SOME CONSTITUENTS OF THE POISON IVY PLANT
(RHUS TOXICODENDRON)

DISSERTATION

SUBMITTED TO THE BOARD OF UNIVERSITY STUDIES OF THE JOHNS HOPKINS UNIVERSITY IN CONFORMITY WITH THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

BY WILLIAM ANDERSON SYME

1906

1906
THE SUN JOB PRINTING OFFICE
BALTIMORE

CONTENTS.

Acknowledgments 4

Literature 5

Introduction 7

Work of Khittel 11

Work of Maisch 12

Work of Pfaff 13

Experimental 14

Gallic Acid 18

Fisetin 20

Rhamnose 23

The Poison 28

Potassium Permanganate as a Remedy for Rhus Poisoning 35

Summary 37

Biography 38

ACKNOWLEDGMENTS.

The author desires to avail himself of this opportunity to tender his thanks to those under whose guidance he has worked while a student at the Johns Hopkins University, namely to Professors Remsen, Morse, Jones, and Andrews, and to Doctors Acree and Tingle for instruction in lecture room and laboratory.

He is especially indebted to Dr. S. F. Acree, at whose suggestion this research work was undertaken, for counsel and assistance in its prosecution.

He would also thank Messrs. Parke, Davis and Co., of Detroit, Mich., for the preparation of the crude material used in this investigation, and the U. S. Department of Agriculture, Washington, D. C., for electrotypes of figures 17, 18, and 19 in Bulletin No. 20, Division of Botany.

LITERATURE.

Acides Gummiques, Garros (Dissertation) 1895.

American Chemical Journal.

American Journal of the Medical Sciences.

American Journal of Pharmacy.

Annalen der Chemie und der Pharmacie (Liebig).

Annales de Chimie et de Physique.

Berichte der deutschen chemischen Gesellschaft.

Biochemie der Pflanzen (Czapek) 1905.

Brooklyn Medical Journal.

Bulletin de la Société Chimique.

Bulletins 20 and 26 U. S. Department of Agriculture, Division of Botany.

Chemie der Zuckerarten, Von Lippmann, 1904.

Chemiker-Zeitung.

Comptes rendus.

Industries of Japan, J. J. Rein.

Journal of the Chemical Society.

Journal of Experimental Medicine.

Les Sucres, Maquenne, 1900.

Manual of Botany, 6th Edition, Gray.

Medical and Surgical Reporter.

New York Medical Record.

Proceedings of the American Pharmaceutical Association.

Treatise on Chemistry, Roscoe and Schorlemmer.

Über Mategerbstoff, Reuchlin (Dissertation) 1904.

SOME CONSTITUENTS OF THE POISON IVY PLANT.

(RHUS TOXICODENDRON)

INTRODUCTION.

Plants belonging to the natural order Anacardiaciæ (Cashew family or Sumach family) are found in all the temperate climates of the world and quite frequently in semi-tropical climates. Many of these plants play important parts in economic botany, yielding dye-stuffs, tanning material, wax, varnish, and drugs. Several species are poisonous. At least three poisonous species of the genus Rhus are found in the United States. These three are all common and well-known plants, but confusion frequently arises concerning them on account of the different names by which they are known in different localities. For example, poison ivy (Rhus toxicodendron or Rhus radicans) probably the best known poisonous plant in America, being found in all the States except those in the extreme West, is often confounded with and popularly called "poison oak." The true poison oak is the Rhus diversiloba of the Western States.[1] The third and most poisonous species of this plant is Rhus venenata or Rhus vernix; it is the Rhus vernicifera of Japan, from which Japanese lac is obtained. It is popularly known in the United States as "poison sumach," "poison dogwood" and "poison elder." It grows in swamps from Canada to Florida.

As the poison ivy is by far the most common of these plants in the Eastern States, a brief description of it is given here:[2] A shrub climbing by rootlets over rocks, etc., or ascending trees, or sometimes low and erect; leaflets 3, rhombic-ovate, mostly pointed, and rather downy beneath, variously notched, sinuate, or cut-lobed; high climbing plants (R. radicans) having usually more entire leaves. It is found in thickets, low grounds, etc. Greenish flowers appear in June.

