Yokukansan, a kampo medicine, protects against glutamate cytotoxicity due to oxidative stress in PC12 cells.
OBJECTIVE
Yokukansan is a traditional Japanese medicine consisted of seven medicinal herbs and has been used for treatment of neurosis, insomnia, and behavioral and psychological symptoms of dementia in Japan. The aim of the present study is to clarify the active compounds responsible for the protective effect of yokukansan against glutamate-induced cytotoxicity in PC12 cells.


METHODS
PC12 cells which is a tool for selective evaluation of test substances against oxidative stress was used in the present study. The cell survival rates or glutathione (GSH) levels were evaluated by a MTT reduction assay or GSH assay based on the GSH reductase enzymatic recycling method, respectively.


RESULTS
Glutamate (1-17.5mM) induced cell death of PC12 cells in a concentration- dependent manner. Yokukansan (125-500μg/ml) inhibited the glutamate-induced PC12 cell death. When the effects of extracts of the seven constituent herbs in yokukansan on the cell death were examined, Uncaria thorn was found to have the highest potency in the protection. To clarify the active compounds in Uncaria thorn, the effects of seven alkaloids (rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine, hirsuteine, and geissoschizine methyl ether) on the cell death were further examined. The protective effects were found in hirsutine, hirsuteine, and geissoschizine methyl ether, which also ameliorated the glutamate-induced decrease in GSH levels.


CONCLUSIONS
These results suggest that yokukansan protects against PC12 cell death induced by glutamate-mediated oxidative stress, i.e., reduction of intracellular GSH level, and the effect may be mainly attributed to a synergistic effect of the hirsutine, hirsuteine, and geissoschizine methyl ether in Uncaria thorn.
Tsumura Research Laboratories, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan. kawakami_zenji@mail.tsumura.co.jp
Zenji
Z
Kawakami
Hitomi
H
Kanno
Yasushi
Y
Ikarashi
Yoshio
Y
Kase
D000818:Animals; D016923:Cell Death; D004365:Drugs, Chinese Herbal / Q000627:therapeutic use*; D018698:Glutamic Acid / Q000187:drug effects* / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism; D018384:Oxidative Stress; D016716:PC12 Cells; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004365:Drugs, Chinese Herbal; C524644:Yi-Gan San; D018698:Glutamic Acid; D005978:Glutathione