LIBRARY
UNIVERSITY OF CALIFORNIA.
Gl FT OF
Class
Japanese Lac- -Ki-urushi
ALVI3O B 9TEVENS
Contribution to the Knowledge of
Japanese Lac '
(Ki-urushi)
THESIS
Presented to the Philosophical Faculty of the University of
Bern, for the Degree of Doctor of Philosophy.
By ALVISO B. STEVENS
University ol Michigan
Ann Arbor, Michigan, United States ol America
ANN ARBOR:
The Ann Arbor Pitts, Printers
1906
JAPANESE LAC (KI-URUSHI)
Doubtless nearly every one has seen and admired the beau-
tiful Japanese vases or boxes without realizing that they were
finished with the most indestructible varnish known to man.
There are at present vases, more than a century old, that l^ave
retained their beautiful luster so perfectly that they look as
though they had been finished but yesterday. The hardened
surface formed by the genuine Japanese lac, is practically un-
affected by the usual reagents, which are so detrimental to most
varnished surfaces, as, alcohol, ether, alkalies and acids. It is
acted upon to some extent by strong sulphuric or nitric acids,
and may be dissolved by continued heating in fuming nitric acid.
Rein 1 states that the Japanese doubtless received their knowl-
edge of the lac industry from the Chinese in the early part of the
third century ; but that its use did not attain great importance
before the middle of the seventh century. Kotoku-Tenno, the
36 Mikado (645 to 654 A. D.) had a ceremonial head covering
of paper, which was covered with black lacquer. There is a
lacquered scarf box in the temple at Nara, which belonged to
a priest in the time of Kinnari Tenno (540 to 572 A. D.).
For centuries its use and production remained a secret. As
late as 1873 wc ^ nc ^ tne statement that "The manner of prepar-
ing the varnish and the mode of applying it, is likely to remain
a secret." 2 In the following year Prof. J. J. Rein made a thor-
1 J. J. Rein's The Industries of Japan, London, 1889, Lacquer Work,
PP- 339-377; Rein, Japan II, Leipzig 1886. This Author has minutely de-
scribed the lac industry and it is to his excellent work that I shall fre-
quently refer.
2 Belfour's Cyclopaedia of India.
ough study of the method of collecting and applying the lac.
He describes the tree, Rhus vernicifera, from which the lac is
obtained, as follows:
' 'Lacquer trees grow up straight and have fairly symmetrical
crowns. The young trees have fine, large, pinnate leaves, which
in good soil often grow to be more than a meter long, and far
exceed all other species of Rhus in size and beauty. The leaves
are unequally pinnate and have long stems. Before falling off
in October they become yellow or reddish brown. There are
from nine to fifteen leaflets, large, oval, pointed and unindented,
which have fine short hairs on the under side.
"In June appear loose, greenish yellow branches of blossoms,
from numerous axils near the end of the thick twigs. The fruit
is ripe in the second half of October, is yellowish green and re-
mains hanging all winter, though usually gathered in November.
"The two sexes are separate. Therefore when the chief ob-
ject of its cultivation is the manufacture of wax from the seed,
the male trees should be avoided, reproduction being obtained by
root sprouts from female specimens. The trees begin to bear
fruit when eight years old and increase in productiveness until
thirty or forty years old."
Shirasawa gives the following: 3 Rhus vernicifera, D. C.,
Syn. Jap. Urushi-no-ki, Fam. Anacardiaceae.
"Cultivated in the countries of the temperate zone, i. e. Shin-
ono, Kai, etc., and the provinces to the northeast of Honshiu.
It does not grow in warm regions, and thrives best in moist soil.
The tree attains its growth quickly and reaches a hight of 10
meters, and the trunk a diameter of four decimeters.
"The buds are of a pyramidal form, short with curved points
and covered with hairs of a brown, ash-gray color and glistening ;
cicatrix of the leaves large, heart shaped ; pith large.
"Flowers, end of May ; fruit, end of October.
"The wood is soft and brittle, with a remarkable difference
in the color between the sap wood, which is white and the heart
3 Iconographie des Essences forestieres du Japan, par, M. Homi
Shirasawa, 1899, P- 94-
wood which is yellow. Air dried 0.51 sp. gr., kiln dried 0.45
sp. gr.
"The wood is used for the manufacture of utensils, furni-
ture, bric-a-brac, wood engraving, etc.
"The lac is gathered in China, of which the provinces Noto,
Iwashivo are renowned. Wax is collected from the fruit."
Doubtless the principal source of vegetable wax is Rhus
succedanea and R. sylvestris. The former is cultivated exten-
sively in the warmer parts of Japan, south of latitude 35 N.
Japan but is cultivated principally between latitudes 35 and 39,
The lacquer-tree grows in nearly all parts of China and
including the provinces of Etschizin, Schmano, Aidzo, Yoshino
and Yamato. The greatest yield is from trees 15 to 20 years old,
but the age of the trees when the lac is collected varies in different
localities, in some places at from five to six years old when the
stem is the size of a man's arm and in other localities at from
nine to ten years old. The time of collecting is from April or
May to the first of November.
The tree and its anatomic relations are given in detail by
Moebius*. He states that schizogenic lacticiferous canals are
present in all parts of the plant. These contain the milk juice
that exudes after incision.
METHOD OF COLLECTING THE LAC, AND ITS PROPERTIES.*
The peasants sell the trees to the lac merchants who employ
collectors to gather the lac. Each workman operates upon from
600 to 800 old trees, or 1000 young trees in a season. He begins at
the bottom of the tree and makes horizontal incisions through the
* D'er Japanishe Lacbaum, Rhus vernicifera D. C. Eine Morphio-
logishe anatomische Studie aphandlungen der senkenbergischen naturfor-
schenden Gasellschaft, Band XX, Heft II.
5 The principal facts have been tv.ken from Rein's Industries of
Japan, but reference is also made to "J a P a m scner Lack" by Dr. Wagner,
Dingler's polyt. Journal, 218, 1875, pp. 361-367.
The above description is nearly the same as that given by Ishimatsu,
which he states was partly taken from an account of the urushi manufac-
ture published for the use of the Japanese primary schools. Manchester
Literary and Philosophical Soc. 3 series, 7, 1882, p. 449.
8
bark about six millimeters wide 8 with the sharp side of the Kaki-
gaina, a hook shaped instrument (No. 10, p. 10). He then repeats
the operation on the other side of the tree about 15 or 20 c.m.
higher. Thus alternating from side to side until he has cut as
high as he can reach, making from six to ten grooves on a side,
which extend about half way round the tree. He then goes to
other trees until about ten or fifteen trees have been cut after
which he returns to the first trees and collects the raw lac. This
is removed with the Natsu-bera, an iron spatula with a curved
point (No. II, p. 10), and scraped into the Go, a small wooden or
bamboo pail, which the workman carries in his left hand. After
about four days he returns to the first group of trees and cuts
grooves parallel to each of the first. These operations are re-
peated at intervals until the tree is literally covered with grooves.
The entire operation requires from 60 to 100 days.
The juice usually fills, but does not flow out of the grooves.
In the spring the lac is thin, gradually becoming thicker as the
season advances. The best is collected in mid-summer. When
collected the juice is in the form of a thick grayish- white emul-
sion, which on exposure to air rapidly changes to brown and
finally to black. If left in an open can it rapidly forms a black
skin over the surface which prevents further oxidation. The
lac collected as above is the best quality, known as "Ki-Urushi"
and has a sp. gr. of 1.002 to 1.0379. When strongly magnified
it appears to be a brownish mass of globules, which consists of
two kinds, one kind small, dark brown and between these a less
number of large, light-colored globules, the former soluble in
alcohol and the latter in water. A second grade known -as
"Seshime-Urushi" is obtained at the close of the season by cut-
ting down some of the trees and cutting and binding the branches
into bundles about one metre long and these, with the trunk are
macerated in warm water when the sap comes to the surface and
is removed. Wagner 7 states that after maceration the branches
are placed in a screw press to remove the juice. This is thin and
dark and, after mixing with some drying oil, is used as an under
varnish. Each tree yields on an average from 27 to 54 Grammes
8 Rein gives 2 m.m. wide, but Wagner gives 6 m.m. Doubtless the
latter is more nearly correct. v
7 Dingler's Polytechnisches Jour. 218, p. 3611875.
//
-' HE
VE^S'TY
of raw lac. In China the yield is said to be much less, in some
districts not more than 10 Grammes.
Yoshida 8 states that Ki-Urushi is never sent to the market
in the form in which it is obtained from the tree but is usually
mixed with about 40% of "Mokuyiki" (wood juice) which close-
ly resembles Ki-Urushi but contains a much larger proportion
of gum and about j4 as niuch substance soluble in alcohol. It
is doubtless an impure form of urushi juice. Before
the raw lac is ready for use it must be strained
through cotton or linen cloth to remove pieces of bark and
foreign particles. It is then stirred in a shallow wooden
pail to remove the grain and give it a uniform consistence.
The varnish makers sometimes add linseed oil ; also from i 10%
of perilla oil is sometimes added. The lac mixed with 1/5 perilla
oil is sometimes used for coating umbrellas and water proofs.
Various colors are made by adding pigments. The red, so fre-
quently used for a part of Japanese decorations is formed by
mixing 70 parts Ki-Urushi, 20 parts linseed oil and 10 parts ver-
million. One per cent of gamboge either in powder or in solution
is sometimes added. The best gloss black is formed by mixing
purified lac with acetate of iron, formed by macerating nails or
iron filings in vinegar or rice beer, and heating or exposing to
the action of the sun. The lac thus prepared contains from 0.5
to 2% of iron. Other substances are sometimes added as indigo,
iron oxide, lead oxide, charcoal, and for decorative purposes, gold
and silver dust, gold, silver, and tin foil are used.
The only substance used by the Japanese to thin the lac is
camphor, which is powdered and mixed with the lac. Rein ob-
served that when water is mixed with lac that it thickens and be-
comes jelly-like, and if applied to wood dries very rapidly.
If the lac is allowed to harden in a dry atmosphere it has
a dull appearance. Hence it must be dried in the presence of
moisture which is necessary to ensure the best action of the
enzyme. Therefore the articles coated with the lac are placed
in a room and wet cloths are hung on the wall or about the lac-
quered articles. A temperature of from 20 to 30 is best adapted
for this process.
8 Jour. Chem. Soc., 18839. 472.
10
INSTRUMENTS USED IN COU,ECTING AND APPLYING THE I<AC.
INSTRUMENTS USED IN COLLECTING AND APPLYING THE LAC.
The accompanying reproduction is from "Rein's Japan,"
and is from original instruments in the Royal Industrial Art
Museum in Berlin.
No. i is a sharp kitchen knife ; No. 2, a gouge or chisel ;
No. 3, shears ; Nos. 4 and 5, wooden spatulas ; No. 6, a bamboo
spatula; No. 7, a surface brush made from human hair; No. 8,
palette made of tortoise shell or buffalo horn, to be carried on the
hand ; No. 9, spoon for putting on the gold or silver dust. The
above, with various sized brushes made of deer and rat's hair
form the implements used in applying the lac, gold, silver leaf,
etc. Nos. 10 and 11 are used in collecting the lac and have al-
ready been described.
RULES TO BE OBSERVED BY THE WORKMEN.
1. The surface must be brushed over equally, first in one
direction and then in the other.
2. The old coat must be thoroughly dried before a new
one is applied.
3. The drying is best conducted in vapor of steam.
4. Only the ground coat can be dried in the open air or
sun, and that only when the coating contains very little or no lac.
5. The peculiar lac properties result from drying without
heat in a chest or closed room with wet cloths placed by the
side of the object or on the sides of the room.
6. Dust, light and air are to be excluded while the varnish
is hardening.
7. For fine finish the varnish must be strained once or twice
through fine porous bast or paper.
8. After every new coat the surface should be polished with
charcoal or burnt horn, usually with free application of water.
9. The finished object must not show the quality or the
condition of the base must be free from ridges or specks. The
complete mirror must not change by contact with hot water.
Wagner states that the first coat is rubbed down with pow-
dered pumice stone and that the final coat is polished with bone
ash and charcoal and finished with the palm of the hand and the
tips of the fingers.
12
CHEMICAL INVESTIGATION OF LAC.
The most important chemical investigations of Japanese lac
have been made by three Japanese chemists. Ishimatsu 9 made
the first chemical investigation. He states that the lac has a
sweetish odor, an irritating taste, burns with a luminous flame
emitting dense black smoke and mixes with fixed oils in all pro-
portions ; hence these oils are frequently used as adulterants.
He states that it was generally supposed that the hardening
was due to the action of light and air, but he proves that light
is practically without effect on the lac, as it is blackened very
rapidly when exposed to moist atmosphere during the night or
when kept in a light-tight box. It is unacted upon even in sun-
light when kept under water, or in carbonic acid in a sealed flask.
It dries very slowly in dry air. This he attributes to the rapid
drying of the surface, which prevents the evaporation of the
volatile constituents, while in moist air the drying takes place
so slowly that the volatile constituents have time to escape. This
theory is not in harmony with the statement which immediately
follows, where he states that the hardening in the atmosphere
is, in all probability, due to the oxygen of the air.
He finds that the fresh lac yields 58.24% of substance soluble
in alcohol, while the perfectly dry powdered lac yields only
18.07% t substance soluble in alcohol. He attributes this differ-
ence to the fact that the alcohol has greater difficulty in getting
at the dry lac. He fails to realize that the difference is due to
a chemical change.
