Dichloromethane Extract from Amburana cearensis (Allemão) A.C. Sm. Seeds and Its Coumarin Reduce ROS Production and Protect PC12 Cells Against Glutamate Excitotoxicity and Oxygen-Glucose Deprivation

<i>Amburana cearensis</i> is a plant native to Brazil used in folk medicine for the treatment of several pathological conditions including stroke. Previous research indicates that a dichloromethane extract of <i>A. cearensis</i> seeds (EDAC), rich in coumarins, protects neural cells against oxygen and glucose deprivation (OGD) and glutamate-induced stress. However, further studies are needed to elucidate the role of coumarin, in the protective effect of EDAC. Glutamatergic excitotoxicity is an important cause of neuronal loss involved in the pathogenesis of Alzheimer's disease, Huntington's disease, Parkinson's disease, and ischemic stroke. Therefore, this study aimed to investigate the protective effects of coumarin isolated from EDAC against glutamate excitotoxicity in regulating MAPK pathway proteins and reactive oxygen species (ROS) production on PC12 cells. Furthermore, we aimed to investigate the protective effects of coumarin against cell death induced by OGD. We characterized the isolated compound from EDAC as coumarin by ¹H and ¹³C-NMR. Thus, PC12 cells were exposed to OGD or glutamate (20 mM) and/or treated with EDAC or coumarin (500 μg/mL) for 24 h. Subsequently, cell viability was assessed by propidium iodide staining or by MTT test. Furthermore, the expression of MAPK pathway proteins was investigated by Western blot analysis and the expression of cleaved caspase-3 by immunofluorescence. Furthermore, reactive oxygen species (ROS) production was assessed by 2',7'-dichlorofluorescein diacetate and CellROX. We observed that EDAC and coumarin were able to protect PC12 cells against OGD conditions. Moreover, EDAC totally inhibited the glutamate toxicity in PC12 cells. Meanwhile, coumarin mitigated the glutamate toxicity. Both were able to downregulate the expression of ERK1/2 and phosphorylated ERK and inhibit caspase-3 activation. EDAC and coumarin also prevented the increase of ROS induced by treatment with H₂O₂ or glutamate. Our results evidenced that coumarin from <i>A. cearensis</i> is antioxidative and is an important cytoprotective compound in EDAC against glutamate excitotoxicity or OGD injury.