Identification of Active Ingredients of Huangqi Guizhi Wuwu Decoction for Promoting Nerve Function Recovery After Ischemic Stroke Using HT22 Live-Cell-Based Affinity Chromatography Combined with HPLC-MS/MS

Objective: The Chinese medicine Huangqi Guizhi Wuwu Decoction (HGWD) has been reported to improve the clinical symptoms and restore nerve function after ischemic stroke; however, its active ingredients are not well-determined. Therefore, this study aimed to investigate the bioactive compounds of HGWD and explore the possible mechanism of action. Methods: The methods, including live HT22 cells, solid-phase extraction, and HPLC-MS /MS were utilized. The potential ingredients were identified through comparisons with literature and monomer compounds. Then, oxygen-glucose deprivation reperfusion (OGD/ R)-treated HT22 cells were utilized to investigate the effect of HGWD components with specific binding affinities. Reactive oxygen species (ROS), superoxide dismutase (SOD), lactate dehydrogenase (LDH), and Tunel staining were used as testing indexes to analyze the protective effects of potential active ingredients on OGD/R-induced damage. Results: Eleven compounds with specific binding affinities were identified as calycosin7-O-glucoside, calycosin, formononetin, cinnamic alcohol, cinnamic acid, betaine, dl-2-phenylpropionic acid, 4-hydroxycinnamic acid, 6-methylcoumarin, wogonin, and paeoniflorin. Among them, six compounds had a protective effect on OGD/R-treated HT22 cells. Furthermore, calycosin-7-O-glucoside, calycosin, paeoniflorin, 4-hydroxycinnamic acid, wogonin, and formononetin could regulate oxidative stress and apoptosis to attenuate the cell damage caused by OGD/R. Conclusion: The mechanism of action of HGWD to promote neurological recovery after ischemic stroke was related to the regulation of oxidative stress and apoptosis. This study suggested that cell membrane affinity chromatography combined with HPLC-MS/MS could be applied to screen potential active components in traditional Chinese medicines (TCM).