SOIffi CONETITUEITTS OF THE POISON IVY PLANT . (RHUS TOZICODMDROn) 000™ BISSERTATION —COO Submitted to the Board of University Studies of the Johns Hopkins University in Conforniity with the Re- quirements for the Degree of Doctor of Philosophy. By William Anderson Symc. 1906. h^^'ll CONTENTS. Page g AclaiowledgTnents - Literature 6' Introduction • Work of Khittel '' /I Work of Maisch- Work of Pfaff '^ Experimental'; Gallic Acid — ^ Fisetin °^^ Rhamnose •* The Poison- ^D Potassium Permanganate as a j-^ Remedy for Rhus Poisoning - Biography ACKNOWIEDGIIEHTS. 0000 The author desires to avail himself of this oppor- tunity to tender his thanks to those under whose guidance he has vrorked while a student at the Johns Hopkins Uni- versity, namely to Professors Remsen» Morse, Jones, An- drews, and to Doctors TJ.ngle and Acree for inct ruction 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 ma- terial used in this investigation, and the U. S. Depart- ment of Agriculture, Washington, D. C. for electrotypes of figures 17, 18, and 19 in Bulletin Ho. 20, Division of Botany, -3- LITERATURE. -_-000 — Acides Gummiques, Garros (Dissertation) 1895. American CShemical Journal. American Journal of the Medical Sciences. American Journal of Pharmacy. Annalen der Ghemie und der Pharmacie (liebig). Annales de Chimie et de Phycique. Beriohte der deutschen chemischen Gesellschaft. Biochemie der Pflanzen (Czapek) 1905. Brooklyn Medical Journal. Bulletin de la Societe Chimique. Bulletins 20 end 26 U. S. Department of Agriculture, Division of Botany, Ghemie der Zuckerarten, Von lippmann, 1906. Chemike r-Zeitung, Comptes rendus. Industries of Japan, J. J. Rein. Journal of the Ghemical Society. Journal of Eperimental Medicine. Les Sucres, Maquenne, 1900, Manual of Botany, 6th Edition, Gray, Medical and Surgical Reporter, ITew York Medical Record. Proceedings of the American Pharmaceutical Association, Treatise on Chemistry, Roscoe and Schorl emmer, Ueber Mategerhstoff , Reuchlin, (Dissertation) 1904, -4- " INTRODUCTION, —coo- Plants belonging to the natural order Anacardiaoiae (Cashew :^ily or Sumach family) are found in all the tem- perate oliinates 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 3tates 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. The third and most poisonous species of this plant is Rhus veneiyata or Rhus vemix; it is the Rhus vemicifera of Japan, from which Japanese lao is obtained. It is popularly known in the United States as "poison elder". It grows in swamps from Canada to Florida, 1. Chesnut. Bull. Ho, 20, U. 3. Dept. of Agr. , Div, of Botany. -5- ,-^ £^.1 As the poison ivy is by far the most coiamon of these plants in the Eastern States, a brief description of it is given here: A shrub climing by rootlets over rocks, etc., xa or ascending trees, or sometimes low and erect; leaflets 3, rhombic-ovate, mostly pointed, and rather downy beneath, variously notched, sinuate, or cut-labed - high climing plants (R radioans) having usually more entire leaves. It is found in thickets, low grounds, etc.. Greenish flowers appear in June. (Space for cuts) -U.:^ 1. Gray, Manual o-f Botany, 6th Edition p. 119. -6- ^ - --. -, ^^^ ^ j^K^^^^f^ ^'yf/jj^ ^8 M ^^^^^^ ^^^^ ^ Wl ^5 X . C ^ ^ '£,. In the general description of the order Anacardlaci\e» Gray says: "Juice or exhalations often poisonous." Whe- ther 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 na- ture there has been more speculation than experimental evidence bearing on this question, although a few investi- gations have been made with the object of isolating the polfson* It is most generally believed that the exhala- 2 tions are poisonous. Dr. J. H. Hunt states that the 1, Gray, Manual of Botany, 6th Edition, p. 119, 2. Brook. Med. Jour. June 1897. exhalations have been collected in a Jar and found to be capable of inflaming and blistering the akin of an arm plunged into it. 1 Prof. J. J. Rein , in his treatise on Lacquer Work, describes the poison of the Japanese lac tree, Rhus vemi- cifera, as being volatile, as do also the Japanese chemist 2 3 Yoshida and the French chemist Bertrand. Recent work by Prof. A. B. Stevens , however, seems to show that this poison is not volatile, and is similar to, if not identi- 5 cal with that obtained by Pfaff from Rhus toxicodendron and Rhus venenata. Sot many cases of internal poisoning by Rhus toxico- dendron are on record in medical literature. Two cases of poisoning from eating the fruit of this plant have been 6 described • 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 1, Rein, The Ind, of Jap., p. 338, et seq. E. H. Yoshida on Urushi Lacquer, Jour. Chem. See. ,1883, p. 472, 3. Ann. de Chem. et de Phys. , Series YII, Vol. 12, p 125, 1897. 4. Amer. Jour, Pharm. 78, p. 53, Feb. 1906. 5. An account of Pfaff's work will be found in another part of this papers. 6. Amer. Jour. Med. 5ci. 51 (1866), p. 560, -8- of soda were given in solution under the belief that it was an antidote to the poison. Otherwise they were treat- ed on general principles. Both children recovered. Another case of internal poisoning is the following : Three children drank an infusion of the root of poison ivy thinlcing 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. 2 Dr. Pfaff explains the few fatal cases that have fol- lowed Rhus poisoning on the assumption that enough of the poison was absorbed through the skin to cause renal compli- cations 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, 3 to a volatile acid, and to a non-volatile oil. Pfaff , 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 1. Med. and Surg. Hep. 17, Nov. 1867. 2. Jour. Exp. Med. 2, (1897), p. 181, 3. Loc. cit. -9- principle, and that one could be poisoned only by actual contact with some part of the plant. He assumed minute quantities of ^Ilen dust to be in the air to account for the cases of Action J-t 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 oases may obviously render mistakes extreme- ly easy as to the occasion when contact with the plant real- ly 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 suf- ficient to produce the dermatitis on a person very suscepti- ble to its action. It is also conceivable that a volatile poison manufactured by a living plant could become non-vola- tileby changes in it consequent upon thp 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. i:. Maisch, a ptormacist, and Dr. Franz Pfaff, of the Harvard University Medical School, to whose work reference has been frequently -10- 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 was made by Khittel in 1857, His work was published in Wittstein's Vierteljahrresschrift for praktische Pharmaoie , VII. 348-359 , Khittel obtained 37-l/E oz. of fresh leaves of poison ivy from the botanical garden in Munich, dried them, and got a residue of 9-1/2 oz. which he analyzed, lot 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 oz, of the powdered leaves were Infused with hot distilled water, after three days strained, expressed, the liquid evaporated to 3 oz. , and with the ad» dition 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 spontaneous- 1. A free translation of thi^; paper ij given in Amer, Jour. Phanri. for 1558, p. 542, ly, distilled with potassa, the alkaline distillate neu- tralized with hydrochloric acid, and a precipitate could now he obtained with chloride of platinum. Want of materi- al prevented further experiments." The editor of the American Journal of Pharmacy inserts the folloT/ing 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." 