Part 2

A portion of the original substance was treated with 50 per cent. alcohol and was found to be partly soluble in this medium. The solution was filtered from insoluble tar. A portion of the yellow filtrate gave a reddish yellow precipitate with lead acetate. The alcoholic solution was distilled in an Anschuetze flask under diminished pressure; a yellow liquid condensed in the arm of the flask while most of the alcohol was collected in a bottle connected with the arm. The yellow liquid was acid to litmus. Water was added, the solution was shaken out with ether and the ether was evaporated. When the small residue was completely dry, it was a yellow solid soluble in dilute alcohol and acid to litmus. The substance was not volatile enough to justify the use of this method for getting it.

Chlorophyll could not be removed from the original substance because the solvents for chlorophyll such as alcohol, ether, fats, petroleum, and carbon bisulphide dissolve large quantities of the mixture.

A precipitate obtained by adding lead acetate to a filtered solution of the original substance in 50 per cent. alcohol was suspended in water, decomposed by hydrogen sulphide, shaken out with ether and the ether evaporated. The residue appeared at first to be a yellow oil, but on complete evaporation of the ether in a desiccator, a yellow solid was obtained--apparently the same as that obtained by vacuum distillation.

A solution of the original material in 50 per cent. alcohol was filtered through bone-black and the filtrate was colorless. Examination showed that everything had been removed by the bone-black and the filtrate was apparently pure alcohol and water.

In precipitating an alcoholic solution of the crude material with a solution of lead acetate, it was noticed that at least two kinds of precipitates were formed. The part that went down first was darker in color than that thrown down later. Pfaff used the last fractions in obtaining his oil and stated that these precipitates consisted of the lead compound of the oil in a pure state. It was found by experiment that the darker part was soluble in ether while the lighter part was not. This indicated that the darker part consisted of tarry matter which was brought down mechanically or separated out when the alcoholic solution was diluted by the lead acetate solution, or was perhaps a lead compound soluble in ether. To test this point an experiment was made as follows: Some of the crude material was thoroughly extracted with 50 per cent. alcohol. The tar insoluble in 50 per cent. alcohol was then treated with 95 per cent. alcohol; most of it dissolved; the solution was filtered and lead acetate in 50 per cent. alcohol was added. A greenish colored precipitate was formed which was filtered off and found to be completely soluble in ether and soluble to a considerable extent in strong alcohol. These experiments suggested that the light colored lead compound which was thought to contain the poison could be purified by extraction with ether in a Soxhlet apparatus more conveniently than by the tedious process of fractional precipitation. Further preliminary experiments showed that 50 per cent. alcohol extracted from the original material all of the substance or substances which gave the light colored precipitate and dissolved only a small amount of the tar.

Two hundred and eighty-eight grams of the crude material were then extracted several times with 50 per cent. alcohol and filtered; the insoluble tar was washed and saved for examination. To the filtrate was added an excess of a solution of lead acetate in 50 per cent. alcohol. The large precipitate, which will be designated as "precipitate A," was filtered and drained by suction in a Buechner funnel. The alcoholic "filtrate A" was saved. Precipitate A was extracted with ether in Soxhlet extractors until the ether came over practically colorless, the operation being interrupted from time to time to stir up the precipitate in the thimble. The green colored ether solution was saved for future examination. The lead precipitate, after extraction with ether and drying, weighed about 116 grams. A portion of this lead compound was decomposed by hydrogen sulphide in a mixture of water and ether which was well shaken during the operation. The ether was separated, filtered, and evaporated under diminished pressure without heat, and there remained a yellow oily looking residue having a pleasant odor. When the ether and water were completely removed in a vacuum desiccator, a hard, brittle, yellow resin weighing about 16 grams was obtained. It was soluble in alcohol, had a strong acid reaction and was free from nitrogen[16] and sulphur. The nitrogen tests were made by the Lassaign and soda lime methods,[17] and the sulphur test was made with sodium nitroprusside after fusing the residue with sodium. The main portion of the lead compound was decomposed under alcohol by hydrogen sulphide, filtered, and the alcoholic filtrate evaporated in vacuo. The same yellow acid resin was obtained. Experiments continuing through several weeks were made in applying solutions of this resin to rats, rabbits and guinea pigs. Finding it to be without effect upon these animals it was tried on the writer and found to be not poisonous.[18] In the meantime the resin was being studied in the laboratory.

