Neuroprotective Effects of Banryong-hwan in Primary Rat Mesencephalic Dopaminergic Neurons

반룡환의 흰쥐태아중뇌에서의 도파민세포 보호효과

Objectives : Oxidative stress has a critical role in neurodegenerative diseases. In this study, we investigated the antioxidant and neuroprotective effects of the ethanolic extract of Banryong-hwan (BRHE) in SH-SY5Y cells and primary rat mesencephalic dopaminergic neurons. Methods : To assess the antioxidant effects, we carried out 1,1-diphenyl-2-picrylhydrazyl(DPPH) free radical scavenging assay, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS) radical cation decolorization assay, and determination of total polyphenolic content. We evaluated the effect of BRHE treatment on neuroprotection against 6-hydroxydopamine(6-OHDA) toxicity using thiazolyl blue tetrazolium bromide(MTT) assay, nitric oxide(NO) assay, reactive oxygen species(ROS) assay in SH-SY5Y cells and tyrosine hydroxylase(TH) immunocytochemistry in primary rat mesencephalic dopaminergic neurons. Results : BRHE showed IC50 values of 328.10 ${\mu}g/mL$ and 43.12 ${\mu}g/mL$ in DPPH assay and in ABTS assay, respectively. Total polyphenolic content was 180.76 ${\mu}g/mL$. In SH-SY5Y cells, BRHE significantly attenuated the toxicity induced by 6-OHDA at the concentrations of 25-100 ${\mu}g/mL$ pre- and post- treatment in MTT assay. While 6-OHDA increased the NO and ROS contents, BRHE decreased them in a dose dependent manner. Moreover, in primary dopaminergic neuron culture, BRHE significantly protect-ed the dopaminergic cell loss against 6-OHDA toxicity up to 136% at the concentration of 75 ${\mu}g/mL$. Conclusions : These results demonstrate that BRHE has neuroprotective effect against 6-OHDA induced neurotoxicity through decreasing NO and ROS generation.