The writer's investigation was undertaken with the object of attempting
to throw more light on the chemical nature of the poisonous substance
found in _Rhus toxicodendron_. Soon after commencing work, however, it
became apparent that the poison could be more intelligently studied if
the substances associated with it in the plant were first identified;
the scope of the work was therefore extended to an investigation of the
other constituents of the plant, and it was hoped that a knowledge of
the properties of these constituents would suggest a more economical way
of getting the poison than the method of fractional precipitation.
The crude material for this work was prepared by Messrs. Parke, Davis &
Co., of Detroit, Mich., according to special instructions submitted to
them: 67-1/2 pounds of fresh leaves and flowers of poison ivy were
collected near Detroit and carefully inspected by a competent botanist.
This material was thoroughly macerated and put into ten-liter bottles
with ether. The mass was thoroughly shaken, water being added to make it
more mobile. The ether was then separated off and the extraction was
repeated three times in the same way to insure complete removal of the
toxicodendrol. The ether extracts were combined, thoroughly dried with
anhydrous sodium sulphate, and the ether was distilled off, the
temperature being kept below 40 deg. C. during the entire distillation.
The residue after the removal of the ether was a thick, black, tar-like
mass, weighing 3 pounds 11 ounces. In extracting the plant, about
twenty-four gallons of ether were used. It is a significant fact in
regard to the volatility of the poison that during the process of
preparing this material none of the employees engaged in the work were
in any way affected, since proper precautions were taken and the
utensils were handled with rubber gloves.
The crude ether extract, which will be designated as the "original
material," was shipped to Baltimore in August and was kept in a cool
place until November when the investigation was begun. When the bottle
was opened, there seemed to be an escape of a vapor, and a nauseating
odor suggesting crushed green leaves pervaded the atmosphere. Some days
later, irregular red patches appeared on the face though a mask of
cotton cloth was worn during the work, and the hands were protected by
rubber gloves.
Assuming from Pfaff's work that this original material contained the
non-volatile oil toxicodendrol, the first experiment was to try to
distil it out under diminished pressure. For this purpose, an Anschuetze
distilling bulb containing ten grams of the tar was connected with a
vacuum pump. After a pressure of 2 mm. had been established the bulb was
gradually heated in a bath of Wood's metal. Nothing distilled over. The
material began to carbonize at a temperature of 140 deg. to 150 deg.
It was then thought that perhaps the oil could be converted into an
ester which might be more volatile and could be distilled out. 20 grams
of the original material were dissolved in 100 cc. of absolute alcohol
containing 3 grains of hydrochloric acid gas, and the mixture was heated
10 hours on a water-bath under a return condenser. After the heating,
the mixture had a delightful ethereal odor. The flask was corked and
left standing several weeks while other work was in progress. The ester
solution was then put in a vacuum desiccator over sulphuric acid and the
alcohol evaporated. A black, tarry, solid mass was left having the ester
odor. It was extracted with warm water and filtered from insoluble tar.
The filtrate had a green color and the ethereal odor. It was shaken out
with ether; the ether layer had a blood-red color while the water layer
was deep green. The extraction with ether was continued until the water
layer was no longer green. The combined ether extracts were evaporated
in a desiccator without heat. A black tar-like solid was left very much
like the original material, but it had the ester odor. It was partly
soluble in water and readily soluble in alcohol. The alcoholic solution
was tested on the skin and found to be not poisonous. The ester, or
mixture of esters, was not investigated further in this connection, but
was later shown to give the reactions for gallic acid and methyl
furfurol. These reactions will be referred to in connection with other
experiments.
After a few other preliminary experiments, it became evident that the
original material was a complex mixture of substances and that it would
have to be fractionated by some means and the fractions studied
separately.
A portion of the original substance was treated with 50 per cent.
alcohol and was found to be partly soluble in this medium. The solution
was filtered from insoluble tar. A portion of the yellow filtrate gave a
reddish yellow precipitate with lead acetate. The alcoholic solution was
distilled in an Anschuetze flask under diminished pressure; a yellow
liquid condensed in the arm of the flask while most of the alcohol was
collected in a bottle connected with the arm. The yellow liquid was acid
to litmus. Water was added, the solution was shaken out with ether and
the ether was evaporated. When the small residue was completely dry, it
was a yellow solid soluble in dilute alcohol and acid to litmus. The
substance was not volatile enough to justify the use of this method for
getting it.