1 Work of Mais oh. The next investigation of this plant was made by Kaisch 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 poison- ous principle must, therefore, be volatile and, at the same time, be naturally in such a loose state of combination as to be continually eliminatr^d and separated with the usual products of vegetable exhJllations. It is natural to sup- pose 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 in- fasion prepared from them, and this infusion evaporated Proc. Amer. Pherm. Assn. 1865, p. 166, and Amer. Jour, Pharm. 1866, p. 4. -IE- down to the original weight of the dried leaves. It is obTious that Dr. Khittel could not have selected a tetter method for obtaining the least possible quantity of the poisonous principle, if, inffeed, it could be obtained by this process at all." Maisch then worked up 8-3/4 oz, 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 him- self 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 Ac id." writes Maisch. He farther says: "That it is the principle to which poison oak owes its effects on the human system -13- 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 €md operating with it, Ceveral persons coming into the room while I was engaged in with it were more or less poison- ed "by the vapours difl\ised in the room; and I even trans- ferred the poisonous effects to some persons, merely by shaking hands with them. "The dilute acid, as obtained by me, and stronger solu- tions of its salts, were applied to several persons, and eruptions were produced in several instances, probably by 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 expo- sure of my hands to the juice of the plant." PFAFF»S WOBK. 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 q_uicker is its action on the skin. Formic acid acts very quickly; acetio 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 Maisoh -14- might have contained seme 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 hy 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-poison- ous 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 poison- ous 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, an.d 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 com- -16- pounds with armnonitUD sulphide, shaking out with ether, and letting the ether evaporate spontaneously, a non- volatile oil was obtained which gave the characteristic skin erup~ tions. The pure lead compounds made in different prepara- tions were analyzed and assigned the formula Gg-j^H^QO^Pb, The oil itself was not analyzed. Pfaff proposed the name Toxicodendrol for the oil. He found that it was not vola- tile, 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 l/lOOO mg. 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 IQius venenata seemed to be identical with that from Rhus toxicodendron. EZPERIMSHTAL. The writer's Investigation was undertalien 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 sub- -16- stances associated with it in the plant were first identified; the scope of the work was therefore extended to an investiga- tion of the other conctituents of the plant, and it was hoped that a knowledge of Ihe properties of these constituents would suggest a more economical way of getting the poison than the method of fractional precipitation. The crude material for thia work was prepared "by Messrs. Parke, Davis and Co., of Detroit, Ilich. according to special instructions submitted to them: 67~l/2 lbs. of fresh leaves and flowers of poiaon 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 msike it more mobile. The ether was then separated off and the extraction was repeated three times in the same way to insure complete ranoval 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, blaok, tar-like mass, weighing 3 lbs. 11 oz. 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, -17- since proper precautions were taken and the utensils were handled wilii rubber gloves. 1 The crude ether extract 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 sug- gesting 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 ex- periment was to distil it out under dimished pressure. For A this purpose, an Anschutze distilling bulb containing ten grams of the tar was connected with a vacuum pump. After a pressure of 2 ram. had been established the bulU was gradually heated in a bath of Wood's metal. Nothing distilled dver. The material began to carbonize at a temperature of 140°-150° It was then thought that perhaps the oil could be converted into an ester which might be more volatile and could be dis- tilled out. 20 grams of the original material were dissolved in 100c, c. of absolute alcohol containing 3 gm. of hydro- chloric acid gas, and the mixture was heated 10 hours on a water-bath under a return condenser. After the heating, the This will be designated as the original material. -18- 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 extrac- tion with ether was continued until the water layer was no longer green. The combined ether extracts were evaporated in a ±±sx desiccator without heat. A black tar-like solid was left very much like the original material but it had the ester odor. It wa- partly oluble 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 in- vestigated 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 -19- this medium* The solution was filtered fron inaoluhle tar. A portion of the yellow filtrate gave a reddish yellow pre- cipitate with lead acetate. The alcoholic solution was distil- led in an Anschutze 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 solu- tion 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 sub- stance 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 substabce in 50';^ 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/o alcohol was filtered through bone-black and the filtrate was colorless. Examination showed that everything had been removed by the -80- ■bone-tlack and the filtrate was apparently pure alcohol and wat er. In precipitating an alcoholic solution of the crude ma- terial with a solution of lead acetate, it was noticed that at least two kinds of precipitates were formed. The part that went down first wa-, darker in color than thatrthJrown down later, Pfaff used the last fractions in ohtaining 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 ccr.alsted of tarry - "-tter which was brought down mechanically or separated out cTv ihe dilution of the alcoholic solution l, une lead acetate solution, or