A portion of the poisonous tar was heated in an open dish with strong
acetic acid. The tar seemed to be decomposed to some extent, giving a
yellow substance. Acetic acid was added from time to time as it
evaporated. After several evaporations, water was added, the mixture was
heated to boiling and filtered. This filtrate No. 1 will be mentioned
later. The residue in the dish consisted of undecomposed tar and an
olive-green flaky substance. This substance was heated with a fresh
portion of glacial acetic acid. Water was added, and the mixture was
boiled and filtered. The filtrate had a deep yellow color suggesting
fisetin. It was shaken out with ethyl acetate which became colored
yellow. A portion of the ethyl acetate solution gave an orange red
precipitate with lead acetate showing the presence of fisetin. The ethyl
acetate was removed from the remainder of the solution by evaporation
and the yellow residue was taken up in alcohol. This alcoholic solution
gave the characteristic reactions for fisetin with stannous chloride,
with potassium hydroxide, with ferric chloride and with Fehling
solution.
Filtrate No. 1 obtained by heating the poisonous tar with acetic acid
and hot water as described above was investigated as follows: A portion
of it gave a reddish colored precipitate with sodium carbonate as in the
case when the tar was hydrolyzed with sulphuric acid. The remainder was
nearly neutralized with sodium carbonate and lead acetate was added in
excess to remove gallic acid. The excess of lead was removed by
sulphuric acid, and the sulphuric acid was removed by barium carbonate.
The solution on evaporation reduced Fehling solution to some extent, but
a white precipitate was also formed.
In this experiment, gallic acid and fisetin and probably sugar were
formed by decomposition of the poisonous gum with acetic acid, the acid
found in the plant by Pfaff. The presence of free gallic acid, fisetin
and rhamnose in the plant can therefore be explained by the natural
hydrolysis of a complex gum or tar or a constituent thereof. The
poisonous property is lost in the general rearrangement which takes
place during hydrolysis.
The poisonous tar was not hydrolyzed by boiling with a dilute solution
of sodium carbonate.
It was found, as has been stated elsewhere, that the lead compound of
the poison could not be precipitated in 95 per cent. alcohol. Further
experiments, however, showed that on extracting the poisonous gum with
50 per cent. alcohol, a portion of it dissolved, and this solution gave
a precipitate with lead acetate which was a true lead compound. The
remainder of the purified tar (about 10 gm.) was treated with 50 per
cent. alcohol and filtered. Very little dissolved in alcohol of this
strength, but on addition of lead acetate in 50 per cent. alcohol to the
solution, a light colored precipitate was formed, which became dark on
standing. It was filtered off, washed free from lead acetate, decomposed
by hydrogen sulphide, and shaken out with ether. The ether left, on
evaporation, a yellow resinous substance having a faint odor like
garlic. By drying in a desiccator, a small quantity of a solid yellow
resin was obtained which was completely soluble in alcohol. A very small
drop of this solution applied to the skin on the end of a glass rod
which had been drawn out to a point caused an eruption in about
thirty-six hours. Following the nomenclature used by Maisch and Pfaff,
this substance will be designated as _Toxicodendrin_, the ending "in"
indicating its glucoside nature.
The filtrate from the lead precipitate just described was freed from the
excess of lead acetate by hydrogen sulphide, was tested for poison, and
was found to be poisonous, showing that the precipitation by lead
acetate was not complete even in 50 per cent. alcohol. On spontaneous
evaporation of the solution, a yellow, sweet smelling resin was left.
A portion of the alcoholic solution of the toxicodendrin gave a dark
coloration with ferric chloride, did not reduce Fehling solution and was
slightly acid to litmus.
To see whether the toxicodendrin could be hydrolyzed, the remainder was
dissolved in alcohol and dilute sulphuric acid was added. A fine, white
precipitate was formed at once which rose to the surface on standing as
a light flocculent substance. The mixture was heated for several days on
a water bath, filtered from unhydrolyzed resin and the filtrate was
neutralized and concentrated in the way already described. The solution
obtained reduced Fehling solution. Not enough was obtained for further
sugar tests, but all the hydrolysis experiments point to the conclusion
that the poisonous substance is a rhamnoside, and is the source of the
sugar in the plant.
The reaction with ferric chloride observed whenever a lead compound of
the poison is decomposed by hydrogen sulphide may be explained by the
formation of traces of gallic acid or fisetin through the action of the
weak acids present.
The supply of purified poisonous tar having been exhausted in the
preceding experiments, further study of the active principle is
postponed until more can be prepared. It is highly desirable to
investigate the white precipitate formed by addition of sulphuric acid
to an alcoholic solution of the toxicodendrin.