Extraction and isolation of acetylcholinesterase inhibitors from Citrus limon peel using an in vitro method

A simple and efficient ultrafiltration-liquid chromatography-mass spectrometry-based method was developed for the rapid screening and identification of ligands from Citrus limon peel, which are suitable acetylcholinesterase inhibitors. Subsequently, the anti-Alzheimer's activity of these compounds was assessed using a PC12 cell model. Six major compounds, viz. neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin, were identified as potent acetylcholinesterase inhibitors. A continuous and efficient online method, which involved the use of a microwave-assisted extraction device, solvent concentration tank, and centrifugal partition chromatography column, was developed for the scale-up of these compounds, and the obtained compounds presented high purity. Next, their bioactivity was evaluated using a PC12 cell model. This novel approach, which was based on ultrafiltration-liquid chromatography-mass spectrometry, microwave-assisted extraction online coupled with solvent concentration tank, and centrifugal partition chromatography along with in vitro evaluation, could represent a powerful tool for the screening and extraction of acetylcholinesterase inhibitors from complex matrices, and could be a useful platform for the large-scale production of bioactive and nutraceutical ingredients.