was perhaps a lead compund soluble in ether. To test this point an experimerit was made as follows: Some of the crude material was thoroughly extracted with 50^ alcohol. The tar insoluble in 5Gfo alcohol was then treated with 25% alcohol; most of it dissolved; the solution was filtered and lead acetate in 50^ alcohol was added. A greenish colored preci- pitate was formed which was filtered off and found to be com- pletely soluble in ether and soluble to a considerable extent in strong alcohol. These experiments suggested that the li^t 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 -21- of fractional precipitation. Farther preliminary experi- ments showed that 50^ alcohol extracted from the original material all of the substajice or substances which gave the light colored precipitate and dissolved only a small amount of the tar. 288 gm. of the crude material were then extracted several times with 50^ 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/S alcohol. The large precipitate, which will be designated as "precipitate A", was filtered and drained by suction in a Buchner 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 inter- rupted 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 pOrtilfton of this lead compound was decomposed by hydrogen sulphide in a mixture of water an. ether which was well shaken during the operation. The ether was separated, filtered, and evaporated under diminished pressure without heat ^ and there rer.ained a yellow oily looking residue having a plea^jant odor. When the ether and water were completely removed in a vacuum desiccator a hard, brittle, yellow resin weighing about 16 grams was -22- obtained. It was soluble in alcohol, had a strong acid 1 reaction and was free from nitrogen and sulphur. The nitrogen teats were made by the Las^^aign and 3oda lime 2 methods , and the sulphur test waj made with sodium nitro- prusside after fusing the rcoidiie 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 solution- of this kix 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 poison- 3 ous • In the meantime the resin was being studied in the laboratory. 1. ITitrogen was found very readily by the soda lime test in the ta.T left after extracting the original material with 50^ alcohol, but was not found by the Lassaign test. 2. 3tevens. Amer. Jour. Pharm. 77, 255, June 1905. 3. Whenever it is stated in this paper that a solution was poisonous or not poisonous, the test was made by the writer upon himself. .23- GALLIC ACID, An alcoholio solution of the resin was Just neutralized with potassium hydroxide. During the titt«tion, the solution rapidly "became dark brown. After neutralization it was shaken with ether; the water solution remained hrown 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 aud 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 pre- cipitate A, and alao slight precipitates with salts of zinc, mercury, copper, an 1 TrkyrrysLXx. 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 in- dicated the presence of a tannin compound. An alcoholic solution of the reain 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 trl-hydroxybenzO'.c acids have in general the same color reactions. The p- isence 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 -24- 1 together in the ssane plant. 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 aa excess of potassium hydroxide gave a black substaijce (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 (E) and (3). At later stages in the work the potassium, barium, and sodium salts of gallic acid were ob- tained, and finally the pure acid was made by decomposing the sodium salt with sulphuric acid and cjrystallizing from water. A portion of the acid so obtained was further purified by dissolving in absolute alcohol and pouring into absolute 2 ether . 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?S alcohol, hydrochloric acid gas was passed in, and the solution was heated an hour on the water bath. It 1. Liebig's Annalen, CXI, p. 215. 2. Uber Kategerbstoff , p. 20. -25- was then evaporated to a small bulk^ neutralized with barium carbonate and extracted with ether. The ether, on evaprration, left the ester which was crystallized from water and dried in a desiccator over sulphuric acid. The anhydrous ester agreed in melting point (156°-159°) and other properties 1 with the ester of gallic acid described by Grimaux • 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 aeries of experiments was in progress. Eleven and one-hsilf grams of the resin obtained from lead precipitate A by decomposition with hydrogen sul- phide were treated with 0. In. potassium hydroxide added from a burette until the acid was exactly neutralized. All went into solution. On shaking with ether some of the po- tassium salt sep&rated 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 potas- sium salt was precipitated out in four fractions by adding for each fraction one-fourth the amount of Bin sulphuric acid required to neutralize the potassium hydroxide used. 1. Bull. 3oc. Chim. (II) Vol. 2, 95 (1864). -26- 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 filter- ed off. The succeeding precipitates were in hetter condi- tion but were not pure. All appeared to he 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 waa shaken several times with ether and saved for further examination, which will be described under "Bhamnose". 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 poison- ous as already moitioned. By this test, all the substfn as 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?S alcohol and filtered from insoluble tar. The filtrate was precipitated in six fractions by lead ace- tate. The last fractions were lighter in color and apparent- ly much purer than the first. The cixth lead precipitate was decomposed by Hydrogen sulphide, the li^t-yellow water solution was tested and found to be not poisonous. It gave -27- 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 o lead compund in the first precipitates* FISETUr, Having identified gallic acid, and not finding any other ph,en©l derivative in the lead precipitate, some of the ori- ginal 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 -E8- 1 Rhus cotinus and nemed "fustin-tannide" by Schinld • He showed that the fustin-tannide could he decomposed hy acetic acid Into tannic acid and a glucoclde, fustln C^gS^g^gl* Pustln, on heating with dilute sulphuric acid, gave fisetln and a sugar supposed to he rhacmose. Fisetln was also found 2 as a glucoslde compound In Rhus rhodanthema by Perkln . The yellow substance which separated from the boiling water solution was farther 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 am- monia. Concentrated acids intensified the yellow color of the alcoholic solution. Fehling solution and amraonlacal silver nitrate ware reduced by it. Potascium 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 cauatlc potash, the red color returned and was permanent. These reactions are 1. Berlchte 19, 1725 (1886). 