RHAMNOSE.
It was stated above that Schmid obtained a sugar solution by the decomposition of a fisetin-glucoside from Rhus cotinus, and Perkin obtained the same from a glucoside in Rhus rhodanthema. These investigators thought that the sugar was isodulcite or rhamnose, but they did not isolate it on account of the small quantities of material at their disposal. Moreover, the sugar is very hard to crystallize in the presence of other soluble substances and is not found in large quantity in plants. Maquenne[27] could obtain only 15 to 20 gm. of rhamnose by working up 1 kilogram of the berries of Rhamnus infectorius. Assuming that the free fisetin found in poison ivy leaves had its origin in the decomposition of a fisetin-glucoside by natural processes, it was reasonable to suppose that the sugar would also be found in the free state, although, according to Roscoe and Schorlemmer:[28] "Isodulcite does not occur in the free state in nature, but is found as a peculiar ethereal salt belonging to the class of glucosides. On boiling with dilute sulphuric acid, this splits up into isodulcite and other bodies...." The more recent works on the sugars and on plant chemistry[29] mention the occurrence of rhamnose only in the glucoside form, with one possible exception. The exception referred to is the occurrence of a free sugar, supposed to be rhamnose, in a certain palm-wine.[30] Czapek says:[31] "The well-known methyl pentoses do not occur in the free state in plant organisms so far as we know."
Since rhamnose forms a lead compound, the sugar, if present, should be found in the first lead precipitate, A, and also in filtrate A in case it is not completely precipitated in the presence of acetic acid and alcohol.
The filtrate A (about two liters) was examined first. It had a light yellow color, contained an excess of lead acetate, and was acid from the acetic acid liberated in the precipitation of the lead compound A.[32] This filtrate was evaporated to dryness under diminished pressure to remove alcohol, water, and acetic acid. The clear distillate had a peculiar odor suggesting both tea and amyl formate. It was saved for examination and was found to be not poisonous. The residue in the dish after evaporation was a tough reddish brown, gummy mass which could be drawn out into fine threads. It had a pleasant sweet odor. It was extracted several times with hot water, each portion being filtered. A brownish yellow powder remained undissolved and was saved. The combined filtrates deposited more of the yellow solid on standing. This powder will be referred to later as "P." The filtered liquid was freed from lead by hydrogen sulphide. The solution then had a lemon yellow color, a sweet odor and was acid from acetic acid. On concentrating the solution by evaporation and making a small portion of it alkaline with sodium hydroxide, the yellow color came out very intense[33]. The alkaline solution reduced Fehling solution and ammoniacal silver nitrate, indicating the presence of a sugar. Another portion of the solution gave a slight precipitate with phenyl hydrazine in the cold. The remainder of the solution was evaporated to dryness, extracted with water, filtered, and again evaporated. A dark sticky syrup was left which was only partly soluble in water. This was treated with water, filtered, and the filtrate was evaporated, the water being replaced from time to time to remove acetic acid. Finally the liquid gave the following tests for rhamnose, besides those already mentioned:
(1) With α-naphthol[34] and sulphuric acid, a purple violet color.
(2) With thymol[35] and sulphuric acid, a red color.
(3) With resorcinol[36] and sulphuric acid, red color.
(4) With orcinol[37] 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[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.
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.