After a long extraction of the lead precipitate in the Soxhlet extractors, the green ether solutions were combined and washed by shaking them with water to remove lead acetate and acetic acid in case any should have been held in the lead precipitate. The ether was distilled off at a low temperature and there remained a soft tar, a portion of which was not completely soluble in 95 per cent. alcohol. The alcoholic solution had a greenish yellow color and was poisonous. The tar was also partly soluble in acetic acid, and this solution was found to contain lead. Thinking that the lead acetate had not been completely washed out, the main part of the tar was dissolved in ether and shaken with water. The wash water continued to give a test for lead as long as the washing was continued. This indicated probably the hydrolysis of an unstable lead compound. Hydrogen sulphide was passed into the ether solution mixed with water to remove the lead. Lead sulphide was filtered off, and the ether was evaporated. A small portion of the tar residue in alcoholic solution gave a color reaction with ferric chloride. As this may have been due to traces of lead gallate dissolved in the extraction with ether and afterwards decomposed by hydrogen sulphide, the main portion of the tar was redissolved in ether and shaken with water until it no longer reacted with ferric chloride. The ether was then evaporated and a soft, black, poisonous tar or gum of uniform consistency was left which was shown by tests to be free from gallic acid and lead. These experiments showed that some of the poison was precipitated as a lead compound soluble in ether and some was brought down mechanically in the free state. To see if the extraction with ether in the Soxhlet apparatus was complete, the residue in the thimbles was decomposed by hydrogen sulphide and shaken with ether. The dark colored ether solution was freed from gallic acid by shaking with water and dilute sodium carbonate solution, and was evaporated. A small quantity of tar was obtained which was added to the main portion.
A solution of the poisonous tar in 95 per cent. alcohol did not reduce Fehling solution and did not give a precipitate with lead acetate except the separation of a small quantity of tar, which was not a lead compound. The lead compound of the poison was apparently soluble in 95 per cent. alcohol as well as in ether, for it would not precipitate in this medium, although it was found in the original precipitate by lead acetate. The alcoholic solution of the tar became turbid on diluting with water.
In order to see if the poison is volatile with vapor of acetic acid, since this acid is found in the plant and it is thought by some that the poison is volatile, a portion of the tar was distilled under diminished pressure with acetic acid. It was soluble to some extent in the acid. The temperature did not go higher than 55° during the distillation. A tube containing cotton wet with sweet oil was placed between the receiver and the water suction so that the uncondensed vapors would have to pass through the cotton. This cotton was rubbed on the skin and was not poisonous. The yellow distillate collected in the receiver was also tested and was not poisonous.
HYDROLYSIS OF THE POISON.
About 5 grams of the poisonous tar free from gallic acid and sugar was dissolved in alcohol, and dilute (2 per cent.) sulphuric acid was added. Some of the tar separated out on diluting the alcohol with the acid. The mixture was heated on a water bath during work hours for four days. A purple and green fluorescent solution was formed, though much tar was left apparently unchanged. The alcohol was evaporated off and the solution was filtered from tar. The fluorescent filtrate was shaken with ether, by which the green substance was removed, leaving the solution purple. The ether left, on evaporation, a small quantity of a green substance having a pleasant ester odor. It was not further examined. A portion of the purple solution was exactly neutralized with sodium carbonate. This solution gave a blue-black color with ferric chloride which became red on addition of another drop of sodium carbonate, indicating gallic acid. It also reduced Fehling solution.
Another portion of the purple solution was made alkaline with sodium carbonate. A reddish-brown flocculent precipitate was formed and was filtered off. The filtrate did not give any color with ferric chloride, but it reduced Fehling solution. It also gave the test for rhamnose with α-naphthol.
The main portion of the purple solution was made alkaline with sodium carbonate; the precipitate was filtered off and dissolved in acetic acid. This solution was yellow and gave a reaction with ferric chloride similar to that of gallic acid. The filtrate from the precipitate by sodium carbonate was concentrated by evaporation until sodium sulphate began to crystallize out. Alcohol was added to precipitate the sodium sulphate completely, the mixture was heated and filtered. The alcoholic filtrate was concentrated to a syrup which reduced Fehling solution and gave the characteristic tests for rhamnose already described. By this hydrolysis, the tar was split up into rhamnose and some form of gallic acid which could be precipitated by sodium carbonate. This compound, whose acetic acid solution was yellow, probably contained fisetin also. The reason for this last statement will appear from the following experiment:
DECOMPOSITION OF THE POISON WITH ACETIC ACID.
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.