All attempts to get the osazone of the sugar by the method of Fischer[38] 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[39] 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[40] 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,[41] 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[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.