Chlorophyll could not be removed from the original substance because the
solvents for chlorophyll such as alcohol, ether, fats, petroleum, and
carbon bisulphide dissolve large quantities of the mixture.
A precipitate obtained by adding lead acetate to a filtered solution of
the original substance in 50 per cent. alcohol was suspended in water,
decomposed by hydrogen sulphide, shaken out with ether and the ether
evaporated. The residue appeared at first to be a yellow oil, but on
complete evaporation of the ether in a desiccator, a yellow solid was
obtained--apparently the same as that obtained by vacuum distillation.
A solution of the original material in 50 per cent. alcohol was filtered
through bone-black and the filtrate was colorless. Examination showed
that everything had been removed by the bone-black and the filtrate was
apparently pure alcohol and water.
In precipitating an alcoholic solution of the crude material with a
solution of lead acetate, it was noticed that at least two kinds of
precipitates were formed. The part that went down first was darker in
color than that thrown down later. Pfaff used the last fractions in
obtaining his oil and stated that these precipitates consisted of the
lead compound of the oil in a pure state. It was found by experiment
that the darker part was soluble in ether while the lighter part was
not. This indicated that the darker part consisted of tarry matter which
was brought down mechanically or separated out when the alcoholic
solution was diluted by the lead acetate solution, or was perhaps a lead
compound soluble in ether. To test this point an experiment was made as
follows: Some of the crude material was thoroughly extracted with 50 per
cent. alcohol. The tar insoluble in 50 per cent. alcohol was then
treated with 95 per cent. alcohol; most of it dissolved; the solution
was filtered and lead acetate in 50 per cent. alcohol was added. A
greenish colored precipitate was formed which was filtered off and found
to be completely soluble in ether and soluble to a considerable extent
in strong alcohol. These experiments suggested that the light colored
lead compound which was thought to contain the poison could be purified
by extraction with ether in a Soxhlet apparatus more conveniently than
by the tedious process of fractional precipitation. Further preliminary
experiments showed that 50 per cent. alcohol extracted from the original
material all of the substance or substances which gave the light colored
precipitate and dissolved only a small amount of the tar.
Two hundred and eighty-eight grams of the crude material were then
extracted several times with 50 per cent. alcohol and filtered; the
insoluble tar was washed and saved for examination. To the filtrate was
added an excess of a solution of lead acetate in 50 per cent. alcohol.
The large precipitate, which will be designated as "precipitate A," was
filtered and drained by suction in a Buechner funnel. The alcoholic
"filtrate A" was saved. Precipitate A was extracted with ether in
Soxhlet extractors until the ether came over practically colorless, the
operation being interrupted from time to time to stir up the precipitate
in the thimble. The green colored ether solution was saved for future
examination. The lead precipitate, after extraction with ether and
drying, weighed about 116 grams. A portion of this lead compound was
decomposed by hydrogen sulphide in a mixture of water and ether which
was well shaken during the operation. The ether was separated, filtered,
and evaporated under diminished pressure without heat, and there
remained a yellow oily looking residue having a pleasant odor. When the
ether and water were completely removed in a vacuum desiccator, a hard,
brittle, yellow resin weighing about 16 grams was obtained. It was
soluble in alcohol, had a strong acid reaction and was free from
nitrogen[16] and sulphur. The nitrogen tests were made by the Lassaign
and soda lime methods,[17] and the sulphur test was made with sodium
nitroprusside after fusing the residue with sodium. The main portion of
the lead compound was decomposed under alcohol by hydrogen sulphide,
filtered, and the alcoholic filtrate evaporated in vacuo. The same
yellow acid resin was obtained. Experiments continuing through several
weeks were made in applying solutions of this resin to rats, rabbits and
guinea pigs. Finding it to be without effect upon these animals it was
tried on the writer and found to be not poisonous.[18] In the meantime
the resin was being studied in the laboratory.