Part 6

(2) With thymol and sulphuric acid, a red color.

(3) With resorcinol and sulphuric acid, red color.

(4) With orcinol and hydrochloric acid, red color.

(5) With ammonium picrate and sodium picrate, yellowish red color.

(6) With phloroglucinol and hydrochloric acid, red color.

(7) It decolorized an alkaline solution of potassium ferricyanide.

(8) It gave a white precipitate with lead acetate.

The filtrate B (p. 20) from which gallic acid was precipitated by sulphuric acid in four fractions was saved to examine for sugar. To remove gallic acid completely, and other vegetable matter, it was shaken out several times with ether, and was kept at a low temperature with salt and ice for a long time. It was left standing for several weeks, during which time more brown matter separated out and was filtered off. The filtrate was evaporated to a small bulk, cooled, and filtered from crystals of potassium sulphate. The filtrate was evaporated to dryness, the residue taken up in water and filtered through bone-black. Addition of alcohol caused complete precipitation of potassium sulphate. The solution then gave the above mentioned characteristic tests for rhamnose.

All attempts to get the osazone of the sugar by the method of Fischer failed, probably on account of the small quantity of the sugar present. The plant, it will be remembered, was originally extracted with ether in which rhamnose is practically insoluble. The above described tests, however, can leave no doubt as to the identity of the sugar.

Additional evidence that the sugar is rhamnose was obtained by a method described by Maquenne as follows:

"The production of methyl furfurol in the dehydration of isodulcite furnishes a very simple means of characterizing this sugar in mixtures which contain it; it is sufficient, for example, to distil 50 gm. of quercitron wood with as much sulphuric acid and about 150 gm. of water, then to rectify the liquid obtained in order to get several drops of the crude furfurol, which on addition of alcohol and concentrated sulphuric acid gives immediately the green coloration characteristic of methyl furfurol. This procedure is applicable to extracts as well as to entire plants, and has the advantage that it does not require the separation of isodulcite, the crystallization of which is often very slow and at times impossible when it is mixed with other very soluble substances."

The experiment was tried with the crude ether extract of the plant according to the directions of Maquenne, and the green color with alcohol and sulphuric acid was obtained from the thicker oily portion of the distillate. This test can be made with hydrochloric acid as well as with sulphuric. Therefore the color test was tried with the ester mixture prepared in one of the early experiments by boiling the original plant material with hydrochloric acid and alcohol. Methyl furfurol was found here also, this method indeed giving better results than that of Maquenne.

The presence of free rhamnose has thus been shown in the original material, in the first precipitate by lead acetate, and in the filtrate from this precipitate. Experiments to be described under "The Poison" showed that the ether extract from the Soxhlet apparatus contained a substance which yielded rhamnose when hydrolyzed by dilute sulphuric acid.

The presence of free gallic acid, fisetin, and rhamnose in the plant can be readily explained by a series of assumptions for which there is a considerable amount of experimental evidence. There is reason to believe that tannin-like bodies are formed at the expense of chlorophyll, that complex tannin bodies can be broken down by acetic acid (also found in Rhus toxicodendron) into a tannic acid and a glucoside (for example, the "fustin-tannide" mentioned above yields tannic acid and fisetin-glucoside); and finally that the glucoside can be hydrolyzed by acids or enzymes giving, in the sumach plants, fisetin and rhamnose.

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 found that diastase, pancreatin, and a number of other enzymes cause hydrolysis of triacetyl glucose with the formation of glucose and acetic acid. Stevens 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.

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.