He reports that the lac consists of a substance soluble in
alcohol, a gum soluble in hot or cold water, a residue insoluble
in alcohol or water, which consists of bark, cellulose, dust, etc.
There is also present a small quantity of volatile poison and water.
His method of separation was to extract the lac with absolute
alcohol, evaporate the alcohol and dry at 100 C. to constant
weight.
The residue insoluble in alcohol was extracted with hot
water, filtered and the filtrate evaporated, dried at 100 C. and
weighed as gum.
9 Chemical investigation of Japanese Laquor, or Urushi. Manches-
ter Literary and Philosophical Soc. 3 series, 1882, p. 249. Communicated
by Professor Roscoe, Read Feb. 18, 1879.
13
The residue insoluble in water was dried at 100 C. and
weighed.
The water and volatile matter were determined by difference.
Yoshida 10 used Ishii-natsu's method for the separation of the
constituents, but reported the part soluble in alcohol as urushic
acid, and that soluble in hot water as gum, identical with acacia,
and that which was insoluble in water or alcohol as diastatic
matter.
He proved that the hardening of the lac was due to the action
of an oxidizing enzyme, acting in the presence of moisture. He
states that the enzyme is an albuminous body, coagulated by
boiling. In this he is mistaken for the enzyme is intimately asso-
ciated with the gum and cannot be separated from it, even though
it is destroyed by boiling. See under Gum, p. 45.
Later Korshelt and Yoshida 11 examined several samples of
lac, using the same method of separation. For comparison the
results of these chemists are given in following tabulated form:
CONSTITUENTS
OF RAW LAC
Yoshino
Prov. Yamato
H. Yoshida
Korschelt and Yoshida
Bought in Tokio
S.' Ishimatsu
Hottamnaa,
Prov. Ilidachi
Southern
Sagaimi
Northern
Echtgo
Hachioji,
Prov. Sagami
Bought in
Tokio
Lac ?cid (Urushic acid)
85.15
3-15
2.28
?
9.42
64.62
5.56
2. ID
O.O9
27.63
68.83
5-02
2.01
O.O6
24.08
66.Q2
4-75
1.72
O.o6
26.55
80.00
4.69
3-31
p
12.00
64.07
6.05
3-43
0.23
26.22
58.24
6-32
2.27
?
33-17
Oum
Nitrogenous residue
Oil
Water
It is claimed that the small quantity of oil reported is not a
natural constituent, but is due to the oil of Perilla that is used
on the knife and spatula to prevent the lac adhering to the iron.
Yoshida states that the Yoshino sample which he analyzed
was collected under official inspection for chemical investigation
and was evidently pure and that the sample analyzed by Ishimat-
10 Chemistry of Lacquer, Ki-Urushi. Hikorokuro Yoshida, Jour.
Chem. Soc. 1883, p. 472.
11 Trans. As. Soc. Japan, 12, pp. 182 to 220.
14
su must have contained a considerable quantity of Mokuyki, an
impure form of urushi juice.
Ishimatsu states that the part soluble in alcohol has the same
odor as the original, but never dries up as that does. It is brown-
ish-black, slightly sticky to the touch. With potassium hydrox-
ide it forms a bluish-black precipitate. The alcoholic solution
was precipitated by lead acetate. The precipitate was washed,
dried and analyzed, with the following results:
MEAN
C 49-84 51-06 50.45
H 5.81 5.60 5.705
O 40.-30 39-84 40.07
PbO 3-50 4-05 3-775
From which he calculated the formula C 20 H 30 O 2 .
When he boiled the alcoholic residue with nitric acid it gave
off brown fumes and formed an orange colored mass, which when
washed was partly soluble in absolute alcohol. This alcoholic so-
lution formed a yellow precipitate with lead acetate or silver ni-
trate. The lead precipitate explodes when heated. It could not
be decomposed by sulphureted hydrogen without decomposition
of the acid, therefore he removed the lead by sulphuric acid, and
again precipitated with lead acetate, washed, dried the precipi-
tate, and estimated the lead as oxide, and the other constituents
by combustion. The results were as follows:
MEAN
C 26.77 27.10 26.93
H 4.10 4.12 4.11
NO 18.16 18.28 18.44
PbO 47.41 47-43 47.42
O 3.12 3.07 3-1
From which he calculated the formula C 11 H 20 (NO 2 ) 2 PbOo
and for the original substance C^H^C^.
The following is an abstract of Yoshida's investigation of
the alcohol soluble portion of the lac, which he calls Urushic acid.
It is readily soluble in benzin, ether, carbon disulphide; less
easily soluble in fusel oil and petroleum of high boiling point ; in-
soluble in water; sp. gr. 0.9851 at 23. Remains unchanged at
1 60. Above 200 it decomposes slowly with carbonization.
From the alcoholic solution many salts can be produced, most of
which are slightly soluble in alcohol but insoluble in water.
15
With silver nitrate it forms a fine black precipitate moderate-
ly soluble in alcohol, which, on boiling is reduced with the for-
mation of a metallic mirror. Platinum chloride, gold chloride,
uranium acetate, and copper nitrate form precipitates varying
in color from brown to black.
He prepared the lead compound by precipitating with ace-
tate of lead, washing with alcohol and then with boiling water,
drying on a water bath and finally over sulphuric acid in an
exsiccator. He obtained by analysis the following results:
THEORETICAL FOR
FOUND (CuH 17 O)jPb.
C 52.08 52.4
H 5-34 5-3
O 10.43 10.01
Pb 32-45 32.29
The lead salt is gray ; on heating to 100 it gives off a pecu-
liar odor, turns dark, at iio-ii5 melts to a brown mass, and
at about 120 ignites spontaneously.
By adding an insufficient quantity of ferric chloride he
formed a voluminous black precipitate, which by analysis gave
the following formula: (C 14 H 17 O 2 ) 3 Fe+9C 14 H 18 O 2 .
By adding a larger proportion of ferric chloride he formed
a compound which on analysis gave results corresponding to the
following formula: (C 14 H 17 O 2 ) 3 Fe+3C, 4 H 18 O 2 . Both salts
melt to a black mass at io5-no and ignite spontaneously at
a somewhat higher temperature.
Free alkalies impart a very dark color to the alcoholic sol-
ution, which looks purplish-black by transmitted light and dark
brown by reflected light. On exposure to air it forms a viscid
compound, rapidly becomes black and dries up.
Soluble salts of mercury, zinc, nickel, cobalt, manganese and
earthy metals do not give any distinct reaction.
To a solution of the acid in carbon disulphide, bromine was
gradually added in excess and the whole evaporated to dryness
on a water bath, the mass extracted with strong alcohol, and the
extract again evaporated, whereupon it yielded a dark semi-fluid
mass. This was examined for bromine by igniting with pure
lime. 0.7060 gm. gave 1.151 gm. AgBr= 69.37% f bromine
agreeing very nearly with a hexabromo derivative of the acid,
C 14 H 12 Br O 2 , which requires 69.36%.
i6
Yoshida subjected his urtishic acid to the continued action
of hydrochloric acid for three days, and obtained a hard brown
mass which he cut into pieces, boiled with water, washed with
alcohol, dried at 100 C. and analyzed. The results obtained were
practically identical with those for urushic acid as will be seen
by the following comparison:
MEAN FOUND FOR
FOUND URUSHIC ACID
C 77-07 77.05
H 8.77 9.01
He considers this as a polymerization product, a molecular
transformation under the influence of strong hydrochloric acid
and names it ^-urushic acid, and states that it is soluble 12 in the
usual solvents for urushic acid. He claims that the substance
obtained by the decomposition of an alkali salt of urushic with
hydrochloric acid is the same body as ^-urushic acid.
By the action of strong nitric acid he obtained a sponge-like
body which he washed with water, dissolved in alcohol, and pre-
cipitated with ferric chloride. The precipitate was washed, dried
and analyzed with the following results :
CALCULATF.D FOR
FOUND [C 1 4H 15 (NO 2 ) 2 O 2 ] s Fe.
C 51.49 51-59
H 4.82 4.61
NO 2 28.16 28.25
Fe 9.77 9-8i
This nitro body was light yellow, and soluble in the usual
solvents for urushic acid.
Yoshida oxidized urushic acid with strong chromic acid.
The product was washed with water and then with absolute alco-
hol and dried at 105 C. It was in the form of a brown powder
which by analysis was found to contain one more atom of oxy-
gen than urushic acid. See results below.
He heated a portion of the fresh juice on the water bath
until the water was entirely removed, and at the same time the
action of the enzyme was destroyed. This was then analyzed.
For results see below.
Another portion of the lac was allowed to harden in the
usual way by the action of the enzyme and then analyzed. For
12 Doubtless this is a typographical error, and should read "insoluble
in the usual solvents for urushic acid."
i/-
convenience of comparison these results are tabulated, as fol-
lows:
Mean raw lac Mean for lac hard- Mean for urushic Calculated for
dried by heat. ened by enzyme. acid oxidized by C, 4 Hi 8 Oa
chromic acid.
C 75-47 70.85 71.52 71-79
H 8.97 8.22 8.23 7-69
N o.n o. 092 .... ....
Ash 0.21 0.032
15.17 20.52 20.25 20.52
He concludes from the above analyses that the lac when
hardened in the usual manner takes up one atom of oxygen for
every molecule of urushic acid, becoming C 14 H 18 O 3 . This com-
pound he names Oxyurushic acid.
More recently Bertrand 13 has worked upon Japanese lac ;
however he has contributed nothing of importance to the knowl-
edge of the alcohol soluble substance. His principal work was
upon the soluble ferment referred to elsewhere.
EXPERIMENTAL INVESTIGATION.
Three samples of lac were used in the following experiments.
The first and second were in glass jars bearing original Japanese
labels. The third was in a tin can. Apparently all were identi-
cal.
The samples were all sent gratuitously to Prof. Tschirch.
The first by forester Shirasawa, in Tokio, and others by the
Rhus Company in Frankfort, a. M. to whom I here extend thanks.
When separated according to the method of Ishimatsu they
gave the following results:
Parts soluble in alcohol 72.40%
Parts soluble in water 4.05%
Insoluble residue 2 . 35%
Water and volatile matter 21 .20%
1 have found, as will appear later, that Yoshida's Urushic
acid may be separated by benzini into
Benzin-soluble 78%
Benzin-insoluble 22%
and that the benzin soluble consists of three substances, one
of which is a non-volatile poison, also that the gum and enzyme
13 Ann. chem. phys. sen XII, 1897, p. 115.
(diastatic matter) cannot be separated, also that the lac contains
acetic acid. The lac is of a grayish color. On exposure to air
it rapidly darkens, but if undisturbed an impervious membrane
soon forms on the surface thus preventing further change. The
blackening of the lac is due to the action of oxydase or "Lac-
case," a soluble oxydizing enzyme, in the presence of moisture.
The lac may also be darkened by other means, as, by the action
of alkalies. For example, a piece of wood was coated with fresh
lac; a second piece was coated with lac, sterilized by suspending
a tube containing lac in boiling water for half an hour; and a
third was coated with sterilized lac containing a small portion
of potassium hydroxide ; each piece of wood was covered with
wet filter paper. The first rapidly changed to a dark brown color
and in 24 hours the coating was black and hard. The second
remained unchanged. The third immediately changed to black
but remained moist for several days.
LACRESINS, THE URUSHIC ACID OF YOSHIDA, THE; LACCOIy OF
BERTRAND.
It was desirous to separate the resinous portion from the
gum and enzyme with the least exposure to the air, therefore
the can containing lac was connected with a flask by means of
a tube extending to the bottom of each. The flask was partially
filled with alcohol and the lac drawn into the alcohol by suction.
The contents of the flask were then agitated, filtered and the
residue exhausted with alcohol. The alcoholic solution was
strongly acid and had a peculiar aromatic odor which upon evap-
oration of the alcohol suggested the odor of acetic acid. The
oily residue left after distillation of the alcohol was washed by
shaking out with water. The watery solution was neutralized
with potassium hydroxide and heated, when a fine black precipi-
tate formed. This was removed by filtration and the filtrate evap-
orated to dryness. The residue with sulphuric acid gave an un-
mistakable odor of acetic acid, also when heated with sulphuric
acid and alcohol the odor of acetic ether was developed. It also
gave the cacodyl odor when heated with alkali and with arsenic
trioxide. To prove that the presence of acetic acid was not due
to the oxidation of the alcohol, a fresh portion of lac was ex-
tracted with ether, and the ether residue treated as above with
the same results. It is therefore evident that the lac contains acetic
19
acid. The black precipitate which separated from the watery solu-
tion on evaporation was washed, and upon adding hydrochloric
acid, changed to a red color, but remained insoluble in all ordinary
solvents. There was not a sufficient quantity for analysis, but
doubtless was the same substance which is formed as often as
the lac is oxidized and henceforth will be designated as oxyurush-
in. Its presence in the water solution was due to the acetic acid,
which is an excellent solvent for the unoxidized lac.
One hundred grammes of the lac was placed in a flask and
steam passed through the lac for several hours. The distillate
was covered with a thin oily film, which was removed by shak-
ing out with ether and evaporating. The residue was not volatile
or poisonous. Besides this the distillate contained acetic acid.
A portion of the alcoholic residue was dissolved in ether
and repeatedly shaken out with one per cent solution of sodium
carbonate. The carbonate solution was at first green,
then brown. This was heated on a steam bath to remove the dis-
solved ether, and acidulated with hydrochloric acid, when a small
quantity of reddish brown precipitate appeared. This was wash-
ed and dried. Only a small part of it was soluble in ether, the
remainder being insoluble in any of the ordinary solvents, and is
evidently oxyurushin. ,. .,.,.
