Anthocyanins Extracted from Black Soybean Seed Coat Protect Primary Cortical Neurons against in Vitro Ischemia

The present study investigated the neuroprotective effects of anthocyanins extracted from black soybean (cv. Cheongja 3, Glycine max (L.) MERR.) seed coat against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays were employed to assess cell membrane damage and viability of primary neurons, respectively. OGD-induced cell death in 7 d in vitro primary cortical neurons was found to be OGD duration-dependent, and approximately 3.5 h of OGD resulted in ≈60% cell death. Treatment with black soybean anthocyanins dose-dependently prevented membrane damage and increased the viability of primary neurons that were exposed to OGD. Glutamate-induced neuronal cell death was dependent on the glutamate concentration at relatively low concentrations and the number of days the cells remained in culture. Interestingly, black soybean anthocyanins did not protect against glutamate-induced neuronal cell death. They did, however, inhibit the excessive generation of reactive oxygen species (ROS) and preserve mitochondrial membrane potential (MMP) in primary neurons exposed to OGD. In agreement with the neuroprotective effect of crude black soybean anthocyanins, purified cyanidin-3-glucoside (C3G), the major component of anthocyanins, also offered dose-dependent neuroprotection against OGD-induced neuronal cell death. Moreover, black soybean C3G markedly prevented excessive generation of ROS and preserved MMP in primary neurons that were exposed to OGD. Collectively, these results suggest that the neuroprotection of primary rat cortical neurons by anthocyanins that were extracted from black soybean seed coat might be mediated through oxidative stress inhibition and MMP preservation but not through glutamate-induced excitotoxicity attenuation.