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.
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.