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}.