In the general description of the order Anacardiaciæ, Gray[3] says: "Juice or exhalations often poisonous." Whether it is contact with some part of the plant, or with the exhalation from the plant, that causes the well-known skin eruption has been a topic for discussion ever since its source was known. On account of its intangible nature there has been more speculation than experimental evidence bearing on this question, although a few investigations have been made with the object of isolating the poison. It is most generally believed that the exhalations are poisonous. Dr. J. H. Hunt[4] states that the exhalations have been collected in a jar and found to be capable of inflaming and blistering the skin of an arm plunged into it.

Prof. J. J. Rein,[5] in his treatise on Lacquer Work, describes the poison of the Japanese lac tree, Rhus vernicifera, as being volatile, as do also the Japanese chemist Yoshida[6] and the French chemist Bertrand.[7] Recent work by Prof. A. B. Stevens,[8] however, seems to show that this poison is not volatile, and is similar to, if not identical with that obtained by Pfaff[9] from Rhus toxicodendron and Rhus venenata.

Not many cases of internal poisoning by Rhus toxicodendron are on record in medical literature. Two cases of poisoning from eating the fruit of this plant have been described.[10] The subjects of these cases were two children who had eaten nearly a pint of the fruit. The symptoms are described in detail, being in general, similar to those of alkaloidal poisoning. Warm water was given to promote emesis; afterwards large quantities of carbonate of soda were given in solution under the belief that it was an antidote to the poison. Otherwise they were treated on general principles. Both children recovered.

Another case of internal poisoning is the following:[11] Three children drank an infusion of the root of poison ivy thinking it was sassafras tea. The first of these cases was diagnosed as measles, but on the appearance of similar symptoms in the sisters of the first patient, the cause of the trouble was found. All recovered.

Dr. Pfaff[12] explains the few fatal cases that have followed Rhus poisoning on the assumption that enough of the poison was absorbed through the skin to cause renal complications in persons having chronic kidney trouble. He showed that the poison, when given internally, produces a marked effect on the kidneys, causing nephritis and fatty degeneration of this organ.

The irritating action of poison ivy has been attributed at different times to the "exhalation," to a volatile alkaloid, to a volatile acid, and to a non-volatile oil. Pfaff,[13] who made the most recent investigation of this poison, obtained from the plant a non-volatile oil having the same action on the skin as the plant itself. He found this oil in all parts of the plant and concluded that it was the active principle, and that one could be poisoned only by actual contact with some part of the plant. He assumed minute quantities of pollen dust to be in the air to account for the cases of "action at a distance" so frequently quoted. Pfaff says: "In my opinion, it is more than doubtful if ever a case of ivy poisoning has occurred without direct contact with the plant or with some article that has been in contact with the plant. The long latent period of the eruption in some cases may obviously render mistakes extremely easy as to the occasion when contact with the plant really occurred." Granting, however, that the active principle is practically non-volatile when isolated from the plant, we cannot say positively that it is not volatile in the juices of the plant, or under the influence of vital forces. It is quite conceivable that the water transpired by the leaves of the plant may carry with it a quantity of the poison sufficient to produce the dermatitis on a person very susceptible to its action. It is also conceivable that a volatile poison manufactured by a living plant could become non-volatile by changes in it consequent upon the death of the plant.

Up to the present time, only three important chemical investigations of the active principle of Rhus toxicodendron have appeared in medical and chemical literature, these being the researches of Dr. J. Khittel, J. M. Maisch, a pharmacist, and Dr. Franz Pfaff, of the Harvard University Medical School, to whose work reference has been frequently made. The chemical work of these investigators and their conclusions are given here in some detail for the sake of completeness.

KHITTEL'S INVESTIGATION.

The first attempt to find the poisonous constituent of this plant was made by Khittel in 1857. His work was published in Wittstein's Vierteljahrresschrift für praktische Pharmacie, VII, 348-359.[14] Khittel obtained 37-1/2 ounces of fresh leaves of poison ivy from the botanical garden in Munich, dried them, and got a residue of 9-1/2 ounces which he analyzed. Not detecting anything to which the poisonous qualities of the plant could be attributed, he made another series of experiments which, as he thought, showed that a volatile alkaloid is the poisonous constituent. It was obtained by the following process: "3 ounces of the powdered leaves were infused with hot distilled water, after three days strained, expressed, the liquid evaporated to 3 ounces, and with the addition of potassa, carefully distilled to one-half. The clear, colorless distillate had an alkaline reaction, and an odor resembling henbane or hemlock. It was saturated with sulphuric acid, evaporated, and treated with a mixture of equal quantities of alcohol and ether which left sulphate of ammonia behind, the solution was evaporated spontaneously, distilled with potassa, the alkaline distillate neutralized with hydrochloric acid, and a precipitate could now be obtained with chloride of platinum. Want of material prevented further experiments."