The ether solution which was separated from the carbonate
solution was next shaken out with one per cent potassium hy-
droxide solution. The resulting solution was very dark green,
changing later to brown. It was treated exactly as in the case
of the carbonate solution and with the same result, i. e. a sepa-
ration of the same insoluble oxyurushin.
The ether solution then received attention. It was shaken
out with 5% potassium hydroxide, producing a black precipitate
which floated in the ether solution. The alkaline solution was
again of the same dark green color changing to brown, and
when acidulated produced the same insoluble oxyurushin. The
black precipitate was removed from the ether by filtration and
the ether shaken with more alkali with the same result. By re-
peating the operation a sufficient number of times, the entire
substance could be removed from the ether. The black precipi-
tate formed by potassium hydroxide was washed free from alkali
and heated with dilute hydrochloric acid, washed, dried and pow-
dered ; a very small part of it was soluble in ether but this became
insoluble on evaporation. The powder was reddish brown and
2O
in every way identical with oxyurushin. The alkaline solutions
from which the oxyurushin was precipitated by acid, were evap-
orated and extracted with ether and alcohol but no organic sub-
stance was obtained.
SEPARATION BY LEAD ACETATE AND SUBACETATE.
Another part of the alcoholic residue, free from acetic acid,
was redissolved in alcohol and an alcoholic solution of lead ace-
tate added as long as it formed a precipitate. The
precipitate which was of a light gray color was washed with
alcohol, mixed with fresh alcohol, decomposed with sulphuric
acid, and the excess of acid removed by shaking with lead car-
bonate. On evaporating the alcohol a thick dark brown oily resi-
due was obtained. The residue was somewhat darker than the
original alcoholic residue, but otherwise similar. To the nitrate
secured from the lead acetate precipitate, lead subacetate was
added as long as a precipitate formed. The precipitate was of
a gray color, but a decidedly lighter gray than that obtained by
lead acetate. On decomposing the precipitate, as above, an oily
residue was obtained, which was also lighter in color than that
obtained from the lead acetate residue.
The filtrate from the subacetate precipitate was still of a
brownish color. The excess of lead was removed by adding a
slight excess of sulphuric acid, and the excess of acid removed
by shaking with lead carbonate and filtering. The filtrate was
concentrated by evaporation and shaken out with ether. Upon
evaporating the ether a residue was obtained which, when dis-
solved in alcohol, was readily precipitated by lead acetate or sub-
acetate. By repeated experiments with the original alcoholic
solution it was found that by precipitation with lead acetate and
removing the lead and acid from the filtrate, reprecipitating and
continually repeating this operation, that a series of oily residues
could be obtained, gradually diminishing in quantity, and each
increasing in fluidity, and becoming a shade lighter than the pre-
ceding. Only the last fractions were poisonous.
Lead subacetate is a better precipitant than the acetate. The
acetic acid liberated evidently aids in preventing complete pre-
cipitation. The fact that the fractions decrease in color and
viscosity, and that only the last were poisonous, indicates that the
alcoholic extract consists of a mixture of two or more substances.
21
But in no case can the above method be considered as a com-
plete separation. In alcoholic solutions each fraction assumed
a green or greenish black color with alkalies, the color varying
with the concentration of the solution and the strength of the
alkali used.
SEPARATION OF THE LACRESINS BY SOLVENTS.
SOLUBILITY OF THE ORIGINAL ALCOHOLIC RESIDUE.
On first trial it appeared as though the alcoholic residue
might be soluble in any of the ordinary solvents for oils and
resins, but investigation proved that it was not completely soluble
in carbon disulphide, methyl alcohol, amyl alcohol or petroleum
benzin in all proportions.
After numerous experiments the following method of pro-
cedure was adopted. The alcoholic residue was dissolved in the
proportion of i part to 7 of petroleum benzin, boiling point not
over 60 C., forming a clear solution, but further addition of ben-
zin caused a precipitate. This was then poured into 55 parts of
benzin which produced the immediate separation of a thick brown
mass from which the still cloudy benzin was decanted.
The brown deposit was dissolved in a small quantity of benzin and
again separated by adding a larger amount. After this operation
was repeated several times the deposit became entirely insoluble
in benzin. It was then washed with benzin until the last washings
were colorless. The washings were added* to the portions pre-
viously decanted and allowed to stand 12 hours when the ben-
zin became clear and was not affected by the further addition of
benzin. The second deposit was dissolved, again precipitated by
benzin and washed by agitation with benzin. The second deposit
was thinner and lighter in color than the first.
By this method the alcoholic residue was separated into two
distinct substances, a benzin-soluble, and a benzin-insoluble.
The first and second deposit from benzin, while differing some-
what in physical appearance could not be said to consist of two
distinct substances but doubtless consisted of mixtures of the
same substances in varying proportions. Each was mixed with
a small quantity of ether and added to methyl alcohol which im-
mediately became cloudy and on standing a short time formed a
deposit. The solution remained clear on the further addition of
methyl alcohol. The residue was separated and washed with
22
methyl alcohol but, as the washings remained turbid for several
days, they were not added to the original solution.
The first benzin deposit contained more substance insoluble
in methyl alcohol than the second deposit, otherwise no differ-
ence appeared, therefore their products were combined as methyl
alcohol-soluble and methyl alcohol-insoluble substances. About
half of the methyl alcohol-insoluble was soluble in ether, the
remainder apparently having undergone some change during
manipulation with the methyl alcohol. This theory is supported
by the fact that the methyl alcoholic solution slowly deposits on
standing. A similar condition was also observed when a por-
tion of the original alcoholic residue was precipitated with pe-
troleum benzin as a small portion of this also remained insoluble
in ether. In fact all of the substances so far separated are evi-
dently slowly oxidized, as all solutions except the petroleum
benzin solution on standing for weeks form a slight insoluble
deposit ; and while the soluble benzin portion apparently remained
unchanged, yet on largely diluting with benzin it again became
cloudy ; whereas previously it remained clear under similar condi-
tions.
Three samples of the benzin soluble substance were placed
in small colorless glass vials. No. I was corked and wrapped
in black paper, No. 2 was merely corked and No. 3 was loosely
closed with cotton. All were placed on a shelf exposed to strong
light and allowed to remain for 10 months. When they were
tested as to their solubilities Nos. i and 2 dissolved readily in ben-
zin but, on diluting with a large amount of benzin, became cloudy
and upon standing, formed a slight deposit. That from No. I,
being a little larger than from No. 2, would indicate that the
reducing action of light has a tendency to prevent the change.
No. 3 became quite thick but was still fluid. Only a small part
was soluble in benzin.
SOLUBILITY OF THE SUBSTANCES SEPARATED FROM THE ALCOHOLIC
RESIDUE.
The part soluble in benzin was also soluble in ether, chloro-
form, alcohol, methyl alcohol, amyl alcohol, carbon disulphide,
toluol, oxylol, acetone, toluidin, pyridin, quinolin, carbon tetra-
chloride, amyl acetate, acetic ether, nitro benzol, turpentine oil,
acetic acid and 80% solution of chloral hydrate.
The part soluble in methyl alcohol was soluble in each of the
above except benzin. The part insoluble in methyl alcohol but sol-
uble in ether, was insoluble in benzol, toluol, oxylol, alcohol, amyl
alcohol, carbon disulphide, turpentine, carbon tetrachloride, ace-
tic acid and chloral hydrate solution.
The part insoluble in ether was also insoluble in any of the
above solvents.
SEPARATION OF THE BENZIN-SOUJBLE PORTION.
One volume of the substance was dissolved in eight volumes
of benzin, four volumes of alcohol added and the whole thor-
oughly agitated. Upon standing two layers appeared. The upper
benzin layer was of a yellowish brown color, the lower reddish
brown. These were separated and the benzin solution washed
with alcohol as long as any thing could be removed. The benzin
was driven off by evaporation leaving a non-poisonous, oily,
brown residue, insoluble in alcohol.
The alcoholic solution was in turn washed by shaking out
with benzin but with no positive result. The solution was next
evaporated and a reddish brown, slightly gelatinous residue ob-
tained. By rapidly washing this with a small quantity of benzin
and evaporating a clear, light reddish brown residue was secured.
Both of these residues proved to be poisonous. By continual
washing with benzin or by employing it in larger quantities, the
entire residue was dissolved. I believe that this residue consists
of a poisonous and a non-poisonous substance but thus far the
separation has not been secured. I hope to be able to separate
them later.
All of the resins separated from the lac were tested for
cholestrin but with negative results.
CHEMICAL REACTIONS.
All of the substances separated gave precipitates with lead
acetate, subacetate, silver nitrate, mercurous nitrate, cupric ace-
tate and ferric chloride. The lead precipitates were of a light
gray color gradually becoming darker on standing. The other
precipitates were black. All were slowly blackened by the action
of concentrated sulphuric acid. In the cold, concentrated nitric
acid colored the benzin-soluble substance red which changed to
brown on heating. The methyl alcohol-soluble substance was
slightly darkened by cold nitric acid but became lighter on heat-
ing, forming a light spongy mass.
The benzin and methyl alcohol soluble substances were
slightly darkened by strong hot hydrochloric acid, but by continual
heating formed a spongy mass of a lighter color. All substances
except the ether-insoluble were at first colored green, then black
by strong alkalies. Various shades of green, and black were ob-
tained, the shades varying with the concentration of the alkali
and of the substance in alcohol or ether. Barium and calcium
hydroxides produced the same effect though in a minor degree.
When heated with dry potassium hydroxide the substances
furnished vapors that changed red litmus to blue, but gave no
odor of ammonia. However the pyrrol reaction was obtained
when a pine shaving was moistened with hydrochloric acid and
held in the vapors. All of the above were tested for nitrogen
by the Lassaigne test but with negative results".
The pyrrol reaction was also obtained by boiling the sub-
stance with a strong solution of potassium hydroxide but the
reaction was not as vigorous, and long-continued heat would be
necessary to convert all the nitrogen into pyrrol. This is proven
by the fact that the black precipitate still gave the pyrrol reaction,
though formed by heating these substances with $% potassium
hydroxide for two hours on the steam bath.
ANALYSIS OF SEPARATED CONSTITUENTS.
The methyl alcohol soluble substance was spread on glass
and placed in the drying oven. After several days it was suffi-
ciently dry to be removed from the glass with a knife. The
shavings were not brittle, but were cut into small pieces and
returned to the oven where they remained for two weeks before
they could be pulverized. The particles were hard and electric
but by constant moistening with a mixture of alcohol and ether
they were finally powdered. The powder was placed in a nar-
row tube and percolated with ether which dissolved a small
amount. The ether was evaporated and the residue again spread
on glass when in a few hours it became insoluble. This was
doubtless part of the substance which had been prevented from
drying by the surrounding particles. Some of the powder was
ignited, after which a little ash was left. The remainder was
" See tests for Nitrogen under gums.
25
repeatedly boiled with hydrochloric acid and washed with hot
water but it was impossible to entirely remove the ash. When
examined this was found to consist of silica, aluminum and
traces of calcium. The powder was dried at a temperature of
105 and analyzed, with the following result:
I 0.283 Gm. gave 0.2045 H 2 O, 0.7452 Gm. CO 2
II 0.3268 Gm. gave 0.2288 H 2 O, 0.8457 Gra. COjj
I 0.351 Gm. gave 5 Cc. N at 20 C. and 716 Mm.
II 0.4619 Gm. gave 69Cc. N at 18.6 C. and7i6 Mm.
I II Mean
C 71 808 per cent 71.51 per cent 71.659
H 8.01 per cent 7.85 per cent 7.93
N 1.57 percent 1.646 per cent 1.608
Ash 0.600
Another sample of the same substance was dissolved in
alcohol, a solution of sodium hydroxide added in excess, and
heated until the alcohol evaporated. The bulky black precipitate
was washed until free from alkali, then boiled with hydrochloric
acid which changed the color to a reddish brown. The precipitate
was washed until free from acid, dried, powdered and analyzed
with the following results:
I 0.2436 Gm. gave 0.1718 Gm. H 2 O, 0.6411 Gm. COg
II 0.222 Gm. gave 0.1534 Gm. H 2 O, 0.5814 Gm. CO 8
I 0.2876 Gm. gave 1.5 Cc. N at 2OC. and 711.5 Mm.
II 0.343 Gm. gave 2.0 Cc. N at 24. 5 C. and 714 Mm.
I II Mean
c 71.72 71.418 71-569
H 7.788 7.773 7.830
N 0.49 0.560 0.525
Ash i. 08 i. 060 1.070
The substance insoluble in methyl alcohol and insoluble in
ether was heated with hydrochloric acid for four hours to remove
ash, but even that did not secure its complete removal. It was
washed until free from acid, dried and analyzed with the accom-
panying results:
0.388 Gm. gave 0.2881 Gm. H 2 O, 1.0232 Gm. CO 8
I 0.554 Gm. gave 8.4 Cc. Nat 23C. and 714 Mm.=i.6 per ct | Mean 1,68
II 0.457 Gm. gave 7. 6 Cc. N at 25C, and 712 Mm. =1.76 perct f percent
C 71.896
H 8.303
N i. 680
Ash 0.400
The substance insoluble in methyl alcohol but soluble in ether
was spread on glass, placed in the drying oven where it dried in a
26
few hours. It was very hard to powder and was insoluble in all
ordinary solvents. When analyzed it gave the following results:
0.3606 Gm. gave 0.295 Gm. H 2 O, 0.971 Gm. CO 2
0.4104 Gin. gave 7.3 Cc. at 25 C. N and 712 Mm.
C 73430
9-J45
N 1.850
Ash 0.451
A second sample of the above was dissolved in ether and
alcohol added, then precipitated with sodium hydrate, heated
for two hours on the steam bath, washed until free from alkali,
again heated for four hours w r ith hydrochloric acid, washed and
dried. Analysis gave the following result:
0.2074 Gm. gave 0.194 Gm. H 2 O, 0.5482 Gin. CO 2
0.2926 Gm. gave 2.2 Cc. N at 21 C. and 712.5 Mm.
C 72.08
H 10.46
N 0.74
Ash 1.02
Part of the benzin-soluble substance was dissolved in alcohol,
a strong solution of sodium hydroxide added and heat applied.
