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 C46H42O21. 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.