Extraction and isolation of potential anti-stroke compounds from black soybean (Glycine max L. Merrill) guided by in vitro PC12 cell model

A simple and efficient method based on ultrafiltration liquid chromatography-mass spectrometry was applied to rapidly screen and identify ligands for lactate dehydrogenase (LDH) from the extracts of black soybeans (Glycine max L. Merrill), and the compounds were further assessed for ant-stroke activity using a PC12 cell model. Six major isoflavones including daidzin, glycitin, genistin, 6″-O-acetyl daidzin, 6″-O-acetyl glycitin and malonyl genistin were identified as potent LDH inhibitors. A continuous online method consisted of a microwave-assisted extraction, online solvent concentration tank and countercurrent chromatography (MAE-SCT-CCC) was newly developed for scaled up production of these compounds with high purity and efficiency. This novel approach using ultrafiltration LC-MS coupled with MAE-SCT-CCC and a PC12 cell model, provides not only a powerful tool for screening, extracting, and separating lactate dehydrogenase inhibitors from complex samples, but also a useful platform for large scale production of functional food and nutraceutical ingredients.