2. Jour. Chem. Loc. 71, 1194 (1897). -29- 1 characteristic for fisetin. Furthermore, fisetin should give protocatechuic acid and phlorogluoinol by fusion with caustic potash under proper 2 conditions • The experiment was carried out as follows: 2 grams of fisetin ■»» gently heated in a nickel crucible with 6 gracjs of caustic potash dissolved in 6 c.o. 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 fil- trate to precipitate protocatechuic acid, while phloroglu- oinol remained in the filtrate from this precipitate. The lead precipitate was suspended in water, decomposed by hydro- gen sulphide, filtered, and evaporated to obtain protocate- chuic acid. That the substan'Je obtained was protocatechuic acid was shown by the following characteristic tests: (l) 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 1. Berichte 19, 1740. 2. Ibid. 1747; Annftlen, 112, 97. -30- color tecame red. (2) A violet color was obtained when a solution of the acid was treated with a drop of sodium carbon- ate 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 reoidue left on evaporating the ether was taken up in water. This solution gave the following reactions characteristic for phloroglueinol: (l) It reduced "both silver nitrate and Fehling solution, (2) It colored pine wood moistened with hydrochloric acid red. (3) It gave a teautifal red color with vanillin and hydrochloric acid, and (4) a deeper red color with oil of cloves and hydrochloric acid, hecoming purple on standing. (5) It gave a violet color with ferric chloride. The substance is then, without doubt, fisetin. The 1 formula of fisetin is supposed to be (C/r^-oO^,) Biochem. Pflan. II, 521. -31- HHAKHOSE, It was stated above that rjcmid obtained a sugar solution by the decomposition of a fisetin-gluooside from Rhus cotlnus, and Parkin obtained the same from a glucoside in Rhus rhodan- thema. These investigators thought that the sugar was iso- dulcite or rharcnose but they did not isolate It on account of the small quantities of material at their disposal. More- over, the sugar is very hard to crystallize in the presence of other soluble substances and ir not found in large quantity 1 ^ in plants. Maquenne could obtain only 15«»20 gm. of rham- nose by working up 1kg. of the berries of Rhamnus infect orius. As-uming that the free fisetin found in poison ivy leaves had its origin in the decomposition of a fisetin-glucoflide by natural processes, it was reasonable to suppose that the sugar would also be found in the free state although according Z to Roscoe and Scharlemmer : "Isodulcite does not occur in the free state in nature, but ir; found as a peculiar ethereal salt belonging to the class of glucosides. On boiling with dilute sulphuric acid, this splits up into isodulcite and other bodies " The more recent works on the sugars 3 and on plant chemistry mention the occurrence of rhamnose only 1, Ann. de Chtm. et de Phys. . 6th Series, ZIII, 76 (1891). 2, Treatise on Chem. Vol III, Pt. Ill, 492. 3, les Sucres; Chem. der Zuck. ; Biochem. der Pflan, -32- in the glucoside form, with one possible exception. The ex- ception referred to is the occurrence of a free sugar, sup- 1 2 posed to he rhamnooe, in a certain palm-wine • Czapek says: "The well known methyl pentoses do not occur in the free state in plant organisms so far as we know." Since rhamno.:e forms a lead compound, the sugar, if present, should be found in the first lead precipitate, A, and also in filtrate A in case it is not completely precipitat- ed in the presence of acetic acid and alcohol. The filtrate A (about two liters) was examined first. It had a light yellow color, contained an excess of lead acetate, and was acid from the acttic acid liberated in the 3 precipitation of the lead compound A • This filtrate was evaporated by dryness under diminished pressure to remove alcohol, water, and acetic acid. The clear distillate had a peculiar odor suggesting both tea and arpyl formate. It was saved for examination and was found to be not poisonous. The reaiiue in the dish after evaporation was a tough reddish brown, gunrcy mass which could be drawn out into fine threads. 1, Chem. Seit. 23, Rep, 177. 2, Log. cit, 1, 209. 3, On standing several weeks, a small quantity of tar separated out on the walls of the vessel, also a brown precipitate which was filtered off. suspended in water ana hydrogen sulphide was being passed In when an accident occurred and it was lost. -33- It had a pleasant sweet odor. It was extracted several times with hot water each portion teing filtered. A yellow trown powder rgnained undissolved and was saved. The combined filtrates deposited more of the yellow solid on standing. This powder will he referred to later as "P". The filtered liquid was freed from lead by hydrogen sulphide. T^e solu- tion then had a lemon yellow color, a sweet odor and was acid from acetic acid. On concentrating the solution by evapora- tion and making a small portion of it alkaline with sodium hydrox- 1 ide» the yellow color came out very intense • The alkaline solution reduced Fehling solution and emmoniaoal silver nitrate indicating the presence of a sugar. Another portion of the solution gave a slight precipitate with phenyl hydrazine in the cold. The remainder of the solution was evaporated to dryness, extracted with water, filtered, and again evaporated, A dark sticky syrup was left which was only partly soluble in water. Thi3 was treated with water, the filtrate filtered, and evaporat- ed, the water being replaced from time to time to remove acetic acid. Finally the liquid gave the following tests for rhamnose, besides those already mentioned: 2 (1) With a-napthol and sulphuric acid, a purple violet color, 1, "By warniing with alkalies or barium hydroxide, rhamnose is colored yellow"- ^ * Von lippmann, Chem. der Zuoker. I, 177, 2, Chem, der Zuek. -34- 1 (2) With thymol and sulphuric acid, a red color. 2 (3) With resorcinol and sulphuric acid, red color, 3 (4) With orcinol ^d hydrochloric acid, red color. (5) With ammonium picrate and sodium picrate, yellow- ish red color. (6) With phloroglucinol and hydrochloric acid, red color. (7) It decolorized an alkaline solution of potassium feri cyanide. (8) It gave a white precipitate with lead acetate. The filtrate B (p. 3.7 of this manuscript) from which gallic acid was precipitated hy sulphuric acid in four fractions was saved to examine for sugar. fo remove gallic acid completely^ and other vegetable matter, it was shaken out several times with ether, and was kept at a low temperature with salt and ice for a long time. It was left standing for several weeks during which time more hrovm matter separated out and was filtered off. The filtrate was eraporated to a small "bulk, cooled and filtered from cjrystals of potassiiun sulphate. The filtrate was evaporated to dryness, the residue taken up in water and filtered through honehlack. Addition of alcohol caused com- plete precipitation of potassium sulphate. The solution then gave the above mentioned characteristic^ for rhamnose. 