[Isolation and determination of an antidote for botulinum neurotoxin from black tea extract]

The botulinum neurotoxin produced by Clostridium botulinum exhibits the strongest neurotoxicity, and causes botulism in mammals. We have found an inactivator for clostridial neurotoxins in black tea extract (thearubigin fraction) as a natural foodstuff. In this study, we have isolated and identified the inactivators. The activity against the neuromuscular blocking action of botulinus neurotoxin type A was examined in mouse phrenic nerve diaphram preparation. The purification procedure of the inactivators was as follows. Tea was extracted with aqueous acetone, and then filtrated and lyophilized. It was also extracted with n-hexane, chloroform, ethyl acetate, n-butylalchol and water, so the activity of the antidote was recognized to be in the n-butylalchol layer (named the thearubigin fraction). A two-step reversed phase HPLC was developed for the thearubigin fraction. Three flavonoids were found to have the major activity. The structural elucidation of the compounds by means of NMR spectrascopy revealed, kaempfenol-3-O-[glc-(6-1)-rha-(3-1)-glc];keampfetrin, kaempferol-3-O-[glc-(6-1)-rha];nicotiflorin and quercetin glycoside.