Studies on the possible mechanisms of protective activity against α-amanitin poisoning by aucubin

Aucubin, an iridoid glucoside, was investigated to determine whether it has a stimulating effect on a-amanitin excretion in a-amanitin intoxicated rats, and whether there is binding activity to calf thymus DNA. High-performance liquid chromatography (HPLC) analysis of a-amanitin in rat urine allowed quantitative measurement of the a-amanitin concentration with a detection limit of 50 ng/ml. In this system, a group treated with both a-amanitin and aucubin showed that a-amanitin was excreted about 1.4 times faster than in the a-amanitin only treated group. Our previous results showed that the toxicity of a-amanitin is due to specific inhibition of RNA polymerase activity and the resultant blockage of the synthesis of certain RNA species in the nucleus. However, no significant activity change on RNA polymerase from Hep G2 cells was observed when aucubin was treated with a-amanitin at any concentration tested. Nevertheless, aucubigenin inhibited both DNA polymerase (IC50, 80.5 mug/ml) and RNA polymerase (IC50, 135.0 mug/ml) from the Hep G2 cells. The potential of both a-amanitin and aucubin to interact with DNA were examined by spectrophotometric analysis. alpha -Amanitin showed no significant binding capacity to calf thymus DNA, but aucubin was found to interact with DNA, and the apparent binding constant (K-app) and apparent number of binding sites per DNA phosphate (B-app) were 0.45 x10(4) M-1 and 1.25, respectively.