An adhesive, black mass formed on the surface with a thick gray-
ish-black mixture underneath. When cold, globules of an oily sub-
stance could be seen. An attempt was made to separate them but
without success. Ether and benzin dissolved a large part of the
precipitate together with the oily globules, forming an insepar-
able emulsion. More sodium hydroxide was added and the whole
again heated for two hours, when a black precipitate separated
leaving a clear solution. The solution was separated as com-
pletely as possible by decantation. Distilled water was added
to the precipitate, when it again formed a homogeneous mixture
as inseparable as before but the addition of more alkali pro-
duced a separation. All attempts to free the precipitate from
alkali by washing failed, as it invariably formed the same homo-
geneous mixture which could not be filtered and refused to sep-
arate on standing. However, the addition of sodium chloride
to the mixture secured a perfect separation. The precipitate was
carefully collected on a filter and washed with solution of sodium
chloride until nearly free from alkali. It was then dried and
heated with hydrochloric acid which changed the color to reddish
brown. The precipitate was washed until free from acid, dried
and powdered. The powder was exhausted with ether which
27
dissolved about one-third. The insoluble part which continued
obstinately insoluble in all ordinary solvents, was dried and anal-
yzed. Result of analysis :
I 0.366 Gra. gave 0.2628 Gm. H 2 O, 0.945 Gm. CO 2
II 0.3016 Gm. gave 0.213 Gm. H 2 O, 0.7855 Gm, CO
I 0.3566 Gm. gave I Cc. N at 21 C. and 717 Mm.
II 0.3006 Gm. gave I Cc. N at 22 C. and 719.5 Mm.
I II Mean
C 70.96 7I-03 2 70.996
H 8.03 7.899 7.965
N 0.24 0.25 0.245
Ash 1.200
The ether solution was darker and more truly red than any
of the other solutions ; on allowing the ether to evaporate spon-
taneously and allowing the residue to stand for a few hours, a
portion became insoluble in all ordinary solvents. This was
exhausted with ether, dried and analyzed with the following
results :
I 0.3104 Gm. gave 0.2387 Gm. H 2 O, 0.7983 Gm. CO 8
II 0.2394 Gm. gave 0.19 Gm. H 2 O, 0.622 Gm. COjj
I 0.415 Gm. gave 3.2 Cc. N at 21 and 720 Mm.
I II Mean
C 71.012 70.897 70-954
H 8.596 8.877 8.736
N 0.850
Ash 0.210
The ether extract from above was allowed to evaporate
spontaneously as before but remained soluble in ether and alcohol
even after standing several days and after repeated solution and
evaporation. On heating the residue at 100 for a short time it
became insoluble and behaved in all respects like the preceding.
Analysis gave these results:
I 0.329 Gm. gave 0.2501 Gm. H 2 O, 0.8874 Gm. CO 2
II o 3374 Gm. gave 0.2588 Gm. H 8 O, 0.9121 Gm. CO 2
I 0.3946 Gm. gave 3.4 Cc. N at 22 C. and 720 Mm.
I II Mean
C 73-554 73-721 73. 6 37
H 8.503 8.579 8.541
N 1x940 0.940
Ash o.i 60
H. Yoshida oxidized the alcoholic extract (Urushic acid)
with chromic acid mixture, and obtained a brownish powder
which he washed with alcohol, dried and analyzed.
28
For comparison his results will be given with the means
from the preceding results:
PART INSOLUBLE IN BENZIN BUT SOLUBLE IN METHYL ALCOHOL.
C
H
N
Ash
I Methyl alcohol soluble (dried)
7i 659
7 93O
I 608
o 600
2 Methyl alcohol soluble precip. by NaOH
7L569
7.830
0.525
1.070
PART INSOLUBLE IN METHYL ALCOHOL.
3
4
5
6
7
8
Ether-insoluble
71.896
73-430
72.080
ZIN.
70.996
70.954
73.637
8.306
9-145
10.460
7.965
8.736
8.541
i. 680
1.850
0.740
0.245
0.85
0.94
0.400
o.45i
1.02
1.200
0.210
0.160
Ether-soluble (dried)
Ether-soluble precip. by NaOH
PART SOLUBLE IN BEN
Benzin-soluble precip. by NaOH and insol-
uble in ether
Benzin-soluble precip. by NaOH, soluble in
ether but insol on evaporation
Benzin-soluble precip by NaOH. soluble
in ether became insoluble with heat
Yoshida's oxy-urushic acid 71.52 8.280
The methyl alcohol and ether soluble substances (Nos. 4 and
5) constitute less than one per cent of the original alcoholic resi-
due, hence not enough material remained after other experiments
for dulpicate analyses. Likewise only a small per cent of the
alcoholic residue is represented by No. 8, that part of the benzin-
soluble which, after treating with sodium hydroxide and hydro-
chloric acid, required heat to convert it into the insoluble form.
These would have no practical effect upon the final product
obtained by drying or oxidizing the lac. The substances from
which the results in numbers I, 2, 6 and 7 were obtained form
the principal part of the lac-resin that is soluble in alcohol.
Yoshida analyzed the dried lac and found C 70.85%,
H 8.22%, N. -0.092, Ash 0.032.
The one quality which has made Japanese lac so valuable is
its power to resist atmospheric action, solvents and chemicals.
The preceding results show that strong chemicals, like alkalies
and acids, convert the lac-resin into its insoluble form. Also that
the final product appears to be the same whether obtained by the
action of the enzyme, as in the usual method of hardening, or by
chemical action. This becomes more apparent by comparison of
the composition of this substance as obtained by various methods.
C H N Ash
Mean from Nos. I and 3 obtained by drying 71.777 8.1 18 1.644 0.5
Mean of Nos. 2, 6 and 7 precip. by NaOH 71.173 8.177 0.56 0.82
Action of chromic acid (Yoshida) 71.52 8.280
Original lac hardened (Yoshida) 70.85 8.22 0.092 0.032
_-2 9
Yoshida has named this substance oxy-urushic acid and giv-
en it the formula C 14 H 18 O 3 , but as it has none of the properties
of an acid I have called it oxy-urushin. Owing to the presence
of nitrogen I am loth to suggest a change in the formula. That
nitrogen is present has been proved (beyond doubt) by the meth-
ods more fully given under gum-enzyme. That nitrogen is in
actual combination is supported by the fact that it is separable
only by fusing with dry fixed alkali, or incompletely by long
boiling with a solution of fixed alkali. Owing to the small amount
of nitrogen present, a very slight error in estimation would ma-
terially affect the results from calculation of a molecular formula.
The Kjeldahl method could not be used for the determina-
tion of the nitrogen, hence the necessity of using the Dumas
method. The principal objection to this method is the difficulty
of completely removing the air from the fine copper oxide. After
numerous experiments the substance was finally mixed with cop-
per oxide in fine powder and placed in a copper boat which was
then placed in the combustion tube and the air removed by car-
bon dioxide, generated from sulphuric acid and potassium car-
bonate by Thiele's method 15 . The results were very concordant.
For calculation of the empirical formula only those results
which were obtained without heating with alkali, can be used,
as alkali causes a loss of nitrogen. Therefore the mean from I
and 3, corrected for ash, was used as follows:
Mean from Calculated for Ash Calculated for
i and 3: Free Substance CiogHiag^O^
C.... 71.777 72.137 72.206
H.... 8.118 8.156 8.202
N.... 1.644 1.652 1.656
Ash.. 0.5
o .*.... 17.936
The above formula was calculated from the benzin-insoluble
portion which represents only 22% of that portion of the lac
that is soluble in alcohol. The remaining 78% which is soluble
in benzin could not be obtained in a dry form, or changed into
its end product without the use of reagents which caused a loss
of nitrogen; hence no attempt was made to calculate a formula
from the results obtained by combustion. The results obtained
in 6, 7 and 8 differ so much from those of 2 and 3 that it would
15 Ann. der chem. 253, 1889, p. 242.
indicate a difference in composition, though the physical proper-
ties are the same.
It is evident that the urushic acid of Yoshida consists of at
least four, if not five substances, differing in their solubilities as
well as in other respects, for I have already shown that some dry
in air to an insoluble substance, while others remain for months
without drying; one is piosonous and the others not. However
it is a remarkable fact that all may be converted into an end pro-
duct having practically the same properties, resisting the action
of all ordinary solvents, are black when heated with alkalies and
red when heated with acids. The product seems to be the same
whether obtained by drying with heat, by the action of the enzyme,
or by the action of alkalies.
ORIGINAL RESIDUE INSOLUBLE IN ALCOHOL OR WATER.
The residue from the lac which was insoluble in alcohol or
water consisted largely of hardened lac. By boiling this with
caustic alkali a dark brown solution was obtained which, on neu-
tralization and evaporation, left a hygroscopic residue, soluble in
water but insoluble in alcohol or ether. All attempts to obtain
a crystalline product failed.
The residue insoluble in alkali was dissolved by continued
heating with fuming nitric acid, concentrated, and precipitated
by pouring into water. The precipitate formed a gummy, plastic
mass, soluble in alcohol but non-crystallizable.
The watery solution contained oxalic acid, but no picric or
styphnic acid.
Oxyurushin when treated with fuming nitric acid gave the
same results as above.
PREVIOUS INVESTIGATIONS OF SOLUBLE FERMENTS.
The first observation recorded upon the color action of Gums
and of guaiac was made in 1809 by Goettling 18 who observed a
bluish-gray color when compounding a mixture of resin of guaiac,
sugar, acacia, and peppermint water. In the same year Boulay 17
observed the same color reaction when syrup, gum arabic and tr.
of guaiac were mixed and by experiments proved that the color
16 Bulletin de Pharmacie t. I. p. 220, 1809.
17 Bulletin de Pharmacie t. I. p. 225, 1809.
was produced by the acacia and guaiac. He also records the fact
that certain toothache remedies containing resin of guaiac colored
the mouth blue or green, and, from experiments with albuminous
substances, concluded that the color was produced by the albumen
in the saliva and the guaiac.
Planche" in 1810 observed that pieces of certain fresh roots
colored tr. of guaiac blue, also that nearly the same effect was
produced by sulphurous acid gas and tr. guaiac.
In 1819 Taddey 19 observed that corn meal and powdered
resin of guaiac when mixed with water and exposed to the air
became blue.
Rudolphi 19 found that in the above mixture the color was
produced by gluten and guaiac.
Planche 20 in 1820 gives a list of about 25 plants, the fresh
roots of which give a blue color with tincture of guaiac, and states
that fresh milk not boiled produces the blue color with tr. guaiac.
He was the first to discover that the power of albuminous sub-
stance to produce the blue color with tr. of guaiac was destroyed
by heat. He experimented upon the action of light and air, suc-
cessively examined and rejected the intervention of oxygen, but
concluded that action was due to a kind of undetermined cyano-
gen.
Schonbein 21 in 1856 observed that the juice of certain mush-
rooms, Boletus luridus and Agaricus sangnincns, colored tincture
of guaiac blue but lost their power when heated to 100 C. In
1868", he reports that the blue color is formed by ozone, pro-
duced by a kind of catalytic action. He arrives at this conclu-
sion after a series of experiments with the fresh juice from
plants, and tincture of guaiac, under varying conditions of light
and air, and with oxygen obtained from other sources.
In 1872 Struve 23 made some experiments upon the change of
pyrogallol to purpurogalline by acacia, saliva and other substances
which produced a blue color with tinct. of guaiac, but did not
arrive at any definite conclusions as to the true action.
In 1877 Traube 24 divided ferments into two groups : a. Oxi-
18 Bulletin de Pharmacia t. II. p. 579, 1810.
18 Journal de Fisice Chim. etc. 2d. Semestre, 1819.
' M Journal de Pharmacie t. VI. 1820, pp. 16-25.
!1 Jour, fur Prakt. Chem. 67, 1856, 496.
"- Jour, fur Prakt. Chem. 105, 1868, p. 198.
23 Ann. d. Chem. u. Pharm. 163, 1872, p. 160.
" 4 Ber. d. Chem. Ges., 10, 1877, p. 1985.
32
dizing ferments, those that take up free oxygen and carry it to
another substance. In this class he places the ferments that pro-
duce a blue color with tincture of guaiac, such as that found in
potatoes and many other plants, b. Reducing ferments ; those
that have the power of changing the combined oxygen, produc-
ing not only an oxidation product but also a reduction product,
as alcohol and carbonic acid from sugar, by the action of yeast.
In 1882 Clermont and Cheutard 25 obtained a considerable
quantity of pur-purgallin by exposing a solution of pyrogallol
containing 10% of acacia for several weeks to the action of air
but failed to recognize the true cause of the change in color.
