Protective effect of Keishi-bukuryo-gan and its constituent medicinal plants against nitric oxide donor-induced neuronal death in cultured cerebellar granule cells

Keishi-bukuryo-gan (Gui-Zhi-Fu-Ling-Wan) (KBG) is a traditional Chinese/Japanese medical (Kampo) formulation that has been administered to patients with "Oketsu" (blood stagnation) syndrome. In the process of neuronal cell death induced by brain ischemia, excessive generation of nitric oxide (NO) free radicals is implicated in the neurotoxicity. In the present study, we examined the protective effects of KBG and its constituent medicinal plants against NO donors, sodium nitroprusside (SNP) and 2,2′- (hydroxynitrosohydrazino)bis-ethanamine (NOC18)-induced neuronal death in cultured rat cerebellar granule cells (CGCs). MTT assay showed cell viability to be significantly increased by the addition of KBG extract (KBGE) (100 μg/ml) Cinnamomi Cortex extract (CCE) (3, 10 and 30 μg/ml), Paeoniae Radix extract (PRE) (100μg/ml) and Moutan Cortex extract (MCE) (10 and 30μg/ml) compared with exposure to SNP (30 μM, 24 h) only. Also, cell viability was significantly increased by the addition of KBGE (100 and 300 μg/ml), CCE (30 and 100 μg/ml), PRE (100 and 300 μg/ml) and MCE (30 and 100 μg/ml) compared with exposure to NOC18 (100 μM, 48 h) only. Persicae Semen extract and Hoelen extract did not protect against NO donor-induced neuronal death. These results suggest that KBG has protective effect against NO-mediated neuronal death in cultured CGCs and that it is derived from Cinnamomi Cortex, Paeoniae Radix and Moutan Cortex.