Part 3

Nitrogenous ferments which can effect the hydrolysis of glucosides and give rise to sugars are frequently found in plants, for example, emulsin in almonds, myrosin in mustard, and erythrozym in madder. Acree and Hinkins[42] found that diastase, pancreatin, and a number of other enzymes cause hydrolysis of triacetyl glucose with the formation of glucose and acetic acid. Stevens[43] obtained a nitrogenous oxidizing enzyme from _Rhus vernicifera_. The close relationship between the poisonous species of _Rhus_ would lead us to suppose that the same soluble ferment exists in poison ivy, though it was not detected in the original material used in these experiments, probably because the plant was extracted with ether in which the enzyme is insoluble. The existence of such a soluble ferment would explain the presence of free sugar and free fisetin.

EVIDENCE OF THE PRESENCE OF A FATTY ACID IN FILTRATE A.

The brown substance P, obtained from filtrate A by evaporation and extracting the residue with hot water, was suspended in warm water and dilute sulphuric was added. A white precipitate was formed and a strong fatty acid odor was developed. After the mixture had been heated for some hours on the water bath a small portion was made alkaline and it reduced Fehling solution. The main solution was filtered and the precipitate supposed to be a fatty acid was saved. The filtrate was neutralized with barium carbonate, filtered, evaporated, freed from caramel, and the solution then gave the tests mentioned above for rhamnose.

A portion of the precipitate supposed to be a fatty acid was ignited in a porcelain spoon. It fused, carbonized, and burned. The remainder was heated with alcoholic potash and reprecipitated with hydrochloric acid. The precipitate was washed and heated with alcohol. Part of it dissolved. The insoluble part was found to be a lead compound. On boiling it with hydrochloric acid and cooling, lead chloride crystallized out. This was confirmed by dissolving the lead chloride in hot water and precipitating as lead sulphide. These experiments were not carried farther on account of the small quantity of material, but they show that the gummy substance obtained from filtrate A contained rhamnose (either as a lead compound of free sugar or as a lead compound of a rhamnoside), and also, most probably, the lead compound of an organic acid.[44]

THE FRAGRANT DISTILLATE.

Several times in the course of this work, extracts of the original plant material in alcohol and in water were distilled under diminished pressure for the purpose of concentrating the solutions. The distillate, in every case, had an ethereal odor suggesting amyl formate in very dilute solution, but was more fragrant. The distillate from a water extract was examined. It was a clear liquid, a little darker than pure water, was not poisonous, was neutral to litmus paper, gave no color with ferric chloride, reduced ammoniacal silver nitrate, but not Fehling solution, and gave a faint red color with dilute ammonium hydroxide and with sodium carbonate.

A small quantity of a finely divided black precipitate separated out from the water distillate on standing.

The substance with the fragrant odor was extracted by shaking the distillate with ether and letting the ether evaporate spontaneously. A very small quantity of a yellow solid was deposited on the sides of the dish. This substance had a strong and persistent odor, so sweet as to be almost nauseating. Not enough was obtained for examination or analysis. This fragrant residue was difficultly soluble in water and the solution reduced silver nitrate in ammonia. A steam distillate of the original plant material had the same fragrant odor as the distillate from a water extract.

THE POISON.

288 grams of the original poisonous material were extracted with 50 per cent. alcohol, and this alcoholic solution was precipitated with lead acetate in the manner already described (p. 17). The lead precipitate so obtained was extracted with ether in Soxhlet extractors and after the extraction was found by test to be free from poison. Therefore the poison, if precipitated by the lead acetate, must have been extracted by the ether. This ether solution had a dark green color, and was acid from acetic acid brought down in the lead precipitate. The ether was evaporated in a vacuum desiccator without heat and there remained a small quantity of an acid mixture of water and a soft tar; the watery part was colored green, showing that the tar was soluble to some extent in dilute acetic acid. The mixture had the peculiar odor of the original material. A small drop of the green watery part was applied to the wrist, allowed to remain a few minutes and was then removed by absorbent paper, but the spot was not washed. Itching and reddening of the skin commenced within twenty-four hours. At the end of forty-eight hours, there was a well developed case of poisoning. How this was cured will be described in another place.