The editor of the American Journal of Pharmacy inserts the following note: "It would have been more satisfactory if the author had given some physiological evidence of the poisonous nature of the alkaloid substance obtained. It is quite interesting to hear that the hitherto intangible venom of this plant has at last been detected."

WORK OF MAISCH.[15]

The next investigation of this plant was made by Maisch in 1864. He criticizes Khittel's experiments as follows: "It is well known that the exhalations of Rhus toxicodendron exert a poisonous influence on the human body; the poisonous principle must, therefore, be volatile and, at the same time, be naturally in such a loose state of combination as to be continually eliminated and separated with the usual products of vegetable exhalations. It is natural to suppose that during the process of drying, the greatest portion of the poisonous principle should be lost. The loss must be still greater if the dried leaves are powdered, a hot infusion prepared from them, and this infusion evaporated down to the original weight of the dried leaves. It is obvious that Dr. Khittel could not have selected a better method for obtaining the least possible quantity of the poisonous principle, if, indeed, it could be obtained by this process at all."

Maisch then worked up 8-3/4 ounces of the leaves of the plant in a way to get the alkaloid, making some improvements on Khittel's method, but failed to find it. Believing that the poison was a volatile acid, he enclosed some fresh leaves of the plant in a tin box with several test papers. The blue litmus paper became red showing the presence of an acid. He concluded from this experiment that the exhalations of the leaves contained a volatile organic acid which he thought was the poisonous substance. To determine this point, he prepared the acid in larger quantity by macerating the leaves with water, expressing and distilling the expressed juice. He was poisoned in doing this work although he had not been affected by handling the living plant and had considered himself immune. He obtained an acid which investigation showed to be somewhat like formic acid, more like acetic acid, but having some reactions different from both. "Taking all the reactions together, it is unquestionably a new organic acid for which I propose the name of Toxicodendric Acid," writes Maisch. He further says: "That it is the principle to which poison oak owes its effects on the human system was proved to my entire satisfaction by the copious eruption and formation of numerous vesicles on the back of my hand, on the fingers, wrists, and bare arms while I was distilling and operating with it. Several persons coming into the room while I was engaged with it were more or less poisoned by the vapours diffused in the room; and I even transferred the poisonous effects to some persons, merely by shaking hands with them.

"The diluted acid, as obtained by me, and stronger solutions of its salts, were applied to several persons, and eruptions were produced in several instances, probably by the former, though not always, which was most likely owing to the dilute state of the acid. Whenever this was boiled, I always felt the same itching sensation in the face, and on the bare arms, which I experience on continual exposure of my hands to the juice of the plant."

Toxicodendric acid was thought to be the active principle from the time of Maisch's work until the investigation by Pfaff in 1895.

PFAFF'S WORK.

By far the most valuable work on Rhus toxicodendron is that of Pfaff. From a clinical study of Rhus poisoning, Pfaff came to the conclusion that the poison must be a non-volatile skin irritant. The more volatile the irritant, the quicker is its action on the skin. Formic acid acts very quickly; acetic acid, less volatile than formic, acts more slowly, but still much more quickly than poison ivy, the latent period of which is usually from two to five days. Pfaff thought that the volatile acid obtained by Maisch might have contained some of the poisonous principle as an impurity, but that it would not produce the dermatitis if prepared in a pure state. He therefore prepared a quantity of the acid by distilling the finely divided fresh plant with steam. The yield was increased by acidulating the mixture with sulphuric acid before the distillation. The acid distillate so obtained was freed from a non-poisonous oily substance by shaking the solution with ether. Barium and sodium salts were made by neutralizing the acid, and were purified by crystallization. Analysis showed them to be salts of acetic acid, and they gave the characteristic tests for this acid. The toxicodendric acid of Maisch was thus shown to be acetic acid, and was therefore not the poisonous principle of the plant.