GALLIC ACID.

An alcoholic solution of the resin was just neutralized with potassium hydroxide. During the titration, the solution rapidly became dark brown. After neutralization it was shaken with ether; the water solution remained brown while the ether layer was nearly colorless and contained practically no dissolved substance. A portion of the water solution of the potassium salt on being acidified with sulphuric acid and standing over night, deposited a slight precipitate. The solution of the potassium salt gave a heavy precipitate with lead acetate somewhat similar to the original lead precipitate A, and also slight precipitates with salts of zinc, mercury, copper, and silver (with reduction). It gave a bluish-black color with impure ferrous sulphate and a dark color with ferric chloride. It reduced ammoniacal silver nitrate and Fehling solution. These experiments indicated the presence of a tannin compound. An alcoholic solution of the resin gave the same color reactions with iron salts as did the potassium salt. To determine which one of the tannin compounds was present was a matter of some difficulty since the di- and tri-hydroxybenzoic acids have in general the same color reactions. The presence of other plant substances in the solution also interferes with the color tests, and finally, a substance which gives a blue color with iron salts and one giving a green color may be found together in the same plant.[19] Further tests with a solution of the resin in dilute alcohol, and with a water solution of the acid precipitated by adding sulphuric acid to a solution of the resin in potassium hydroxide, led to the conclusion that the acid is gallic acid. These tests were the following:

(1) Boiling with an excess of potassium hydroxide gave a black substance (tauromelanic acid).

(2) The acid was not precipitated by gelatin.

(3) On addition of potassium cyanide a transitory red color appeared which reappeared on shaking with air.

Gallic acid is distinguished from tannic acid by tests (2) and (3). At later stages in the work the potassium, barium, and sodium salts of gallic acid were obtained, and finally the pure acid was made by decomposing the sodium salt with sulphuric acid and crystallizing from water. A portion of the acid so obtained was further purified by dissolving in absolute alcohol and pouring into absolute ether.[20] The melting point behavior of the acid corresponds with that of gallic acid; it melted with decomposition at about 230 deg. For further identification, some of the acid was converted into an ester by the following process: it was dissolved in 80 per cent. alcohol, hydrochloric acid gas was passed in, and the solution was heated an hour on the water bath. It was then evaporated to a small bulk, neutralized with barium carbonate and extracted with ether. The ether, on evaporation, left the ester which was crystallized from water and dried in a desiccator over sulphuric acid. The anhydrous ester agreed in melting point (156 deg. to 159 deg.) and other properties with the ester of gallic acid described by Grimaux.[21] For the sake of comparison, an ester was made from gallic acid obtained from another source and the two agreed in properties. A mixture of the two esters melted within the limits given for the ester of gallic acid.

While the tests leading to the identification of gallic acid were being made, another series of experiments was in progress. Eleven and one-half grams of the resin obtained from lead precipitate A by decomposition with hydrogen sulphide were treated with 0.1 n. potassium hydroxide added from a burette until the acid was exactly neutralized. All went into solution. On shaking with ether, some of the potassium salt separated out and was saved for examination. The solution became brown on exposure to air and got darker as the work proceeded. The acid in solution as a potassium salt was precipitated out in four fractions by adding for each fraction one-fourth the amount of 0.1 n. sulphuric acid required to neutralize the potassium hydroxide used. The precipitates were filtered off and examined. The first was small in amount, gummy and hard to filter. The solution was shaken with ether after each precipitate had been filtered off. The succeeding precipitates were in better condition, but were not pure. All appeared to be impure gallic acid which had become brown by absorption of oxygen. They were saved, however, to be tested for poison. After the last fraction had separated, the filtrate was shaken several times with ether and saved for further examination, which will be described under "Rhamnose." This filtrate is designated as B.

At this stage of the work a portion of the resin obtained from lead precipitate A was tested and found to be not poisonous as already mentioned. By this test, all the substances contained in the lead precipitate A after its extraction with ether in the Soxhlet apparatus, were eliminated from the possible poisonous substances. The poison must therefore have been extracted by the ether.

