- no.50, pp.415-420, 2008-09-01
Accidental ingestion of a toadstool, Russula subnigricans causes lethal poisoning to human. In the 1950's, the first poisoning caused by this mushroom was reported. Since then there have been no reports about lethal poisoning for 50 years, which was enough to raise doubts about its existence. However, in these three years, 2005 to 2007, the poisoning accidents were continuously happened and four people died. Although chemical studies on this fungus were reported using mushrooms distributed in Miyagi prefecture, the isolated compounds, russuphelins, russupherol, and hydroxybaikiain, have no toxicity on mouse. Accordingly, we studied the isolation of the toxic constituent of R. subnigricans. One of the reasons that such a strong toxin has not been revealed until now is the incomplete classification of this mushroom, that is, there are many resemble species distributed in Japan. We collected three species in Kyoto, Miyagi, and Saitama prefectures. The aforementioned compounds were found only in the Miyagi species. All three species show toxicity on mouse by intraperitoneal injection of the water extract; however, only the Kyoto species exhibits toxicity by oral injection. Accordingly, we estimated that the Kyoto species is the genuine R. subnigricans. During the separation steps, we found that the toxicity was remarkably decreased after concentration to dryness; therefore, all manipulations were carefully performed. The water extract was successively separated through ODS column chromatography, ion exchange chromatography, and gel filtration to give an aqueous solution of the toxic compound. The toxic compound was revealed to be unstable under concentration to dryness (polymerization occurs) and volatile, which was turned out to be the cause of decrease in toxicity after evaporation. The unstable toxin was converted to a stable derivative using diphenyldiazomethane. Taking ^1H, ^<13>C NMR and MS spectral analyses of the toxic compound and its derivative into consideration, the structure of the toxic compound was determined to be cycloprop-2-ene carboxylic acid. This compound was found only in the Kyoto species.