Pfaff obtained the active principle by the following process: The plant was extracted with alcohol, the alcohol was distilled off, and the residue was taken up in ether. The ether solution was washed with water and dilute sodium carbonate solution, and the ether was evaporated. An oily, black, poisonous substance partly soluble in alcohol was obtained. To get the active principle in a pure state, this residue was extracted with alcohol and filtered and the filtrate was precipitated fractionally by lead acetate. The final precipitates consisted of the lead compound of the poison in a pure state. On decomposing the lead compounds with ammonium sulphide, shaking out with ether, and letting the ether evaporate spontaneously, a non-volatile oil was obtained which gave the characteristic skin eruptions. The pure lead compounds made in different preparations were analyzed and assigned the formula C₂₁H₃₀O₄Pb. The oil itself was not analyzed. Pfaff proposed the name Toxicodendrol for the oil. He found that it was not volatile, was decomposed by heat, was soluble in alcohol, ether, chloroform, benzene, etc., but insoluble in water. Its effects upon the human skin were studied in many experiments upon himself and others. It was shown that an exceedingly minute quantity of the poison will produce the dermatitis, even 1/1000 milligram applied in olive oil being active. The oil was given internally to rabbits, its effects being most marked on the kidneys.

The oil obtained by Pfaff from Rhus venenata seemed to be identical with that from Rhus toxicodendron.

EXPERIMENTAL.

The writer's investigation was undertaken with the object of attempting to throw more light on the chemical nature of the poisonous substance found in Rhus toxicodendron. Soon after commencing work, however, it became apparent that the poison could be more intelligently studied if the substances associated with it in the plant were first identified; the scope of the work was therefore extended to an investigation of the other constituents of the plant, and it was hoped that a knowledge of the properties of these constituents would suggest a more economical way of getting the poison than the method of fractional precipitation.

The crude material for this work was prepared by Messrs. Parke, Davis & Co., of Detroit, Mich., according to special instructions submitted to them: 67-1/2 pounds of fresh leaves and flowers of poison ivy were collected near Detroit and carefully inspected by a competent botanist. This material was thoroughly macerated and put into ten-liter bottles with ether. The mass was thoroughly shaken, water being added to make it more mobile. The ether was then separated off and the extraction was repeated three times in the same way to insure complete removal of the toxicodendrol. The ether extracts were combined, thoroughly dried with anhydrous sodium sulphate, and the ether was distilled off, the temperature being kept below 40° C. during the entire distillation. The residue after the removal of the ether was a thick, black, tar-like mass, weighing 3 pounds 11 ounces. In extracting the plant, about twenty-four gallons of ether were used. It is a significant fact in regard to the volatility of the poison that during the process of preparing this material none of the employees engaged in the work were in any way affected, since proper precautions were taken and the utensils were handled with rubber gloves.

The crude ether extract, which will be designated as the "original material," was shipped to Baltimore in August and was kept in a cool place until November when the investigation was begun. When the bottle was opened, there seemed to be an escape of a vapor, and a nauseating odor suggesting crushed green leaves pervaded the atmosphere. Some days later, irregular red patches appeared on the face though a mask of cotton cloth was worn during the work, and the hands were protected by rubber gloves.

Assuming from Pfaff's work that this original material contained the non-volatile oil toxicodendrol, the first experiment was to try to distil it out under diminished pressure. For this purpose, an Anschütze distilling bulb containing ten grams of the tar was connected with a vacuum pump. After a pressure of 2 mm. had been established the bulb was gradually heated in a bath of Wood's metal. Nothing distilled over. The material began to carbonize at a temperature of 140° to 150°.

It was then thought that perhaps the oil could be converted into an ester which might be more volatile and could be distilled out. 20 grams of the original material were dissolved in 100 cc. of absolute alcohol containing 3 grains of hydrochloric acid gas, and the mixture was heated 10 hours on a water-bath under a return condenser. After the heating, the mixture had a delightful ethereal odor. The flask was corked and left standing several weeks while other work was in progress. The ester solution was then put in a vacuum desiccator over sulphuric acid and the alcohol evaporated. A black, tarry, solid mass was left having the ester odor. It was extracted with warm water and filtered from insoluble tar. The filtrate had a green color and the ethereal odor. It was shaken out with ether; the ether layer had a blood-red color while the water layer was deep green. The extraction with ether was continued until the water layer was no longer green. The combined ether extracts were evaporated in a desiccator without heat. A black tar-like solid was left very much like the original material, but it had the ester odor. It was partly soluble in water and readily soluble in alcohol. The alcoholic solution was tested on the skin and found to be not poisonous. The ester, or mixture of esters, was not investigated further in this connection, but was later shown to give the reactions for gallic acid and methyl furfurol. These reactions will be referred to in connection with other experiments.

