.87:1.0:1.33 and 5.62:1.0:2.42:5.25, v/v/v/v, which were optimised by a mathematical model. CONCLUSION: Therefore, a useful platform for the large‐scale production of bioactive and nutraceutical ingredients was developed herein. With the on‐line system developed here, we present a feasible, selective, and effective strategy for rapid screening and identification of enzyme inhibitors from complex mixtures.