In 1883 H. Yoshida 26 was the first to discover that it was an
enzyme that acted as an oxidizing agent. He proved that the
milky juice of Rhus vernicifera was converted into a hard insolu-
ble black varnish by the action of a peculiar diastatic substance
contained in the juice and that the change took place in the pres-
ence of moisture, but more rapidly in moist oxygen. Also that
the change did not take place at all in the presence of dry car-
bonic acid gas, or in a solution that had been previously boiled,
thus proving that the color change was due to a distase which
was destroyed by heat. He also proved that the substance acted
upon by the enzyme had taken up oxygen, but did not give off
carbonic acid.
Bouffard 27 states that the disease of wines which causes a
skin to grow on the surface of the wine is due to an enzyme and
that if the action is allowed to continue the wine becomes color-
less or light yellow. Later 28 he states that this action can be pre-
vented by heating the wine to 60 or by adding a very small
quantity of sulphuric acid.
Gouirand 29 found that by filtering diseased wine through
porous tile and precipitating the filtrate with alcohol he obtained
a substance which, when added to sound wine, changed it rapidly
to the diseased condition.
Martinand 30 obtained a substance in ripe grapes, pears and
25 Compt. rend. 97, 1882, p. 1254.
20 Jour. Chem. Soc. 43, 1883, p. 472.
27 Compt. rend. 118, 1894, p. 827.
28 Compt. rend. 124, 1897, p. 706.
29 Compt. rend. 120, 1895, p. 887.
33 Compt. rend. 121, 1895, p. 502. Also 124, 1897, p. 512.
33
apples which gives the reactions of laccase but does not seem
to be identical with it.
Cazeneuve 31 found that laccase produced only a very slight
action in wines. He therefore attributed the disease to a partic-
ular enzyme and named it "Oenoxydase." He obtained it in the
form of a gum by precipitating it from the wine by the addition
of a large amount of alcohol. It colors guaiac blue, is active at
oC. and is not entirely destroyed at 65 C. He finds that the col-
oring matter of wines is a phenol-like body which is oxidized by
oenoxydase.
Laborde 32 attributes the secretion of Oenoxydase to a mould,
Botrytis cinerea (sweet rot), which is present at the root of the
vine.
Eduard SchaY 3 has especially examined the enzyme in Phyto-
lacca decandra. He used an extract prepared by macerating the
fresh parts of the plant in glycerin containing not more than 5 or
10% of water, for a few days, then filtering. He states that an
extract so prepared scarcely loses any of its activity for a year
and a half. He found the extract from the leaves to be the most
active, that from the root less active, and that from the flowers
the least active.
Schar 34 states that the blue color produced by enzymes upon
tincture of guaiac depends upon the formation of a peculiar oxy-
gen combination with the resinous constituent of guaiac, the
guaiaconic acid. To the blue compound he gives the name ozon-
ized-guaiaconic acid. He also states that guaiaconic acid is very
sensitive to the action of acids, alkalies, light, air and water and,
that when the tincture is to be used as a reagent, it should be
prepared fresh, of the strength of 2 to 3 per cent, of resin free
from wood.
The most valuable contributions regarding the action of sol-
uble ferments have been given in a series of articles by G. Bert-
rand 33 . He has given the name "Laccase" to the enzyme first
found in Japanese lac by Yoshida but since found in many plants.
81 Compt. rend. 124, 1897, p. 406 and 781.
82 Compt. rend. 126, 1898, p. 536.
33 Vierteljahrsschrift d. Naturf. Ges. Zurich, XU (1896) 233.
84 66 Versammlung deutscher Naturforscher und Aertze & Wien, 1904.
85 Bull. Soc. 3d. Series to 51, p. 159, 1891.
Compt. rend. 119, p. 1012, 1894.
34
Bertrand named the gummy substance that was separated
from Japanese lac, "Laccase". He has since used a somewhat
different method of separation in order to prepare it from other
plants, like potatoes, turnips, beets, artichokes, asparagus, apples,
pears, etc. The fresh parts of the plants are crushed, the juice
expressed and after saturating with chloroform allowed to stand
24 hours when a coagulum forms and the juice is separated and
the gum is then precipitated by alcohol.
Bourquelot and Bertrand 36 examined about 200 species of
mushrooms of which they give the following:
GENERA EXAMINED ACTIVE INACTIVE
Russule 18 18 o
Lactarius 20 18 2
Psalliota 5 4 i
Boletus 18 10 8
Clitocybe 9 5 4
Marasmius 6 o 6
Cortinarius 12 i n
Inocybe 6 i 5
Amanite 7 2 5
Hygrophorius 6 o 6
The parts of plants which contain the least chlorophyl con-
tain the most laccase.
Bertrand 37 has shown by experimenting upon such substances
as hydroquinone, pyrogallol, gallic acid, etc., that the soluble
ferments like laccase act by direct oxidation and that under its
influence these bodies, in the presence of air, take up oxygen and
give off carbon dioxide. He found that the phenols most easily
acted upon are those having hydroxyl in the ortho or para posi-
tion. When in the meta position they are oxidized with great
difficulty.
On adding laccase to a solution of hydroquinone it changes
to a deep red color and after some time green crystals are formed
and the solution has the characteristic odor of quinone.
Bertrand 88 states that the darkening of certain substances
as the dahlia, beet, etc., is due to the oxidation of ty rosin under
the influence of soluble ferments. But that tyrosin resists in-
definitely the action of gaseous oxygen in the presence of laccase,
even in strong solutions ;therefore the blackening of the tyrosin
30 Compt. rend. 121, p. 166 & 783, 1895.
37 Compt. rend. 120, p. 226, 1895.
38 Compt. rend. 122, p. 1215, 1896.
35
in the dahlia and beet is due to a peculiar oxidizer. This he has
been able to separate and has called it "Tyrosinase". It exists
not only in the dahlia and beet but in several varieties of mush-
rooms which do not contain ty rosin.
Tyrosinase is very unstable. It is best prepared from Rus-
sules.
One can either use juice of the mushrooms at once, or pre-
serve them for future use by cutting in thin slices and drying in
a vacuum. When wanted for use the dried residue is macerated
for some time in cold water and then filtered.
If a solution of tyrosinase is mixed with a solution of ty-
rosin and the liquid frequently shaken to introduce air the liquid
will first turn red, then black. Tyrosinase is not so frequently
found in plants as laccase, but may be found in many fungi such
as Russula, Lactarius, Hebeloma, Boletus, Amonita, and many
others.
The two enzymes frequently exist in the same plants. Bert-
rand found that tyrosinase was more easily destroyed by heat
than laccase.
Bertrand 39 gives the following as the theory of the action of
oxydizing enzymes. That the manganese, which is present in all
enzymes, even as high as 2%, exists in the albuminous substance
as a manganous compound, and plays the part of oxygen carrier.
The oxygen molecule is broken by the manganous compound to
form manganese dioxide and the remainder of the oxygen mole-
cule acts upon the oxydizible body present. Finally through the
acid character of the albumen radical, the manganese dioxide is
decomposed and the original manganese compound restored. He
believes that the activity of the enzyme is proportional to the
manganese present.
Bourquelot 40 reports upon the action of tyrosinase on phenols,
etc. He states that the tyrosinase of mushrooms forms with gua-
iacol a red precipitate.
Bokorny 41 after giving the apparent similarity of enzymes
and protoplasm, especially with reference to the action of light
and temperature, concludes that one can scarcely think that they
originate from the same source.
39 Compt. rend. 124, 1356, 1897.
40 Repertoire de Pharmacie, 1897, p. 136.
41 Allgeminen Brauer und Hopfen Zeitung, No. 74, 1901.
- 3 6-
Loew 42 reports two kinds of enzymes, an insoluble and a
soluble form a- and ^-catalase respectively. The former is prob-
ably a compound of the soluble catalase with a nucleoproteid.
while the soluble catalase is an albumose and can be liberated by
the action of very dilute alkaline media upon the insoluble cata-
lase. He has studied these enzymes with special reference to the
tobacco plant.
Catalase does not color tincture of guaiac blue, but changes
hydroquinone into quinone. Traces of acids increases its action
while alkalies destroy it.
Chodat and Bach 43 state that catalase is not a true enzyme
like oxydase or peroxydase, as its function is only to decompose
hydrogen peroxide.
Kastle and Shedd 44 have shown that phenolphthalin is oxi-
dized to phenolphthalein by oxidizing ferments and that it forms
a very sensitive reagent for the presence of soluble enzymes.
They have tested this reagent upon a number of enzymes from
plants and on a few from animals. These results were com-
pared with those obtained by tincture of guaiac and found to be
practically identical, i. e., all those enzymes which gave a blue
color with guaiac, gave a pink color with phenolphthalin, the
colors in both cases increasing or decreasing with the activity
of the enzyme.
In i8c)8 45 L,aborde proposed to measure the activity of en-
zymes by comparing the color produced by the enzyme when act-
ing upon an alcoholic tincture of guaiac, with a standard color
formed by adding 0.5 gramme of iodine to 20 cc. of the tincture
of guaiac.
Alliot and Pozzi-Escot 48 found it impossible to estimate oxy-
dases colorometrically either by Laborde's guaiac method or by
Kastle and Shed's phenolphthalein method.
Kastle and Shed found that the only enzyme obtained from
animal source, which acted as an oxidizing enzyme was the
human saliva.
About this time Cavazzani 47 found a soluble oxidizing en-
42 U. S. Dept. of Agriculture, Report No. 68, 1901.
M
Am. Chem. Jour. 26, 1901, p. 526.
45 Compt. rend. 126, 1898, p. 536.
** Ann. Chem. Anal. 7, 1902, p. 210.
41 Cent. Physiol. 14, 1901, p. 473.
37
zyme in the cerebro-spinal fluid of dogs and calves, and Vitali 48
reported oxydase in pus, which he obtained by triturating the pus
with glass, and then extracting with water, dilute acetic acid, or
with equal parts of glycerin and water. It imparted a blue color
to tincture of guaiac. This action was destroyed by hydrocyanic
acid, chloroform, hydroquinone, pyrogallol, and hydroxylamine,
but not by phenol, thymol or mercuric chloride.
Gersard 49 found in the ink sac of the cuttle-fish laccase, tyros-
inase and an oxidizing diastase which is more resistant to heat
that laccase.
Kastle and Loevenhart 50 while studying the action of oxidiz-
ing enzymes have established the fact that the organic peroxides
like benzol, phthalyl and succinyl peroxides will color tincture of
guaiac blue.
The same authors have very thoroughly studied the poison-
ous action of a variety of substances upon enzymes, and also the
effect of the same substance upon organic peroxides, and find
that those substances which destroyed the power of enzymes
to color guaiac blue also prevented the coloration by organic
peroxides.
They made many experiments with the juice from the potato
which colored guaiac blue, and oxidized phenolphthalin, but the
enzyme could not be precipitated by absolute alcohol as most en-
zymes. They arrive at the following conclusions:
"i. That oxygen is absolutely essential to the production of
the guaiacum-bluing ferment of the potato.
"2. That this so-called oxidizing ferment is in all probability
not a true ferment, but an organic peroxide.
"3. That the oxidation phenomena occurring in the plant,
and probably in the animal organism also, can be satisfactorily
explained upon the supposition that the readily autoxidizable
substance which they contain is oxidized to the peroxide condi-
tion by molecular oxygen, and that the peroxides thus formed
in turn give up part of their oxygen to other less oxidizable sub-
stances present in the cell. In other words, that the process of
rendering oxygen active, by the living cell, is probably brought
4S L'Orosi, 24, 1901, p. 263, from Jour. Chem. 800.4. ii, 672.
4J Compt. rend. 136, 1903, p. 631.
60 Am. Chem. Jour. 26, 1901, p. 539.
-38-
about in essentially the same way that is accomplished by phos-
phorus, benzaldehyde and other oxygen carriers."
Hunger 51 states that the guaiac reaction is interfered with
by tannins, certain sugars, hydrogen sulphide, pyrogallol and
other reducing agents.
Pozzi-Escof' 2 reports that when living tissues of animal or
vegetable origin do not affect guaiac, but decompose hydrogen
peroxide, that reductase must be looked for, which may be done
by treating them, out of contact with air, with a solution of in-
digo, or litmus, or ferric ferricyanide and note if any reduction
has taken place. They also liberate hydrogen sulphide from a
mixture of sulphur and potassium fluoride, if protected from the
air.
Bach and Chodat 53 find that oxydase is always accompanied
by peroxydase, which they have found in about 25 plant families.
The oxydase has the power of forming peroxides in the presence
of free oxygen, which can be detected by the liberation of iodine
from hydriodic acid. When parts of plants containing these en-
zymes are heated to 80 C. the power to liberate iodine or color
guaiac blue is destroyed. This is also true of the expressed juice.
The power to liberate iodine disappears more quickly than that
which colors guaiac. When the substance ceases to act it may
be made active again by the addition of a small quantity of hydro-
gen peroxide.
Bach 54 showed that a large number of substances when sub-
mitted to slow oxidation in the air, formed peroxides, and that
the peroxide formed helped to continue the oxidation. In the
blood the easily oxidizable substances first form peroxides and
these aid in the oxidation of the more difficultly oxidisable bodies.
The oxidation does not seem to be influenced by light.
Wender 55 thought that the action of the oxydase in the living
cell is to cause the oxygen of the aeroxydase to oxidize the easily
oxidizable bodies so that intermediate bodies are formed, then
these are again decomposed by the action of catalase. The free
oxygen by the action of anaeroxydase (peroxides) becomes active
and oxidizes difficultly oxidizable substances.