1. Chem. der Zuck. 2. Eayman, 3ur I'Isodulcite, Bull. Soc. Chim. 47, 668 (1887). 3. Acides Gummigues. -35- All attempts to get the osdzone of the sugar ty the method of Fischer failed^ pr oh uuly on account of the small (luantity of the sugar present. The plant, it will he rememher- ed was originally extracted with ether in which rhamnose is practically insoluble. The above described testa, however, can leave no doubt as to the identity of the sugar. Additional evidence that the sugar is rhamnose was ob- 2 tained by a method described by Maquenne as follows: "The production of methyl furfurol in the dehydration of isodulcite furnishes a very simple means of charac- terizing this sugar in mixtures which contain it; it is sufficient, for example, to distil 50gm. of q.uercitron wood with as much sulphuric acid and about 150gm. of water, then to rectifj- the liquid obtained in order to get several drops of the crude furfurol, which on addition of alcohol and concentrated sulphuric acid gives immedi- ately the green coloration characteristic of methyl furfurol. This procedure is applicable to extracts as well as to entire plants, and has the advantage that it dibes not require the separation of isolulcite, the cjrystallization of which is often very slow and at times impossible when it is mixed with other very soluble substances," The experiment was tried with the crude ether extract of the plant according to the directions of Maquenne, and the green color with alcohol and sulphuric acid was obtained from the thicker oily portion of the distillate. This test can 3 be made with hydrochloric acid as well as with sulphuric. 1. Berichte XX, pp. 1089 » 1091, 1188, 2566, 2. Ann. de Chim. et de Phys. (6) XXII, 93 (1891), 3. Biochem. der Pflan. I, 210. -36- Therefore the color test was tried with the ester mixture prepared in one of the early experiments by lolling the ori- ginal plant material with hydrochloric acid and alcohol. Methyl fuxfurol was found here also, this method indeed giv- ing hetter results than that of Maquenne, ^ The presence of ■I free ramnose has thus heen shown in the original material, in the first precipitate hy lead acetate, and in the filtrate ftom this precipitate. Txperiments to he described under "The Poison" showed that the ether extract from the Soxhlet apparatus contained a substance which yielded rhamnose when hydrolyzed by dilute sulphuric acid. The presence of free gallic acid, fi set in, and rhamnose in the plant can be readily explained by a series of assump- tions for which there is a considerable amount of experimental evidence. There is reason to believe that tannin-like bodies 1 are formed at the expense of chlorophyll , that complex tannin bodies can be broken down by acetic acid (also found in Rhus toxicodendron) into a tannic acid and a glucoeide (for example, the "fustin-tannide" mentioned above yields tannic acid and fisetin-glucoside) ; and finally that the glucoside can be hydrolyzed by acids or enzymes giving, in the sumach plants, fisetin and rhamnose. Hitrogenous ferments which can effect the hydrolysis of glucosides and give rise to sugars are freq^uently found in 1. Comptes rendus CIV, 892, -37. plants, for example, emulsifn in almonds, myrosin in mustard, 1 and erytlrrozym in madder, Acree and Hinklns found that diastase, pancreatin, and a number of other enzymes hydro lyze o with the formation of glucose and acetic acid. Stevens obtained a nitrogenous oxidizing enzyme from Rhus vernicifera. The close relationship between the poisonous species of Rhus would lead us to suppose that the same soluble ferment exists in poison ivy, though it was not detected in the original material used in these experiments, probably because the plant was extracted with ether in which the enzyme is insolu- ble. The existence of such a soluble ferment would explain the presence of free sugar and free fisetin* EVIDMCE OF THE PRESMCE OF A FATTY ACID IK FILTRATE A, ooco The brown substance P, obtained from filtrate A by evapora. tion and extracting the residue with hot water, was suspended in warm wa^er and dilute sulphuric was added. A white pre- cipitate was formed and a strong fatty acid odor was developed. After the mixture had been heated for some hours on the water bath a small portion was made alkaline and it reduced Fehling 1. Amer. Ghem. Jour. 28, 370. E, Amer. Jour. Pharm. 77, 255 (June 1905); 78,53 (Feb, 1906), -38- solution. The main solution was filtered and the precipitate supposed to be a fatty acid was saved. The filtrate was neutralized with Ijarium carbonate, filtered, evaporated, freed from caramel, and the solution then gave the tests mentioned above for rhamnose, A portion of the precipitate supposed to be a fatty acid was ignited in a porcelain spoon. It fused, carbonized, and burned. The remainder was heated with alcoholis potash and reprecipitated with hydrochloric acid. The precipitate was washed and heated with alcohol. Part of it dissolved. The insoluble part was foujid to be a lead compound. On boiling it with hydrochloric acid and cooling, lead chloride crystal- lized out. This was confirmed by dissolving the lead chloride in hot water and precipitating as lead sulphide. These experiments were not carried farther on account of the small (luantity of material, but they show that the gummy substance obtained from filtrate A contained rhamnose (either as a lead compund of free sugar or as a lead compound of a rhamnoside), and also, most probably, the lead compound of an organic acid. THE F^GRAUT DISTILLATE. Several times in the course of this work, extracts of the original plant material in alcohol and in water were dis- tilled under diminished pressure for the purpose of concentrat- 1. A wax obtained from Rhus succedanea was .;hown by Stahmer to contain palmitic acid and glycerol in the form of glyceryl palmitate. Annalen 43, 343, (1342). ing the solutions. The distillate, in every case, had an ethereal odor suggesting anjyl formate in very dilute solution, but was more fragrant. The distillate from a water extract was examined. It was a clear liq^uid, a little darker than pure water, was not poisonous, was neutral to litmus paper, gave no color with ferric chloride, reduced ammoniacal silver nitrate hut not Fehling solution, ana gave a faint red color with dilute ammonium hydroxide and with sodium carbonate. A snail q^uantity of a finely divided black precipitate separated out from the water distillate on standing. The substance with the fragrant odor was extracted by shaking the distillate with ether and letting the ether evaporate spontaneously. A very small quantity of a yellow solid was deposited on the sides of the dish. This substance had a strong and persistent odor, so sweet as to be almost nauseating. Hot enough was obtained for examination or analysis. This fragrant residue was difficultly soluble in water and the solution reduced silver nitrate in ammonia. A steam distillate of the original plajit material had the same fragrant odor as the distillate fron. a water extract. THE POISOH. 