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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 OCT281998 
 
 BERKELEY 
 
 20 
 
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 l/n-6,