51 Ber. d. Bot. Ges. 19, 1901, p. 648.
62 Ann. Chem. anal. 7, 1902, 260.
53 Ber. d. Chem. 35, 1902, p. 2466.
54 Compt. rend. 124, 1897, p. 951.
55 Chem. Zeit. 26, p. 1217 & 1221, 1902.
39
Chodat and Bach 56 prepared peroxydase from cucumbers and
horseradish but on account of the large amount of water in the
former, they preferred to prepare it from the horseradish as the
yield was much larger. Their method was to reduce it to as fine
a condition as possible and allow it to stand for an hour to permit
the glucoside-splitting enzyme to act, then to express the juice
and precipitate with absolute alcohol. The precipitate was ex-
tracted with 40% alcohol, the alcoholic solution concentrated in
a vacuum at 30 C. and precipitated with absolute alcohol and
dried in a vacuum.
The product is a yellowish gummy mass which in solution
reduces Fehling's solution ; however they claim that this is not
due to the enzyme, for by repeated precipitation an enzyme may
be obtained which will not reduce Fehling's solution.
The purest peroxydase obtained from horseradish con-
tained 6 per cent, of ash of which aluminum amounted
to 0.8 to 1.4 per cent, and manganese from 0.2 to 0.6 per cent.
They found that the peroxydase from Russule and Lactar-
ius was much more active than that from the horseradish or
cucumber ; on the other hand with hydrogen peroxide the activity
was reversed. By heating a mixture of oxydase and peroxydase
to 70 C. the former is destroyed while the latter is only weaken-
ed but seems to regain its strength on standing" but if subjected
to a second heating its activity is destroyed completely. In alco-
holic solution it is destroyed by boiling. Peroxydase does not
possess the power to oxidize except in the presence of peroxides.
The same authors state 08 that peroxydase and catalase
are present in nearly all parts of plant and animal
bodies, and apparently are antagonistic, as the first is
active with hydrogen peroxide while the other is destroyed
by oxygen. They experimented upon a mixture of oxygenase,
peroxydase and pyrogallol alone, and in the presence of catalase,
and found that catalase had no effect upon the amount of oxygen
absorbed. In another experiment catalase was mixed with oxy-
genase and peroxydase, and the mixture allowed to stand over
night, then hydrogen peroxide was added and the amount of gas
liberated was found to be the same as without the catalase. Hence
M Ber. d. Chem. 36, 1903, p. 600.
57 Compare U. S. Dept. of Agric., Bull. No. 8, 17.
BS Ber. d, Chem. 36, 1903, p. 1756.
40
they conclude that peroxydase and catalase can exist in the same
plants without interfering with the functions of either. They
also prove that catalase and reductase are not identical.
Kunz-Krause 59 and Dr. Richard Fibras 60 believe that one of
the causes of the deposit in tinctures is due to the action of en-
zymes.
Bourquelot 81 studied the ferments which cause hydrolisis
of the various polysaccharides, and finds that two and in some
cases three ferments are required to completely hydrolise poly-
saccharides. In another paper, with Herissey 62 he deals with the
ferment of milk, almonds, peach and cherry laurel leaves and finds
that emulsine as obtained from almonds, is a mixture of several
ferments, emulsin, lactase and probably gentiobias, and frequently
invertin. He concludes:
1. That lactase accompanies emulsin in the different al-
monds of rosaceae.
2. That emulsin exists without laccase, as in Perquillus
niger leaves.
3. That lactase exists without emulsin, as in the yeast of
kaphis.
Chodat and Bach 63 review the researches of soluble ferments
(oxydase) and give the following theory: The oxidizing fer-
ments called oxydase, are bodies of a peroxide character, there-
fore they are organic peroxides which heat decomposes. Their
action is accelerated by a second class of bodies which act as
catalytics, and have the power to bring back the peroxides to
their normal condition. They state that there is present in the
living cell a diastase (peroxydase) which acts on hydrogen per-
oxide similar to ferrous sulphate, and with hydrogen peroxide
produces a blue color with guaiac.
Chodat and Bach give the following characteristics for per-
oxydase : M
If free from other enzymes, it does not liberate oxygen from
59 Pharm. Centralhalle, 1902, No. 52.
90 Pharm. Post, 35, 1902, p. 548.
81 Jour. d. Chim. et d. Pharm., 1903.
Also Compt. rend. 136, p. 762, 1903.
62 Jour. d. Chim. et d. Pharm., 1903.
Also Compt. rend. Soc. Biol. 55, p. 219, 1903.
" Ber. d. Chem. 35, P- 1275, 1902.
* Ber. d. Chem. 37, p. 1342, 1904-
41
hydrogen peroxide as Catalase, or oxydize pyrogallol as Oxy-
genase ; or liquefy starch paste, forming substances which de-
duce Fehling's solution, as Amylase; or invert cane sugar, as
Invertase ; or break up glucosides, as Emulsin ; or digest coagu-
lated albumen, as the Proteolytic enzymes.
Chodat and Bach" state that Bertrand found that, by frac-
tional precipitation of laccase with alcohol, he obtained a
substance poor in manganese and weak in oxidizing power, also
a substance rich in manganese and strong in oxidizing action,
but did not suspect that the decrease in power of oxidation was
connected with the separation of peroxydase. Five years later
fractional precipitation was brought forward as a method of sepa-
ration of peroxydase from oxydase, by Aso. M
The authors have used this method to obtain two end frac-
tions, one with weak oxidizing power and the other without any
oxidizing action. The first was practically insoluble in 40% alco-
hol while the other was soluble, and was active with hydrogen
peroxide, and behaved as a true peroxydase. The weak oxidizing
fraction which principally took the part of an oxygen carrier
they designate as Oxygenase. They add that it is comparatively
easy to prepare peroxydase free from oxygenase, but have not
succeeded in preparing oxygenase free from peroxydase. A par-
tial separation may be made by extracting a mixture of the two
with 30 to 60 per cent, alcohol, or by dialysis with pure water
when the peroxydase passes into the dialysate. 67
In 1892 Tschirch 68 called attention to the fact that the differ-
ence between the color of black and green tea was due to fer-
mentation. The black tea is prepared by allowing the fresh leaves
to undergo partial fermentation at a relatively low temperature,
while in the case of green tea the ferment is destroyed by heat.
Aso 69 states that the color of black tea is produced by the
action of oxydase on the tannin in the first stage of preparation,
while in the green tea the ferment is destroyed.
Tschirch and Oesterle 70 state that the formation of cola-red
co Ber. d. Chem. 36, 606, 1903.
50 Bull. Coll. Agric. Tokio, 5, 2, 1902, p. 233.
67 Compare Engler and Wild, Ueber die sogenannte activirung des
Sanerstofrs und iiber Superoxyd bildung. Ber. d. Chem. 30, 1669, 1897.
m Indische Heil-und Nutzpflanzen und deren Kultur, Berlin, 1892.
" Bull. Coll. Agric., Tokio, 4, p. 254, 1901.
" Anatom. Atlas d. Pharmacognosie, p. 350.
42
in cola nuts is produced by fermentation, and that the nuts may
be preserved colorless by heating to 65 degrees, or by immersing
in boiling alcohol.
Tschirch 71 observed that cinchona bark does not become col-
ored, if the fresh branches are immersed in hot water before re-
moving the bark.
The same author states that it is the action of oxydases upon
the tannin group, which produces the strong red-brown products
that he has grouped together as, cinchona-red, tannin-red, cinna-
mon-red, kino-red, colo-red, etc. 7 "
Wender 73 attributes the brown color of bread to the action
of an enzyme, and adds that the brown color of many trees is
due to the action of enzyme on the tannin.
Browne 74 found lipase in rice bran and tested its hydrolytic
action on castor oil and upon the oil from rice bran.
Mohr 75 finds that lipase acts as a hydrolizer in the decompo-
sition of esters but that the decomposition is not complete if only
alcohol and acid are present. After a time the action is reversed
and esters are formed from the acid and alcohol present.
Wender and Lewin 76 state that the enzyme of grain which has
a catalytic action is not increased during germination. Also that
the diastatic action may be destroyed by carefully increasing the
heat , without destroying the catalytic action. Also that the outer
thin brown seed coat contains the strongest enzyme, and decreases
in strength toward the center.
Bourquelot and Marchadier 77 find that oxydase and peroxy-
dase are active in 10% alcohol, and that both are destroyed by
hydrocyanic acid. With vanilla, peroxydase and hydrogen per-
oxide act the same as oxydase with air, and suggest that per-
oxydase consists of two enzymes, one hydroperoxydase which,
in the presence of air, is capable of converting water into hydro-
gen peroxyde, or forming peroxides with certain substances, and
the other an indirect oxydase, capable of decomposing the per-
oxides with the liberation of oxygen.
71 Schweiz. Wochschr. fiir Pharm., No. 10, 1905.
72 Angew. Pflanzenanatomie, p. 127.
73 Chem. Zeit, 26, p. 1217, 1902.
74 Jour. Am. Chem. Soc., 25, p. 950, 1903.
75 Chem. Centr., 1902, ii, 1424, from Woch. Braii. 19, p. 588.
78 Chem. Centr., 1904, I, p. 1530, from Oest. Chem. Zeit., 7, p. 173, 1904.
77 Compt. rend. 138, p. 1432, 1904.
^V:
f UNIVERSITY )
-43- ^^ ^ J
^^c. '. ;
GUM AND ENZYME.
After extracting the lac with alcohol the residue was extract-
ed with cold water and the gum-enzyme precipitated by pouring
into strong alcohol. The precipitate was dissolved in a small
quantity of water and reprecipitated with alcohol. By repeating
the operation several times and finally washing with ether and
drying in an exsiccator, it was obtained perfectly white and easily
reduced to powder. In physical appearance it is similar to pow-
dered acacia. When so prepared the gum-enzyme is very active,
rapidly changing tincture of guaiac to a deep blue color. If
an emulsion is made with the gum-enzyme, water and the sepa-
rated resin, it soon changes from yellowish-white to black. A
solution of gum-enzyme with naphtol formed a purplish blue
color, and with guiacol a red color in half an hour but produced
no effect upon a solution of vanillin in hydrochloric acid. If a
solution of the gum-enzyme is boiled with water, it becomes en-
tirely inactive.
TESTS FOR NITROGEN.
It is a generally conceded fact that all enzymes contain nitro-
gen. The Lassaigne test for the detection of nitrogen is un-
doubtedly considered the most reliable. It consists in heating the
substance with metallic potassium or sodium and converting the
cyanide so formed into Prussian blue. This test was applied to
the gum-enzyme, but failed to detect the presence of nitrogen.
According to Kehrer the Lassaigne test must be modified for
certain pyrrol derivatives, 78 and cannot be applied to diazocom-
pounds. 79 In view of the certainty of the presence of nitrogen
and the general reputation of the test, it was repeatedly tried with
various modifications. The gum-enzyme was previously mixed
with dry sodium carbonate and carefully ignited. The rapidity
of the heating was varied. In another experiment the sub-
stance was placed in a narrow tube closed at one .end, and the
tube drawn out to contract the opening, small pieces of sodium
were then introduced and the tube again contracted, thus :
c
GUM-ENZYME SODIUM
78 Berichte, 35, 2,525 ; 1902.
79 Berichte, 17, 1,178; 1884.
44
The sodium was first heated, then the gum-enzyme slowly heated
so that the gases would pass over the glowing sodium. This test
was repeated in the same manner, except that the gum-enzyme
was first mixed with dry potassium hydroxide. In another ex-
periment the substance was heated with a small quantity of con-
centrated sulphuric acid until a dry charred mass was obtained,
then mixed with metallic iron and sodium and ignited, and fin-
ally tested for cyanide. In another experiment a modification of
the Kjeldahl quantitative method was tried. The gum-enzyme
was heated with concentrated sulphuric acid and a little mercuric
oxide until a colorless solution was obtained. The solution was
then mixed with an excess of potassium hydroxide and distilled.
The distillate was passed through a tube containing a piece of
red litmus paper into a mixture of chloroform, alcohol and po-
tassium hydroxide to convert the ammonia into cyanide. The
litmus paper remained red throughout the distillation. All at-
tempts to convert the nitrogen into cyanide failed.
Another test for nitrogen which is considered less reliable
than the Lassaigne test, is to convert the nitrogen into ammonia
by heating the substance in a tube with soda-lime or potassium
hydroxide. This test was applied to the gum-enzyme when the
red litmus paper placed over the end of the tube rapidly changed
to blue, but no odor of ammonia could be detected. The paper
was evenly colored as if produced by some gaseous substance.
The test was repeated with a pledget of cotton inserted in the
tube below the paper to prevent the possibility of potassium hy-
droxide being mechanically carried to the litmus paper. The re-
sult was the same as in the previous test. A blank test was next
made under exactly the same conditions, but with negative re-
sults. These experiments indicated the presence of a volatile
base. Professor Tschirch thought the odor similar to pyrrol. I,
therefore, repeated the test, placing in the top of the tube a pine
shaving moistened with hydrochloric acid. This was rapidly
colored red, thus strongly indicating, if not conclusively proving,
the presence of pyrrol, or a pyrrol derivative. This was further
confirmed by placing 5 grammes each of powdered potassium
hydroxide and gum-enzyme in a flask and distilling. The
vapors were passed through a condenser connected with a dry
flask, and this again connected with a second flask by means of
a tube which passed to the bottom of the flask, into a small quan-
tity of water. At the end of the reaction the first flask contained
45
a small quantity of colorless, strongly alkaline liquid, sparingly
soluble in water, but readily soluble in alcohol and ether. The
solution was tested with the following results:
On warming with hydrochloric acid and allowing to stand
a short time a fine red precipitate separated. With sulphuric
acid and quinone a green precipitate formed ; with phosphomo-
lydic acid, first a yellow, then a blue precipitate ; with potassium
ferrocyanide, dark green ; with quinone alone, violet red. The
contents of the second flask was also alkaline. The distillate
was also tested by the Lassaigne test, but no Prussian blue ob-
tained. As pyrrol gives the Lassaigne test it must be that the
distillate did not consist of pyrrol, but was a pyrrol derivative.