A small portion of the poisonous mixture was dissolved in alcohol, and this solution was divided into three parts. The first part was treated with ferric chloride, but it gave no color reaction. Another portion of the alcoholic solution was diluted with water. It became turbid. The third portion gave a dirty-green precipitate with lead acetate, which seemed to come down more readily when the solution was diluted with water. The main portion of the poisonous mixture was then dissolved in 95 per cent. alcohol and lead acetate in 50 per cent. alcohol was added. The precipitate was filtered, washed, and decomposed by hydrogen sulphide in a mixture of water and ether. The ether solution was filtered and evaporated. The residue was a tar which, on standing in a desiccator for some time, became dry enough to break into sticky lumps. An alcoholic solution of this substance gave a dark color with ferric chloride and a light colored precipitate with lead acetate.

To get more of the poisonous tar for study, 233 grams of original material were extracted with 95 per cent. alcohol. Strong alcohol was used in order to dissolve as much of the tar as possible. The solution had a dark greenish color, but was somewhat yellow in thin layers. The undissolved tar was filtered off and extracted twice again in the same way. The tar left after the third extraction was only slightly soluble in alcohol, and its solution was not poisonous. The three filtrates from these three extractions were precipitated separately by lead acetate in 50 per cent. alcohol. The first precipitate was largest, darkest in color, and carried down more tarry matter. The second was light green, and the third was quite small, black, and was not a lead compound at all, but some of the tar which separated out on diluting the strong alcohol with the weaker grade containing lead acetate. It was soluble in ether and less soluble in alcohol. The alcoholic solution of this third lot gave no precipitate with hydrogen sulphide. The first and second lead precipitates were filtered by suction and washed with water. They were kept a day or two in a desiccator over sulphuric acid, but did not become completely dry. The weight of these two moist precipitates together was 172 grams. They were combined and extracted with ether in Soxhlet extractors which were kept in operation during work hours for three days.

In the meantime the alcoholic filtrates from these lead precipitates were combined and concentrated on the water bath by distilling off two liters of alcohol. The alcohol obtained had the peculiar odor of the original material, but was not poisonous.

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 deg. 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 alpha-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.

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.

OXIDATION OF THE PURIFIED TAR WITH NITRIC ACID.

When the purified poisonous material (p. 32) was extracted with 50 per cent. alcohol, only a small quantity was dissolved as was stated above. The insoluble residue was treated with fuming nitric acid. Violent reaction took place at once with copious evolution of red fumes and heat. When the reaction was over, a sticky red gummy mass was left which was slightly soluble in cold water and readily soluble in warm alcohol. The water extract was yellow, and the alcoholic solution was red. That the water extract contained picric acid was shown by the following experiments:

(1) A portion was gently warmed with a few drops of a strong solution of potassium cyanide and two drops of sodium hydroxide. The red color of potassium isopurpurate was formed.

(2) A portion of the water solution was heated with glucose and a few drops of sodium hydroxide. The deep red color of picraminic acid was produced.

(3) A few drops of an ammoniacal solution of copper sulphate was added to the water extract. A yellow-green precipitate was formed.

(4) The water extract dyed silk, but did not dye cotton cloth.

DISTILLATION OF THE TAR WITH SODA LIME.

About 25 gm. of the tar left after extracting the original material with hot water was dissolved in ether and poured into a glass retort containing soda lime. The ether was distilled out, leaving the tar intimately mixed with the soda lime. The retort was then gradually heated. Vapors and liquid were given off, both of which turned red litmus blue and had a strong odor like tobacco smoke. No odor of ammonia was detected.[45] At the high temperature of the triple burner, a semi-solid, red, greasy substance collected in and closed the condenser tube. This substance had the same powerful odor as the liquid portion of the distillate. The clear, watery portion of the distillate was separated from the thicker parts, and was found to contain pyrrol and pyridine derivatives by the following characteristic tests:

(1) Wood moistened with hydrochloric acid was turned red by it.

(2) Colorless fumes were formed when brought near hydrochloric acid; mixed with hydrochloric acid, a red insoluble substance was formed.

(3) It precipitated the hydroxides of iron, gave a light blue precipitate with copper sulphate, and a white precipitate with mercuric chloride.

The greasy, semi-solid mass was extracted with 10 per cent. hydrochloric acid and filtered. On addition of a solution of mercuric chloride to the red filtrate, a brown flocculent precipitate was formed. It was filtered off and distilled with caustic soda, but the distillate did not contain pyridine.

POTASSIUM PERMANGANATE AS A REMEDY FOR RHUS POISONING.[46]