288 gm. of tne original poisonous material were extract- ed with 50;'o alcohol, and this alcoholic solution was precipitat- ed with lead acetate in the manner already described (p. ^^ -40- of this manusoript) . The lead precipitate so obtained was extracted with ether in Soxhlet extractors and after the extrac- tion was foixnd "by test to be free from poison. Therefore the poison, if precipitated by the lead acetate, must hare been extracted by the ether. This ether solution had a dark greeA-color, ana was acid from acetic %cid brought down in the lead precipitate. The ether was evaporated in a vacuum desiccator without heat and there remained a small quantity of an acid mixture of water and a soft tarj the watery part was colored green showing that the tar was solu- ble to some extent in dilute acetic acid. The mixture had the peculiar odor of the original material, A small drop of the green watery part v/as applied to the wrist, allo7;ed to remain a few minutes and was then removed by absorbent paper but the spot was not washed. Itching and reddening of the skin commenced within twenty- four hours. At the end of forty-eight hours, there was a well developed case of poison- ing. How this was cured will be described in another place, A small portion of the poisonous mixture was dissolved in alcohol, and this solution was divided into three parts. The first part was treated with ferric chloride but it gave no colorreaction. Another portion tf the alcoholic solution was diluted v/ith water. It became turbid. The third portion gave a dirty-green precipitate with lead acetate, which seemed to come down more readily when the solution was diluted with -41- water* The main portion of the poisonous mixture was then dissolved in 95;o alcohol and lead acetate in 50jx> alcohol was added. The precipitate was filtered, washed, decomposed by- hydrogen sulphide in a mixture of water and ether. The ether solution was filtered aad evaporated. The residue was a tar which on standing in a desiccator for some time became dry- enough to break into sticky lumps. An alcoholic solution of this substance gave a dark color with ferric chloride aad a light colored precipitate with lead acetate. To get more of the poisonous tar for study, E33 gm. of original material were extracted with 95^ alcohol. Strong alcohol was used in order to dissolve as much of the tar as possible. The solution had a dark greenish color but was somewhat yellow in thin layers. The undissolved tar was filtered off and extracted twice again in the same way. The tar left after the third extraction was only slightly soluble in aloohol, and its solution was not poisonous. The three filtrates from these three extractions were precipitated separately by lead acetate in 50^ alcohol. The first preci- pitate was larger, darker in color, and carried down more tarry matter. The second was not as large as the first and was light green. The third was quite small, was black, and was not a lead compound at all but some of the tar which separated out on diluting the strong alcohol with the weaker -42- grade containing lead acetate. It was soluble in ether and less soluble in alcohol. The alcoholic solution of this third lot gave no precipitate with hydrogen sulphide. The first and second lead precipitates were filtered hy suction and washed with water. They were kept a day or two in a desiccator over sulphuric acid but did not become completely- dry. The weight of these two moist precipitates together was 172gm, They were combined and extracted with ether in Soxhlet extracttrs which were kept in operation during work hours for three days. In the meantime the alcoholic filtrates from these lead precipitates were combined and concentrated on the water-bath by distilling off two liters of alcohol. The alcohol obtained had the peculiar odor of the original material, but was not poisonous. After a long extraction of the lead precipitate in the Soxhlet extractors, the green ether solutions were combined and washed by shaking them with water to remove lead acetate and acetic acid in case any should have been held in the lead precipitate. The ether was distilled off at a low temperature and there reir&ined a soft tar, a portion of which was not com- pletely soluble in 95% alcohol. The alcoholic solution had a greenish yellow color and was poisonous. The tar was also partly soluble in acetic acid, and this solution was found to -43- contain lead. Thinking that the lead acetate had not heen completely washed out, the main part of the tar was dissolved in ether and shaken with watjer. The wash water continued to give a test for lead as long as the washing was continued. This indicated probably the hydrolysis of an unstable lead compound. Hydrogen sulphide was passed into the ether solution mixed with water to raiiove the lead. Lead sulphide was filtered off, and the ether was evaporated, A small portion of the tar residue in alcoholic solution gave a color reaction with ferric chloride. As this may have been due to traces of lead gallate dissolved in the extraction with ether and afterwards decom~ posed by hydrogen sulphide, the main portion of the tar was redissolved in ether and shaken with water until it no longer reacted with ferric chloride. The ether was then evaporated and a soft, black, poisonous tar or gum of uniform consistency was left which was shown by tests to be free from gallic acid and lead. These experiments showed that some of the poison was precipitated as a lead compound soluble in ether and some was brought down mechanically in the free state. To see if the extraction with ether in the Soxhlet apparatus was complete, the residue from in the thimbles was decomposed by hydrogen sulphide and shaken v/ith ether. The dark colored ether solu- tion was freed from gallic acid by shaking with water and dilute sodium carbonate solution, and was evaporated, A small -44- quantity of tar was obtained which was added to the main portion. A solution of the poisonous tar in 95;^ alcohol did not reduce Fehling solution and did not give a precipitate with lead ace- tate except the separation of a small quantity of tar, which was not a lead compound. The lead compound of the poison wsis apparently soluble in 95^ alcohol as well as in ether, for it would not precipitate in this medium, although it was found in the original precipitate by lead acetate. The alcoholic solution of the tar hecaDie turbid on diluting with water. In order to see if the poison is volatile with vapor of acetic acid, since this acid is found in the plant and it is thought by some that the poison is volatile, a portion of the tar was distilled under diminished pressure with acetic acid. It was soluble tn some extent in the acid. The temperature did not go higher that 55°C. during the distillation. A tube containing cotton wet with sweet oil was placed between the receiver and the water suction so that the uncondensed vapors would have to pass