Another evidence of the presence of nitrogen was obtained
as follows : An ordinary open combustion tube was filled with
copper oxide and ignited in a current of oxygen. After partially
cooling, a platinum boat containing the gum was introduced
and the gum burned in a current of oxygen. The products
of combustion were conducted through potash bulbs containing
a solution of potassium hydroxide, prepared from metallic po-
tassium, and water distilled with potassium permanganate. Just
before the combustion the solution was tested and found to be
free from nitrogen compounds. After the combustion the solu-
tion was tested with diphenylamine when it gave the blue color
characteristic of nitrates. With brucine and sulphuric acid a red
color and with sulphuric acid and sulphate of iron the brown ring
appeared.
This proves conclusively that the gum contained nitrogen in
some form, which is converted into pyrrol, or a pyrrol derivative,
by heating with potassium hydroxide.
ATTEMPTS TO SEPARATE THE GUM FROM THE ENZYME.
Hikorokuro Yoshida states that by removing his so-called
urushic acid with alcohol and extracting the residue with cold
water, and then boiling the solution, a white precipitate is formed.
He assumes that it is the enzyme, but does not prove it, except
that the solution was active before boiling and inactive after-
wards, and that the precipitate contained nitrogen. It may have
been an inactive vegetable albumen, although he states that it
contained less nitrogen than these bodies usually contain. I have
found, however, that a solution of the purified gum obtained by
- 4 6-
repeated precipitation with alcohol remained perfectly clear on
boiling; yet, previous to boiling, the same solution was strongly
active, rapidly changing tincture of guaiac to dark blue, and the
clear brown resin from the lac to a hard, black, insoluble sub-
stance. ^
Solutions of the gum were treated with acetic, hydrochloric,
nitric and sulphuric acids of various strengths and with varying
degrees of heat, but each failed to separate the nitrogenous sub-
stance from the gum. In one experiment the solution was boiled
for half an hour with a dilute sulphuric acid, precipitated with
alcohol, dissolved in water and reprecipitated with alcohol,
washed until free from sulphuric acid, and dried in an
exsiccator. This still gave the pyrrol reaction. Fractional pre-
cipitation was tried without apparent change in the relation of
gum to nitrogen. Cold saturated solutions of magnesium sul-
phate, ammonium sulphate and sodium phosphate were tried in
vain. Various modifications of Almen's solution of tannic acid
were tried, but in no case was there any separation of nitrogenous
from non-nitrogenous substance. Numerous precipitates were
obtained, but in every case the precipitate contained both gum
and nitrogen in apparently the same proportion as before. The
dry powdered gum was heated for two hours at temperatures
varying from 100 to 160 C. and tested both by boiling alone
and with acids, but no separation occurred.
EXAMINATION OF OTHER GUMS FOR NITROGEN.
A number of the following samples were prepared by stu-
dents and kindly furnished by Professor Tschirch from his col-
lection. The remainder were prepared by the writer. In the case
of the gum-resins the resin was removed by extracting with alco-
hol, the gum dissolved in water and precipitated by alcohol, puri-
fied by repeated precipitation and dried in an exsiccator. The
acids were prepared by the same method, with the exception that
the solutions were acidulated with hydrochloric acid each time
before precipitation, and the precipitate finally washed with alco-
hol until free from hydrochloric acid. Nos. 14 and 15 were pre-
pared by dissolving the tragacanth in a warm solution of sodium
hydroxide, precipitating with alcohol and dissolving in water,
and reprecipitating with acidulated alcohol.
Each sample prepared without heat was tested for enzyme,
47
and all were tested by heating with potassium hydroxide and
testing the vapor for alkalinity and by the pyrrol reaction. The
enzyme's activity is indicated by the time required from the addi-
tion of the tincture of guaiac to the first appearance of color and
afterwards to time required to produce a given shade. In each
case o.i gramme of the gum was dissolved in 4 cc. of water and
three drops of tincture of guaiac added.
NO. GUM FROM
PREPARED BY
PYRROL
REACTION
LITMUS
ENZYME
i Japanese Lac .
Stevens
Oscar Halbey
Knitl
Dr. Saal
Bergniann
Stevens
Schereschewski
Stevens
Halbey
Knitl
Stevens
Very strong
Weak
Medium
Strong
Weak
Strong
Weak
Medium
Medium
Weak
Medium
Strong
B
ue
Immediately
30- 60 Minutes
15- 60
8- 13
I- 4
I- 4
60-120
60-
30-
30 Sec. 12 Min.
15 Minutes
15
Inactive
3 Ammoniac select
4 Acacia
5 Asafoetida
6 Asafoetida select
7 Olibanum_
8 Olibanum
9 Opoponax
10 Tacamohaca
ii Myrrh
12 Galbanum __
13 Chicle
14 Tragacanth, white
15 Tragacanth yellow
16 Tragacanth, white
ACIDS PREPARED FROM
18 Opoponax
19 Acacia __
20 Asafoetida
21 Japanese lac
Nos. 8, 9, ii and 12 did not become as dark as standard,
even after standing twenty-four hours. Heat was used in the
manufacture of No. 13, which would have destroyed the enzyme
had it been present.
As the acids prepared from active gums did not give the en-
zyme reaction, it is evident that the hydrochloric acid used in
their preparation destroyed the enzyme, but did not remove the
nitrogen.
The enzyme in a solution of the gum from Japanese lac was
rapidly destroyed by boiling, but the powder, after heating for
two hours at 100 C., was still more active than any of the other
gums examined. The color with tincture of guaiac appeared at
once and in five minutes became dark blue. Another sample,
when heated for two hours at 120 C., required ten minutes to
produce the same deep blue shade. A third sample, heated for
two hours at 140 C., required ten minutes to produce any color,
but became dark blue in thirty minutes. A fourth sample, after
heating for two hours at 160 C., was inactive.
- 4 8
OXIDATION PRODUCTS.
The gum-enzyme was oxidized by heating I part of gum
with 12 parts of nitric acid 1.15 sp. gr. on a water bath for one
hour then evaporating to 2 parts and adding water 2 parts.
After 24 hours the white crystalline deposit was washed with
water and alcohol, and recrystallized from boiling water. The
melting point was the same as for Mucic acid 210 C. When
analyzed :
0.222 Gm. gave 0.1085 Gm. H 2 O, 0.2844 Gm., CO 2 .
Calculated for Mucic acid
C H 10 Os
C .............. 3448% 34-278
H ............. 4-644 4796
O ............. 60.926
100.000
After removing the mucic acid with hot water from the first
crystalline deposit, there remained a white powder insoluble in
hot water, alcohol or acetic acid but soluble in hydrochloric acid.
This was calcium oxalate which had been formed by the union
of the calcium of the gum with the oxalic acid formed by oxida-
tion. The mother liquor was evaporated to dryness, washed with
ether, which on evaporation left well defined crystals of tartaric
acid.
HYDROLYSIS OF LACGUM.
The gum-enzyme was heated with 2% sulphuric acid for
8 hours and the acid removed with barium hydroxide and carbon-
ate. The solution was evaporated under diminished pressure,
when it formed a very light yellow syrup, non-crystallizable, non-
fermentable, reduced Fehling's solution and was dextrorotary.
Alcohol dissolved only a small part of it. The remainder could
be dissolved by using a large quantity of hot alcohol but deposited
on cooling.
One part of the syrup was heated one hour with two parts
of phenylhydrazine, 3 parts of sodium acetate, and 20 parts of
water. On cooling an abundant yellow crystalline deposit formed.
This was several times recrystallized from hot alcohol, when the
M. P. remained constant beginning at 162 C., and was complete
at 164 without liberation of gas. The crystals were in small
spheroidal clusters, which under the microscope appeared to con-
49
sist of aggregations of needles. This corresponds exactly with
the description and melting point given for phenylsorbinosazone. 80
A second crop of crystals was obtained by concentrating the moth-
er liquor. These were somewhat darker than the first and had a
M. P. of 157 C., but with the production of gas. This corres-
ponds with the description given for the osasone obtained from
the inactive sorbin.
Furfurol was estimated from the gum according to Tollen's
method 81 and calculated as pentosan but the results were not sat-
isfactory. The method is designed for the estimation of furfurol
in food products, and has very little value for scientific investigation.
An analysis of the gum-enzyme gave the following results :
I 0.2626 Gm. gave 0.1424 H S O, 0.402 Gm. CO 2 .
II 0.3464 Gm. gave 0.1812 H 2 O, 0.529 Gm. CO 2 .
I 0.35 Gm. gave 2 cc. N at i8C. & 714.7 Mm.
II 0.452 Gm. gave 2.3 cc. N at i6C. & 713.7 Mm.
I II MEAN
C 4L742 41-645 -41.693
H 6.067 S-58i 5.958
N 0.630 0.587 0.608
Ash 5.180 5.200 5.190
O 46.551
IOO.OOO
Bertrand 82 when working with soluble oxidizing ferments
used the gum-enzyme from Japanese lac under the name "Lac-
case". He reports that it contained 0.44% of nitrogen which he
determined by heating with soda-lime and estimating the am-
monia formed by titrating with decinormal sulphuric acid. From
this he calculated the amount of enzyme present by assuming
that it has the elementary composition of albuminous substances.
He then gives the composition of the gum-enzyme as :
Water 74.00%
Gum 84.95%
Laccase 2 . 50%
Ash S- 1 7%
From the preceding work I think that I am justified in say-
ing that what he estimated as ammonia was not ammonia, but
pyrrol.
80 Vaubel Quantitative Bestimmung. Organ. Verbindungen II, Band
3, 304.
81 Lunge. II Bel. 460.
8J Bull. Soc. Chim. 3 series, 51, p. 259, 1891.
IMPURITIES IN ZINC DUST
Before using zinc dust in some experiments upon the gum
obtained from Japanese lac, I wished to be sure that it was free
from nitrogen. I therefore subjected the zinc dust to the follow-
ing tests, the results of which may be of interest to those who
frequently use it in connection with organic substances:
When heated with potassium hydroxide it formed ammonia.
When heated alone it also gave off ammonia. This led to the
belief that nitrogen in some form had been absorbed from the
atmosphere, and might be removed by heat. A small quantity
was therefore placed in a loosely covered crucible, and strongly
heated for half an hour. When cold it was tested for nitrogen
by heating with potassium hydroxide. Its vapors rapidly changed
litmus paper from red to blue. Upon the suggestion of Profes-
sor Tschirch a sample was thoroughly washed with water acid-
ulated with hydrochloric acid, but this failed to completely re-
move the nitrogen.
As zinc dust is manufactured by heating zinc oxide with coal,
it was believed that part of the nitrogen might consist of conden-
sation products from the coal. Therefore a sample was placed
in a long tube and percolated with ether. The ether when evap-
orated left a yellow, non-saponifiable oil, with an odor and fluor-
escence similar to petroleum. The oil, when heated with dry
potassium hydroxide, gave off alkaline vapors, and the zinc in
the percolator was still found to contain nitrogen. The greater
portion of the oil appeared to be removed with the first portion
of ether, but after continued percolation the ether left a residue
upon evaporation, and it was evident that a much larger amount
of ether was necessary for complete exhaustion ; therefore a small-
er sample, from a can of zine dust which had been in the labora-
tory for more than ten years, was treated with ether in the same
manner, and the powder tested from time to time. After using
a large amount of ether the zinc was practically free from nitro-
gen, yet by taking a large amount of the zinc and heating with
potassium hydroxide in a tube partially closed at the top so that
all of the vapors came in contact with the litmus paper, the color
was slightly changed, thus showing a mere trace of nitrogen.
This sample was then allowed to stand in an open flask for a few
days when it gave a decided ammonia reaction, thus showing that
zinc dust rapidly absorbs nitrogen from the air.
The fact that only a portion of the nitrogen in zinc dust is
removed by heat indicates that the nitrogen is present in more
than one form. This theory is also supported by the following
experiments :
A fresh sample of zinc dust was washed with water ,the
washings giving a decided ammonia test. The washing was
continued as long as traces of nitrogen could be detected in the
washings. It was then treated in the same manner with very
dilute hydrochloric acid. By adding potassium hydroxide in ex-
cess to the acid solution and allowing to stand a few minutes un-
til the precipitate settled, decanting the clear solution and boil-
ing, the vapors gave the odor of ammonia and rapidly changed
litmus from red to blue. Washing with acid was continued until
the washings no longer gave a test for nitrogen. The zinc was
then washed with water until free from acid, and rapidly dried
in a drying oven, and at once extracted with ether, the ether
evaporated and tested for nitrogen as above. Nitrogen was found
to be present, though not in as large amounts as in the oil from
the first sample examined, which was, however, directly treated
with ether.
Three samples were examined : one from a large closely
covered can which had been in use in the laboratory as above
stated ; another from a glass bottle which had been in the museum
about fifteen years, and a third which was ordered by Professor
Oesterle for these experiments. Practically the only difference
found in the three samples was that the oil from the fresh sample
was decidedly yellow, while that from the laboratory sample was
somewhat lighter, and that from the museum sample was colorless.