After a few other preliminary experiments, it became evident that the original material was a complex mixture of substances and that it would have to be fractionated by some means and the fractions studied separately.

A portion of the original substance was treated with 50 per cent. alcohol and was found to be partly soluble in this medium. The solution was filtered from insoluble tar. A portion of the yellow filtrate gave a reddish yellow precipitate with lead acetate. The alcoholic solution was distilled in an Anschütze flask under diminished pressure; a yellow liquid condensed in the arm of the flask while most of the alcohol was collected in a bottle connected with the arm. The yellow liquid was acid to litmus. Water was added, the solution was shaken out with ether and the ether was evaporated. When the small residue was completely dry, it was a yellow solid soluble in dilute alcohol and acid to litmus. The substance was not volatile enough to justify the use of this method for getting it.

Chlorophyll could not be removed from the original substance because the solvents for chlorophyll such as alcohol, ether, fats, petroleum, and carbon bisulphide dissolve large quantities of the mixture.

A precipitate obtained by adding lead acetate to a filtered solution of the original substance in 50 per cent. alcohol was suspended in water, decomposed by hydrogen sulphide, shaken out with ether and the ether evaporated. The residue appeared at first to be a yellow oil, but on complete evaporation of the ether in a desiccator, a yellow solid was obtained—apparently the same as that obtained by vacuum distillation.

A solution of the original material in 50 per cent. alcohol was filtered through bone-black and the filtrate was colorless. Examination showed that everything had been removed by the bone-black and the filtrate was apparently pure alcohol and water.

In precipitating an alcoholic solution of the crude material with a solution of lead acetate, it was noticed that at least two kinds of precipitates were formed. The part that went down first was darker in color than that thrown down later. Pfaff used the last fractions in obtaining his oil and stated that these precipitates consisted of the lead compound of the oil in a pure state. It was found by experiment that the darker part was soluble in ether while the lighter part was not. This indicated that the darker part consisted of tarry matter which was brought down mechanically or separated out when the alcoholic solution was diluted by the lead acetate solution, or was perhaps a lead compound soluble in ether. To test this point an experiment was made as follows: Some of the crude material was thoroughly extracted with 50 per cent. alcohol. The tar insoluble in 50 per cent. alcohol was then treated with 95 per cent. alcohol; most of it dissolved; the solution was filtered and lead acetate in 50 per cent. alcohol was added. A greenish colored precipitate was formed which was filtered off and found to be completely soluble in ether and soluble to a considerable extent in strong alcohol. These experiments suggested that the light colored lead compound which was thought to contain the poison could be purified by extraction with ether in a Soxhlet apparatus more conveniently than by the tedious process of fractional precipitation. Further preliminary experiments showed that 50 per cent. alcohol extracted from the original material all of the substance or substances which gave the light colored precipitate and dissolved only a small amount of the tar.

Two hundred and eighty-eight grams of the crude material were then extracted several times with 50 per cent. alcohol and filtered; the insoluble tar was washed and saved for examination. To the filtrate was added an excess of a solution of lead acetate in 50 per cent. alcohol. The large precipitate, which will be designated as "precipitate A," was filtered and drained by suction in a Büchner funnel. The alcoholic "filtrate A" was saved. Precipitate A was extracted with ether in Soxhlet extractors until the ether came over practically colorless, the operation being interrupted from time to time to stir up the precipitate in the thimble. The green colored ether solution was saved for future examination. The lead precipitate, after extraction with ether and drying, weighed about 116 grams. A portion of this lead compound was decomposed by hydrogen sulphide in a mixture of water and ether which was well shaken during the operation. The ether was separated, filtered, and evaporated under diminished pressure without heat, and there remained a yellow oily looking residue having a pleasant odor. When the ether and water were completely removed in a vacuum desiccator, a hard, brittle, yellow resin weighing about 16 grams was obtained. It was soluble in alcohol, had a strong acid reaction and was free from nitrogen[16] and sulphur. The nitrogen tests were made by the Lassaign and soda lime methods,[17] and the sulphur test was made with sodium nitroprusside after fusing the residue with sodium. The main portion of the lead compound was decomposed under alcohol by hydrogen sulphide, filtered, and the alcoholic filtrate evaporated in vacuo. The same yellow acid resin was obtained. Experiments continuing through several weeks were made in applying solutions of this resin to rats, rabbits and guinea pigs. Finding it to be without effect upon these animals it was tried on the writer and found to be not poisonous.[18] In the meantime the resin was being studied in the laboratory.