through the cotton. This cotton was rubbed on the skin and was not poisonous. The yellow distillate collected in the receiver was also tested and was not poisonous. HYDROLYSIS OF THE TAR. About 5gm. of the tar free from gallic acid and sugar was dissolved in alcohol and dilute (Z/)) sulphuric acid was added. Some of the tar separated out on diluting the alcohol with tne acid. The mixture was heated on a water-bath during work hours for four aays. A purple -45- and green fluoreaoent solution was formed, though much tar was left apparently unchanged. The aloohol was evaporated off and the solution was filtered from tar. The fluorescent filtrate was shaken with ether, by which the green substance was roi^.oved, leaving the solution purple. The ether left, on evaporation, a srrall quantity of a green substance having a pleasant ester od©r. It was not examined* A portion of the purple solution was exactly neutralized vath sodium car- bonate. This solution gave a blue-black color with ferric chloride which becarce red on addition of another drop of sodium carbonate, indicating gallic acid. It also reduced Fehling solution. Another portion of the purple solution was made alkaline with sodium carbonate. A reddish-brown flocoulent precipi- tate was formed and was filtered off. The filtrate did not give any color with ferric chloride, but it reduced Fehling solution* It also gave the test for rhannose with d-naph- thol. The main portion of the purple solution was made alkaline with sodium carbonate; the precipitate was filtered off and dissolved in acetic acid. This solution was yellow and gave a reaction with ferric chloride simmilar to that of gallic acid. The filtrate from the precipitate by sodium carbonate was concentrated by evaporation until sodium sulphate began to crystallize out. Alcohol was added to precipitate the sodium sulphate completely, the mixture was heated and filtered. -46- The alcoholic filtrate waa concentrated to a syrup which reduc- ed i'ehling solution and gave the cloaract eristic tests for rhamnose already described. By this hydrolysis, the tar was split up into rhamnose and some form of gallic acid which could be precipitated by sodium carbonate. This compound, whose acetic acid solution was yellow, probably contained fisetin also. The reason for this last statement will appear from the following experiment. Decomposition of the 'far with Acetic Acid. A portion of the poisonous tar was heated in an open dish with strong acetic acid. The tar seemed to be decomposed to some extent giving a yellow substance. Acetic acid waa added from time to time as it evaporated. After several evaporations, water waa added, the mixture was heated to boiling and filtered. This filtrate Ho. 1 will be mentioned later. The residue in the dish consisted of undecomposed tar and an olive-green flaky substance. This substance was heated with a frsh portion of glacial acetic acid. V/ater was added, and the mixture was boilid and filtered. The filtrate had a deep yellow colir suggesting fisetin. It waa shaken out with ethyl acetate which became colored yellow. A portion of the ethyl acetate solution gave an orange red precipitate with lead acetate showing the presence of fisetin. The ethyl acetate was removed from the remainder of the solu- tion by evaporation and tlie yellow residue was taken up in -47- alcohol. This alcoholic solution gave the characteristic reactions for fisetin with stannous chloride, with potassiuir. hydroxide, with ferric chlride and with Fehling solution. Filtrate Uo. 1 obtained by heating the poisonous tar with acetic acid and hot water as described above was in- vestigated as follows: A portion of it gave a reddish color- ed precipitate with sodium carboaatc as in the case when the tar was hydrolyzed with sulphuric acid. The remainder was nearly neutralized with sodium carbonate and lead acetate was added in excess to remove gallic acid. The excess of lead was removed by sulphuric acid, and the sulphuric acid by barium carbonate. The solution on evaporation reduced Fehling solution to some extent but a white precipitate was also formed. In this experiment, gallic acid and fisetin and probably sugar were foimed by decomposition of the poisonous gum with acetic acid, the acid found in the plant by Pfaff. The presence of free gallic acid, fisetin and rhamnose in the plant can be explained by the natural hydrolysis of a complex gum or tar or a constituent thereof. The poisonous property is lost in the general rearrange- ment which takes place during hydrolysis. The poisonous tar was not hydrolyzed by boiling with a dilute solution of sodium carbonate. It was found, as has been stated elsewhere, that the lead compound of the poison could not be precipitated in 95/o alcohol. -48- Further experimenta» however, showed that on extracting the poisonous guai with 50/o alcohol, a portion of it dissolved, and this solution gave a precipitate with lead acetate which was a true lead compound. The remainder of the purified tar (about lOgm. ) was treated with 5O7S alcohol and filtered. Very little dissolved in alcohol of this strength, hut on addition of lead acetate in BOji alcohol to the solution, a light colored precipitate was formed, which hecame dark on standing. It waS filtered off, washed free from lead acetate, decomposed by hydrogen sulphide, and shaken out with ether. The ether left, on evaporation, a yellow resinous substance having a faint odor like garlic. By drying in a dewiccator, a small quantity of a solid yellow resin was obtained v;hich was completely soluble in alcohol. A very small drop of this solution applied to the skin on the end of a glass rod which had been dr\¥n out to a point caused an eruption in about thirty-six hours. Follov/ing the nomen- clature used by liaiach and Pfaff, this substance will be designated as Toxicodendrin. the ending "in" indicating its glucoside nature. The filtrate from the lead precipitate just described was freed from, the excess of lead acetate by hydrogen sulphide, was tested for poison, and was found to be poisonous, showing that the precipitation by lead acetate was not complete even in 50^ alcohol. On spontaneous evaporation of the solutioa., -49- a yellow, sweet smelling resin was left. A portion of the alcohol solution of the toxicodendrin gave a dark coloration with ferric chloride, did not reduce Pehling solution ana was slightly acid to litmus. To see whetrjer the toxicodendrin coula be hydrolyzed, the reirainder was dissolved in alcohol and dilute sulphuric acid was added. A fine, white precipitate was forired at once Y/hich rose to the surface on standing as a light floc- culent substance. The mixture was heated for several days on a water-hath, filtered from unhydrolyzed resin ana the fil- trate was neutralized and concentrated in the way already described. The solution obtained reduced Fehling solution. Hot enough was obtained for further sugar tests, but all the hydrolysis experiments point to the conclusion that the poi- sonous substance is a rhamnoside, and is the source of the sugar in the plant* The reaction with ferric chloride observed whenever a lead compound of the poison is decomposed by hydrogen sulphide may be explained by the formation of traces of gallic acid or fisetin through the action of the weak acids present. The supply of purified poisonous tar having been exhausted in the preceding experiments, further study of the active principle is postponed until more can be prepared. It is highly desirable to investigate the white precipitate formed by addition of sulphuric acid to an alcoholic solution of the -504 toxicodendrin, OZIDATIOl OF THE PURIFIED TAR 7/ITH HITRIC ACID. When the purified poisonous material (p. ^ extracted with 5O70 alcohol, only a small quantity was dis- solved as was stated above. The insoluble residue was treated with fuming nitric acid. Violent reaction took place at once v/ith copious evolution of red fumes and heat. ^en the reaction was over, a sticky red gummy mass was left which was slightly soluble in warm alcohol. The ?