Dr. Victor Steger ("Metalldampfe in Zinkhiitten," Chem-
ischer und Chemischtechnischer Vortrage) gives the results of
several analyses of zinc dust, some of which contain considerable
insoluble residue consisting principally of carbon. To determine
to what extent this was present, a large amount of zinc dust
was treated with hydrochloric acid. At first the reaction was
rapid, but after a time ceased. The solution was decanted and
fresh acid added, but as the reaction was very weak the mixture
was heated. Even then a large amount remained undissolved.
A few drops of copper sulphate solution were added and digested
for several days, but a large amount remained insoluble. This
was washed with water until free from acid, dried, and per-
52
colated thoroughly with ether, which upon evaporation left a col-
orless oil. Upon removing the ether the zinc dissolved without
difficulty in hydrochloric acid, conclusively proving that this
sample contained no carbon, and that the insolubility was due
to the presence of the oil.
LAC POISONING.
Goertz* 3 gives the following description of lac poisoning in
which the idiosyncrasy of the individual plays an important
part. A few hours after the poisoning the patient complains of
an unpleasant tension of the skin, usually of the face, head and
extremities. Soon after there forms an oedema of the affected
parts. Small red points become visible, which look like fine rash.
These grow larger forming on the points small blisters containing
a watery fluid. The parts of the skin affected are restricted to
the head and extremities.
Ishimatsu states: 84 "It gives off a certain kind of volatile
acid, poisonous in its property, and some persons are seriously
attacked by it, producing great swellings on the face especially,
and even the whole body where the acid comes in contact. Dur-
ing my examination in the laboratory, one day one of the appara-
tus-keepers came in and was violently attacked by it, producing
ugly swellings all over his face. He told me at the time that it
was exceedingly itchy, and by using solution of acetate of lead,
chloride of potash and carbonate of soda, was said to have recov-
ered from his suffering within a week."
"The poison that is evolved from urushi acts only on certain
persons. I had to work with it for many days, yet never had any
attack of the kind nor felt any uneasiness by it."
Prof. J. J. Rein 86 describes the lac poisoning as follows :
"It is a peculiar, not very painful, and not at all fatal, but
always very disagreeable disease, always attacking one new to
the work, whether he be lac tapster, dealer, or lacquerer. It
appears in a mild reddening and swelling of the back of the
hands, the eylids, ears, the region of the navel and lower parts
of the body, especially the scrotum. In all these parts great heat
83 Ueber in Japan vorkommende Fish- und Lackvergiftungen. St.
Petersburger medicinische Wochenschrift, 1878, No. 12.
* Manchester Literary and Philos. Soc., 3 Ser. 7, p. 254.
85 The Industries of Japan, p. 349.
53
is felt and violent itching and burning, causing many sleepless
nights. In two or three days the crisis is reached, and the
swelling immediately subsides. In severe cases, small festering
boils form also. This lacquer disease is not only caused by
handling of the lac, but by its evaporation chiefly, especially that
of the sharp Se-shime, to which I owe my own illness."
"The poison, however, is a volatile substance, and has nothing
to do with the lac-acid and its higher oxidation, as Korschelt
believed. If the poisonous property disappears in the drying of
the plant, this amounts to nothing save that the volatile poison
fully escapes in this manner. A considerable part of it is driven
off in the preparation of the several kinds of lacquer, and by
stirring in open vessels. For this reason, the lacquers mixed
with colors are regarded far less dangerous than the raw lac
and its direct derivatives."
ForneP compares the above symptoms with those of poison-
ing by poison ivy and anarcardium, as well as the cases of poison-
ing of the laborers in the vanilla depot of Bordeaux" where
nearly all, even from the first day, experienced strong itching
accompanied with a burning sensation, especially on the face and
hands. He finds a remarkable similarity in all of these cases and
believes that he is justified in assuming that all are produced by
cardol. He also states that anacardium is used in the preparation
of Japanese lac, but I have been unable to find confirmation of
this fact.
Dr. Andreas 88 reports the case of a gardener in the botanical
garden at Vienna, who was poisoned while collecting and trans-
planting Rhus vernicifera. On the same day the face became
red, the skin inflamed, the eyelids, nose and cheeks swollen. The
reddening extended to the neck, breast, hands and forearms.
The genitals were also red and swollen. By dusting with starch
the inflammation disappeared in fourteen days.
When opening the cans care must be exercised to prevent the
vapors accumulated in the top of the can from coming in contact
with the face or hands, as the poisonous part of the lac is volatile
and may be removed by heating or by distillation. Yoshida also
states that the lac contains a volatile poison which is dissolved
with the urushic acid by alcohol but is almost completely driven
Archiv. fur Dermatologie und Syphilis, L,X, p. 249.
Revue d'Hygiene, Paris, 1883, p. 718.
Therap. Monotshifte, 1903, p. 165.
54
off by drying the acid at 105 to 110 C. Bertrand 89 says that
the lac must be handled with the greatest precaution because the
least traces in the state of vapor produce on the face, hands and
arms an intense rubrefaction accompanied by intense itching, and
adds that these malicious properties make the study of lac very
unpleasant, and he was obliged to interrupt his studies on account
of individual sensibilities.
With these statements before me it was not without mis-
givings that I undertook the study of lac, and these were not
allayed by my first experience. The first sample received was
in a glass can with metal top which had become sealed by the
lac, and was difficult to remove, but when finally started was
accompanied by a slight sound of escaping gas. In about thirty-
six hours an inflamed spot about 2 cm. by 5 cm. appeared on my
wrist ; it itched intensely for about a week and then disappeared.
Laboring under the supposition that I was dealing with a volatile
poison, I was extremely cautious not to come in contact with the
vapors in any form, but supposed that I was practically safe after
the alcohol had been distilled and the residue had been heated
for some time. While shaking out an ether solution of the
alcoholic residue with sodium carbonate solution, it was difficult
to keep the hands entirely free from the solution and no especial
pains were taken to remove it except to carefully wash with soap
and water. However, after working some time with it my face
began to swell and continued until my eyes were nearly closed.
It extended over hands, arms and limbs to the knees ; the desire
to scratch was very great so that it was almost impossible to
sleep. This was also true of the face and ears to some extent,
but here the sensation was more that of burning. After about a
week the face became normal and I was able to resume my work
but the limbs continued to itch and remained covered with a fine
rash. After several weeks I became convinced that the underwear
had absorbed some of the poison and though frequently washed
still retained it. Soft gauze underwear was then worn next the
skin, when the flesh soon became normal.
Various remedies were tried, such as, ointment of zinc
oxide ; a mixture of oxide of zinc, bismuth subnitrate, starch and
solution lead subacetate, tincture of iodine with glycerin ; solu-
tion of potassium permanganate ; solution of oxalic acid. None
Annales de Chimie et de Physique, Series XII, 1897.
55
of these seemed to give anything more than a temporary relief.
The best results were obtained by rubbing the surface with a little-
petrolatum and then scraping it off with a knife and washing the
surface with a weak solution of sodium hydroxide or carbonate.
The burning on the face was relieved by keeping it moistened
with a saturated solution of boric acid. >
Dr. Jadassohn, Professor of Skin Diseases in the University
of Bern, stated that the above symptoms did not prove that the
poisonous principle was volatile, and kindly volunteered to make
the physiological tests for me in order to determine whether the
poisonous principle is volatile or not. He found that the rabbit
was very sensitive to the poison. The method of testing was to
rub a small quantity of the substance on the inside of the ear for
2 or 3 minutes. If poisonous, inflammation appeared in from
i to 5 days and the surface soon became covered with watery
blisters followed, in severe cases by necrosis of the superficial
layers of the skin. This condition lasted about 14 days when it
gradually disappeared.
The following are the most important results obtained from
the tests :
1. Sterilized lac, prepared by suspending a tube of the lac in
boiling water for half an hour, was poisonous.
2. An alcoholic solution of the lac was distilled and the dis-
tillate tested but was not poisonous.
3. After the alcohol was removed, the distillation was con-
tinued when a small quantity of aqueous distillate was obtained,
but this was also inactive.
4. The residue in the retort was extremely poisonous.
5. A fresh can of lac was thoroughly cooled to prevent the
escape of gas while opening, two small openings made, and tubes
introduced. A small quantity of absorbent cotton was placed
in the tube, used for the exit of vapor, to prevent particles of the
fluid from being forced through. The vapor was then slowly
forced out of the can upon the ear of a rabbit. Part of the ear
had previously been moistened. The vapor was entirely without
action. Since then I have worked over the lac, while evaporating
it under all conditions without the slightest inconvenience.
6. The alcoholic residue was later separated into two parts,
one soluble and the other insoluble in benzin. The first was
- 5 6-
poisonous and the second non-poisonous. A thin layer of the
first was left in an open crystallizing jar for four months when
it was found to be still poisonous.
Another sample of five grams was left in an open vial on a
laboratory shelf for ten months, including the hot summer months.
This was then tested on my arm and was found to be still active.
These facts are sufficient to prove that the poisonous principle
is non-volatile. Doubtless the cases of poisoning that have oc-
curred from opening retainers have been due to minute particles
of the lac being forced out with the vapor.
The poison is extremely active even in minute quantities and,
as it forms a part of the resinous body, it is very difficult to remove
from the skin or clothing. Washing with soap and water is not
sufficient to insure its removal. If the hands after contact with
the lac are thoroughly washed with soap and water until they
are to all appearances clean, and then wet with a solution of caus-
tic alkali, black spots will appear wherever the lac has been in
contact. A mixture of powdered soap, pumice stone and sodium
carbonate gives the best result. However, to insure safety
I have usually followed this with soap and sand. The poison
seems to have little or no effect upon the thick skin on the inside
of the hand, but, to prevent its transmission to other parts, it should
be removed as soon as possible. For example, by accident some
of the benzin solution was thrown into one eye and over one
hand. The eye was thoroughly washed with benzin and alcohol,
but in my anxiety for the eye, the hand was forgotten for twen-
ty or thirty minutes, when it was thoroughly washed with ben-
zin and alcohol followed by soap and sand. The eye escaped
without further inconvenience than that caused by the benzin,
but in thirty-six hours the surface of the hand became slightly
swollen, itched considerably for a week and then appeared to
be covered with a thin dry scale, which finally disappeared. Since
then I have tested different parts of the substance to determine
whether or not they were poisonous, by cutting a hole 6 mm. in
diameter in a piece of gum paper, pasting this on the arm and
applying the substance to the opening. In from thirty minutes
to one hour the paper was removed and the spot washed with
ether or benzin. When the substance was poisonous the spot
became red and began to itch within 30 hours. From three to
five vescicles usually appeared. The itching was not intense,
57
usually lasting only a few minutes at a time. A dry scale formed
over the surface and remained for several weeks after all irrita-
tion ceased.
In no case did the poisonous action extend beyond the sur-
face to which it was applied, thus proving that the action is en-
tirely local. If the surface, which has been in contact with the
poison, is not thoroughly washed with some solvent like alcohol,
benzin, ether or kerosene, the poison will be transmitted to other
parts of the body As all kinds of fats and oils are solvents for the
poison they should not be used as remedies. Should pustules form,
the surface should be frequently washed to prevent the serum from
being conveyed to other parts, as it is quite possible that it may
be active. Experiments to determine this fact will be conducted
in the near future.
The poison has not at the present time been isolated in a
pure condition.
Dr. Jadassohn and his assistants, Drs, Winckler and Schulz,
made 26 tests with parts of the lac obtained under different con-
ditions.
Only that portion which is completely soluble in benzin is
poisonous, and this, we have previously seen, was separated
by shaking out the benzin solution with alcohol, into two parts,
one soluble in alcohol and poisonous, the other insoluble in alco-
hol but soluble in benzin and non-poisonous. I have elsewhere
stated that by fractional precipitation with lead acetate a partial
separation of the poison was obtained, but that I did not consider
it a practical method.
After the above experiments with the poison were made I
received from Dr. F. Pfaff a reprint of his article "On the Ac-
tive Principle of Rhus Toxicodendron and Rhus Venenata." 90 As
the poisonous action of these plants is practically identical with
that of Rhus vernicifera, his work is of special interest in this
connection. He has conclusively proved that the poisonous prin-
ciple of poison ivy is non-volatile, thus> shatter'"^ the false idea
that has existed for so many years. He claims to have separated
the poisonous principle in a pure form by fractional precipitation
with lead acetate as an oil. Dr. Pfaff gives the composition of
his lead compound as C 21 H 30 O 4 Pb 6 and proposes the name "Tox-
10 The Journal of Experimental Medicine, Vol. II, No. 2, p. 181,
1897.
- 5 8-
icodendrol" as the name of the poisonous principle. The poison-
ous principle of Japanese lac is so intimately associated with the
resin of the lac that I have not considered the method of fraction-
al precipitation to be a complete separation. Preceding investiga-
tions indicate that the poisonous principles of these plants are
identical but further investigation is necessary before this can be
accepted as conclusive. I hope during the coming year to be able
to separate the poison from both these plants and to determine
their relation.
The present researches in Japanese Lac were undertaken in
the Laboratory of the Pharmaceutical Institute of Bern under
the guidance of my most highly esteemed director, Professor A.
Tschirch. To him and also to Professor Oesterle I desire to ex-
press my warmest and sincerest thanks for the inspiration and
the friendly interest and advice which has ever been so freely
and so kindly given.
I am also thankful to Dr. Jadassohn and his assistants, Drs.
Winckler and Schulz for the physiological tests which they so
kindly made.
The lac for this investigation was kindly presented by fores-
ter Shirasawa of Tokio, Japan, and the Rhus Company, Frank-
fort, Germany. To them I extend sincere thanks.
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