A fresh portion of the original poisonous material was treated with 50 per cent. alcohol and filtered from insoluble tar. The filtrate was precipitated in six fractions by lead acetate. The last fractions were lighter in color and apparently much purer than the first. The sixth lead precipitate was decomposed by hydrogen sulphide, the light-yellow water solution was tested and found to be not poisonous. It gave the characteristic reactions for gallic acid. The poison, if precipitated at all by lead acetate, must have gone down in one of the preceding fractions. Later experiments showed that it is brought down partly mechanically and partly as a lead compound in the first precipitates.

FISETIN.

Having identified gallic acid, and not finding any other phenol derivative in the lead precipitate, some of the original material was extracted with hot water to remove gallic acid and filtered from tar while hot. The filtrate had a deep yellow color. On cooling over night, an olive green precipitate separated out which was dried and found to be a light powder. It was practically insoluble in cold water, soluble with great difficulty in boiling water from which it separated in yellow flakes, slightly soluble in ether and in acetic acid, but readily soluble in alcohol. The solutions were not acid to litmus, gave a dark color with ferric chloride, an orange-red precipitate with lead acetate which was easily soluble in acetic acid, and an orange-yellow precipitate with stannous chloride. These properties and reactions indicated that the substance was the dye-stuff fisetin and that it occurs in the free state in this plant though it is usually found as a glucoside of fisetin combined with tannic acid. A compound of this kind was found in _Rhus cotinus_ and named "fustin-tannide" by Schmid[22]. He showed that the fustin-tannide could be decomposed by acetic acid into tannic acid and a glucoside, fustin C_{46}H_{42}O_{21}. Fustin, on heating with dilute sulphuric acid, gave fisetin and a sugar supposed to be rhamnose. Fisetin was also found as a glucoside compound in _Rhus rhodanthema_ by Perkin.[23]

The yellow substance which separated from the boiling water solution was further purified by dissolving in a small quantity of hot alcohol and adding hot water. On cooling, the yellow substance separated out in a flocculent condition. Examined under the microscope, the flakes appeared to be made up of masses of fine crystals.

An alcoholic solution of the substance gave a black color with ammonia which became red on addition of more ammonia. Concentrated acids intensified the yellow color of the alcoholic solution. Fehling solution and ammoniacal silver nitrate were reduced by it. Potassium hydroxide added to an alcoholic solution gave at first a deep red color accompanied by a green fluorescence which disappeared, leaving a yellow liquid. With an excess of caustic potash, the red color returned and was permanent. These reactions are characteristic for fisetin.[24]

Furthermore, fisetin should give protocatechuic acid and phloroglucinol by fusion with caustic potash under proper conditions.[25] The experiment was carried out as follows: 2 grams of fisetin were gently heated in a nickel crucible with 6 grams of caustic potash dissolved in 6 cc. water. An inflammable gas, apparently hydrogen, was evolved during the fusion. The pasty mass was dissolved in water, acidified with sulphuric acid, and filtered. The filtrate was shaken out with ether containing one-fourth its volume of alcohol. The ether was evaporated and the residue was extracted with warm water and filtered. Lead acetate was added to the filtrate to precipitate protocatechuic acid, while phloroglucinol remained in the filtrate from this precipitate. The lead precipitate was suspended in water, decomposed by hydrogen sulphide, filtered, and evaporated to obtain protocatechuic acid. That the substance obtained was protocatechuic acid was shown by the following characteristic tests:

(1) It gave a greenish brown color with ferric chloride; on addition of one drop of a dilute solution of sodium carbonate, the color became dark blue; on adding more sodium carbonate the color became red.

(2) A violet color was obtained when a solution of the acid was treated with a drop of sodium carbonate solution and then with a drop of ferrous sulphate.

(3) It reduced ammoniacal silver nitrate.

(4) It did not reduce Fehling solution.

The filtrate supposed to contain phloroglucinol was treated with hydrogen sulphide to remove lead, filtered, and shaken with ether. The residue left on evaporating the ether was taken up in water. This solution gave the following reactions characteristic for phloroglucinol:

(1) It reduced both silver nitrate and Fehling solution.

(2) It colored pine wood moistened with hydrochloric acid red.

(3) It gave a red color with vanillin and hydrochloric acid, and

(4) A deeper red color with oil of cloves and hydrochloric acid, becoming purple on standing.

(5) It gave a violet color with ferric chloride.

The substance is then, without doubt, fisetin. The formula[26] of fisetin is supposed to be C_{15}H_{10}O_{6}.

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 alpha-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.