The Neuroprotective Potential of Cyanidin-3-glucoside Fraction Extracted from Mulberry Following Oxygen-glucose Deprivation

Abstract

In this study, cyanidin-3-glucoside (C3G) fraction extracted from the mulberry fruit (Morus alba L.) was investigated for its neuroprotective effects against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Cell membrane damage and mitochondrial function were assessed by LDH release and MTT reduction assays, respectively. A time-course study of OGD-induced cell death of primary cortical neurons at 7 days in vitro (DIV) indicated that neuronal death was OGD duration-dependent. It was also demonstrated that OGD for 3.5 h resulted in approximately 50% cell death, as determined by the LDH release assay. Treatments with mulberry C3G fraction prevented membrane damage and preserved the mitochondrial function of the primary cortical neurons exposed to OGD for 3.5 h in a concentration-dependent manner. Glutamate-induced cell death was more pronounced in DIV-9 and DIV-11 cells than that in DIV-7 neurons, and an application of 50µM glutamate was shown to induce approximately 40% cell death in DIV-9 neurons. Interestingly, treatment with mulberry C3G fraction did not provide a protective effect against glutamate-induced cell death in primary cortical neurons. On the other hand, treatment with mulberry C3G fraction maintained the mitochondrial membrane potential (MMP) in primary cortical neurons exposed to OGD as assessed by the intensity of rhodamine-123 fluorescence. These results therefore suggest that the neuroprotective effects of mulberry C3G fraction are mediated by the maintenance of the MMP and mitochondrial function but not by attenuating glutamate-induced excitotoxicity in rat primary cortical neurons.

Cyanidin-3-glucoside inhibits glutamate-induced Zn2+ signaling and neuronal cell death in cultured rat hippocampal neurons by inhibiting Ca2+-induced mitochondrial depolarization and formation of reactive oxygen species

“…All these data suggest that C3G can induce neuroprotection against glutamate-induced cell death by Ca 2þ -dependent [Zn 2þ ] i increase through mitochondrial depolarization and formation of ROS. C3G has been reported to have a neuroprotective effect against neuronal cell death in oxygen glucose deprivation-induced cell death in rat cortical neurons (Bhuiyan et al, 2011(Bhuiyan et al, , 2012Kang et al, 2006). However, in Bhuiyan et al's studies (2011), C3G (10 μg/ml) was reported to have no inhibitory effects on glutamate-induced neuronal cell death in cultured cortical neurons.…”

“…All these data suggest that C3G inhibits glutamate-induced [Zn 2þ ] i increase by inhibiting formation of ROS through Ca 2þ -dependent mitochondrial depolarization, but not by ROS-dependent mitochondrial depolarization. In fact, the mulberry C3G (Bhuiyan et al, 2011) and black soybean C3G (Bhuiyan et al, 2012) have been reported to prevent OGD-induced mitochondrial depolarization in primary cortical neurons. In this study, while intracellular Zn 2þ chelator TPEN markedly inhibited glutamateinduced [Zn 2þ ] i increase, it tended to inhibit, but not significantly, glutamate-induced mitochondrial depolarization.…”

“…Cyanidin-3-glucoside (C3G) is a component of anthocyanin which belongs to the flavonoid family. C3G has been reported to have a neuroprotective effect against neuronal cell death in vitro (Bhuiyan et al, 2011;Tarozzi et al, 2008) and in vivo (Kang et al, 2006;Ke et al, 2011). Glutamate is a major excitatory neurotransmitter in the central nervous system.…”

Anthocyanins Extracted from Black Soybean Seed Coat Protect Primary Cortical Neurons against <i>in Vitro</i> Ischemia

“…Immunocytochemical staining with anti-NeuN and anti-GFAP antibodies revealed that the cell cultures contained approximately 99% neurons. 29) Preliminary experiments showed that DIV-10 neurons seemed to be more resistant to OGD injury than DIV-7 neurons (data not shown). Therefore, we performed a time course study of OGD-induced cell death in DIV-7 cells, and neuronal injury was quantified by MTT reduction assay 24 h after OGD.…”

Expression and Activity of the Na-K ATPase in Ischemic Injury of Primary Cultured Astrocytes

“…MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Duchefa Biochemie, Haarlem, The Netherlands] is converted to the blue formazan product by metabolically active mitochondria, and the reaction is proportional to the number of viable cells [20,21]. Astrocytes were cultured in 96-well culture plates for 24 h and chemical ischemia was induced for 1, 2, 3, and 6 h. MTT (0.5 mg/ml) was added to the control and the ischemia-treated wells of the culture plate.…”