Part 5

(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. 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. 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. 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.

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.

Furthermore, fisetin should give protocatechuic acid and phloroglucinol by fusion with caustic potash under proper conditions. 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 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 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: "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 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. Czapek says: "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. 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. 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 and sulphuric acid, a purple violet color.