GALLIC ACID.

An alcoholic solution of the resin was just neutralized with potassium hydroxide. During the titration, the solution rapidly became dark brown. After neutralization it was shaken with ether; the water solution remained brown while the ether layer was nearly colorless and contained practically no dissolved substance. A portion of the water solution of the potassium salt on being acidified with sulphuric acid and standing over night, deposited a slight precipitate. The solution of the potassium salt gave a heavy precipitate with lead acetate somewhat similar to the original lead precipitate A, and also slight precipitates with salts of zinc, mercury, copper, and silver (with reduction). It gave a bluish-black color with impure ferrous sulphate and a dark color with ferric chloride. It reduced ammoniacal silver nitrate and Fehling solution. These experiments indicated the presence of a tannin compound. An alcoholic solution of the resin gave the same color reactions with iron salts as did the potassium salt. To determine which one of the tannin compounds was present was a matter of some difficulty since the di- and tri-hydroxybenzoic acids have in general the same color reactions. The presence of other plant substances in the solution also interferes with the color tests, and finally, a substance which gives a blue color with iron salts and one giving a green color may be found together in the same plant.[19] Further tests with a solution of the resin in dilute alcohol, and with a water solution of the acid precipitated by adding sulphuric acid to a solution of the resin in potassium hydroxide, led to the conclusion that the acid is gallic acid. These tests were the following:

(1) Boiling with an excess of potassium hydroxide gave a black substance (tauromelanic acid).

(2) The acid was not precipitated by gelatin.

(3) On addition of potassium cyanide a transitory red color appeared which reappeared on shaking with air.

Gallic acid is distinguished from tannic acid by tests (2) and (3). At later stages in the work the potassium, barium, and sodium salts of gallic acid were obtained, and finally the pure acid was made by decomposing the sodium salt with sulphuric acid and crystallizing from water. A portion of the acid so obtained was further purified by dissolving in absolute alcohol and pouring into absolute ether.[20] The melting point behavior of the acid corresponds with that of gallic acid; it melted with decomposition at about 230°. For further identification, some of the acid was converted into an ester by the following process: it was dissolved in 80 per cent. alcohol, hydrochloric acid gas was passed in, and the solution was heated an hour on the water bath. It was then evaporated to a small bulk, neutralized with barium carbonate and extracted with ether. The ether, on evaporation, left the ester which was crystallized from water and dried in a desiccator over sulphuric acid. The anhydrous ester agreed in melting point (156° to 159°) and other properties with the ester of gallic acid described by Grimaux.[21] For the sake of comparison, an ester was made from gallic acid obtained from another source and the two agreed in properties. A mixture of the two esters melted within the limits given for the ester of gallic acid.

While the tests leading to the identification of gallic acid were being made, another series of experiments was in progress. Eleven and one-half grams of the resin obtained from lead precipitate A by decomposition with hydrogen sulphide were treated with 0.1 n. potassium hydroxide added from a burette until the acid was exactly neutralized. All went into solution. On shaking with ether, some of the potassium salt separated out and was saved for examination. The solution became brown on exposure to air and got darker as the work proceeded. The acid in solution as a potassium salt was precipitated out in four fractions by adding for each fraction one-fourth the amount of 0.1 n. sulphuric acid required to neutralize the potassium hydroxide used. The precipitates were filtered off and examined. The first was small in amount, gummy and hard to filter. The solution was shaken with ether after each precipitate had been filtered off. The succeeding precipitates were in better condition, but were not pure. All appeared to be impure gallic acid which had become brown by absorption of oxygen. They were saved, however, to be tested for poison. After the last fraction had separated, the filtrate was shaken several times with ether and saved for further examination, which will be described under "Rhamnose." This filtrate is designated as B.

At this stage of the work a portion of the resin obtained from lead precipitate A was tested and found to be not poisonous as already mentioned. By this test, all the substances contained in the lead precipitate A after its extraction with ether in the Soxhlet apparatus, were eliminated from the possible poisonous substances. The poison must therefore have been extracted by the ether.

A fresh portion of the original poisonous material was treated with 50 per cent. alcohol and filtered from insoluble tar. The filtrate was precipitated in six fractions by lead acetate. The last fractions were lighter in color and apparently much purer than the first. The sixth lead precipitate was decomposed by hydrogen sulphide, the light-yellow water solution was tested and found to be not poisonous. It gave the characteristic reactions for gallic acid. The poison, if precipitated at all by lead acetate, must have gone down in one of the preceding fractions. Later experiments showed that it is brought down partly mechanically and partly as a lead compound in the first precipitates.

FISETIN.

Having identified gallic acid, and not finding any other phenol derivative in the lead precipitate, some of the original material was extracted with hot water to remove gallic acid and filtered from tar while hot. The filtrate had a deep yellow color. On cooling over night, an olive green precipitate separated out which was dried and found to be a light powder. It was practically insoluble in cold water, soluble with great difficulty in boiling water from which it separated in yellow flakes, slightly soluble in ether and in acetic acid, but readily soluble in alcohol. The solutions were not acid to litmus, gave a dark color with ferric chloride, an orange-red precipitate with lead acetate which was easily soluble in acetic acid, and an orange-yellow precipitate with stannous chloride. These properties and reactions indicated that the substance was the dye-stuff fisetin and that it occurs in the free state in this plant though it is usually found as a glucoside of fisetin combined with tannic acid. A compound of this kind was found in Rhus cotinus and named "fustin-tannide" by Schmid[22]. He showed that the fustin-tannide could be decomposed by acetic acid into tannic acid and a glucoside, fustin C₄₆H₄₂O₂₁. Fustin, on heating with dilute sulphuric acid, gave fisetin and a sugar supposed to be rhamnose. Fisetin was also found as a glucoside compound in Rhus rhodanthema by Perkin.[23]

The yellow substance which separated from the boiling water solution was further purified by dissolving in a small quantity of hot alcohol and adding hot water. On cooling, the yellow substance separated out in a flocculent condition. Examined under the microscope, the flakes appeared to be made up of masses of fine crystals.

An alcoholic solution of the substance gave a black color with ammonia which became red on addition of more ammonia. Concentrated acids intensified the yellow color of the alcoholic solution. Fehling solution and ammoniacal silver nitrate were reduced by it. Potassium hydroxide added to an alcoholic solution gave at first a deep red color accompanied by a green fluorescence which disappeared, leaving a yellow liquid. With an excess of caustic potash, the red color returned and was permanent. These reactions are characteristic for fisetin.[24]

Furthermore, fisetin should give protocatechuic acid and phloroglucinol by fusion with caustic potash under proper conditions.[25] The experiment was carried out as follows: 2 grams of fisetin were gently heated in a nickel crucible with 6 grams of caustic potash dissolved in 6 cc. water. An inflammable gas, apparently hydrogen, was evolved during the fusion. The pasty mass was dissolved in water, acidified with sulphuric acid, and filtered. The filtrate was shaken out with ether containing one-fourth its volume of alcohol. The ether was evaporated and the residue was extracted with warm water and filtered. Lead acetate was added to the filtrate to precipitate protocatechuic acid, while phloroglucinol remained in the filtrate from this precipitate. The lead precipitate was suspended in water, decomposed by hydrogen sulphide, filtered, and evaporated to obtain protocatechuic acid. That the substance obtained was protocatechuic acid was shown by the following characteristic tests:

(1) It gave a greenish brown color with ferric chloride; on addition of one drop of a dilute solution of sodium carbonate, the color became dark blue; on adding more sodium carbonate the color became red.

(2) A violet color was obtained when a solution of the acid was treated with a drop of sodium carbonate solution and then with a drop of ferrous sulphate.

(3) It reduced ammoniacal silver nitrate.

(4) It did not reduce Fehling solution.

The filtrate supposed to contain phloroglucinol was treated with hydrogen sulphide to remove lead, filtered, and shaken with ether. The residue left on evaporating the ether was taken up in water. This solution gave the following reactions characteristic for phloroglucinol:

(1) It reduced both silver nitrate and Fehling solution.

(2) It colored pine wood moistened with hydrochloric acid red.

(3) It gave a red color with vanillin and hydrochloric acid, and

(4) A deeper red color with oil of cloves and hydrochloric acid, becoming purple on standing.

(5) It gave a violet color with ferric chloride.

The substance is then, without doubt, fisetin. The formula[26] of fisetin is supposed to be