/ater ex- tract was yellow, and the alcoholic solution was red. That the water extract contained picric acid was shown by the following experiments: (1) A portion was gently v/armed with a few drops of a strong solution of potassium cyanide and two drops of sodium hydroxide. The red color of potassium isopurpurate was formed. (2) A portion of the water solution v/as heated v;ith glucose and a few drops of sodium hydroxide. The deep red color of picramiinic acid was pro- duced. (3) A few drops of an aramoniacal solution of copper sulphate was added to the water extract. A yellow-green precipitate was formed. (4) The water extract dyed silk, but did not dye cotton cloth. -51- DISTILLATION OF THE TAR WITH SODA Lllffl. About 25grii. of the tar left after extracting the original material with hot v/ater was dissolved ia ether and poured into a glass retort containing soda-lime. The ether was ^yi«e distilled out leaving the tar intimately mixed with the soda-lime. The retort was then gradually heated. Vapors and liquid were given off, "both of which turned red litmus Dlue and had a strong odor like tobacco smoke. Ho odor of 1 ammonia was detected • At the high temperature of the triple burner, a seiri-solid, red, greasy substance collected in and closed the condenser tube. This substance had the same po7irerful odor as the liquid portion of the distillate. The clear, watery portion of the distillate was separated from the thicker parts, and was found to contain pyrrol and pyridine derivatives by the following characteristic tests: (1) Vrf'ood moistened by hydrochloric acid was turned red by it. (E) Colorless fumes were formed when brought near hy- drochloric acid; idcEX mixed vd-th hydrochloric acid, a red insoluble substance was formed. f3) It precipitated the hydroxideSof iron salts, gave a light blue precipitate v/ith copper sulphate, and a white precipitate with mercuric chloride. The greasy, semi-solid mass was extracted with 10^ hy- drochloric acid and filtered. On addition of a solution of 1. See Amer. Jour, Pharm. 77,256. -52- mercuric chloride to the red filtrate, a bro-/m flocculent precipitate was formed. It was filtered off and distilled with caustic soda but the distillate did notcontain pyridine* 1 POTASSIUK PERI.IMGMATE A3 A EEMEDY iK)R EKUS POISOHIIG , In the early stages of this v/ork some experiments virere made to see if potassium penr.anganate could he used, to purify the lead precipitate hy oxidizing the tar "brought down in precipitation. It was found that the permanganate attacked the lead precipitate as well as the other organic matter in the vessel. This fact and the well knovm value of permanganate in treating akin diseases, its use as an antidote for some 2 kinds of alkaloid poisoning , as an antidote given to cattle z a poisoned by plants, and as an antidote for snake bites, sug- 5 gested its use as a remedy for Rhus poisoning. Maisch men- tioned that he had used it with success, but it nevei* came into general use, probably on account of its staining the skin and clothing. In carrying out this work abundant opportunities for testing its value as a remedy for the dermatitis caused 1. This section is added in the hope that it may be of use to others who are subject to this forrr. of poisoning. 2. Lloor, 1. Y. Lied. Rec. 45 (1894), 20C. 3. Bull. llo. 26, U. S, Dpt. Agr. , I)iv. of Bot. 47. 4. Lacerda, Comptes rendus 93 (1881), 465-469, 5. Amer. Jour. Ked. oci. 52, (1866), 285. -55- by poison ivy were afforded, by maaiy cases of acci(ient| and intentional poisoningo The best example of the latter was obtained v/ith. the ether solution from the extraction of the lead precipitate in the Soxhlet apparatus (psige // / of this manuscript). After removing the ether, a small drop of the residue was applied to the wrist as described. An itching red spot about the size of a dime was noticed in thirty-six hours which steadily increased in size. Nearly two days after the application of the poison, a dilute solution of potassium permanganate containing a little caustic potash was rubbed into the spot until the pimples were destroyed. A little black spot was left wherever there had been a pimple showing that the permanganate had been reduced to oxide in the skin. The place was washed and nothing more v/as thought of it until the morning following when it was noticed that the Y/rist had commenoed to swell during the night, and the characteristic watery secretion was running from the poisoned spot. IJore permanganate solution was applied without potash and the wrist was bandaged thinking that this would prevent the spread- ing of the eruption, but it really facilitated spreading by becoming saturated v/ith the poisonous fluid and keeping it in contact with a larger surface of skin. In the meantime the swelling and inflammation had extended nearly to the elbow. The arm now had the appearance of having been bitten by a snake. -54- To reduce the swelling it was iirmersed. in hot water. This seemed to bring out the eruption very quickly and the "blisters were treated with permanganate as fast as they appeared. The swelling was reduced but returned during the night. On the evening follcv/ing, the forearm was immersed in a bowl of hot permanganate solution containing a little caustic potash. The solution was kept as hot as could be borne for about half an hour. After this bath, the poison was compltely oxidized^ for the swelling v/as reduced and did not return, nor was there any fresh ei'uption. TiTiiat appeared to be a severe case of poisoning was thus cured very quickly. The use of hot water not only reduce^ 'jhe swelling but also helps to destroy the poison. The action of permanganateiis also more rapid at high temperatures. The oxidizing power of permanganate, as is v/ell kno'ym, is greater in acid solution than in alkaline, five atoms of oxygen being available in the I'ormer and three in the latter according to these equations: 2KMn04f- 3H2S23D4s:i^204 ^ ^lin^O^fSK^O -f 50 SKMnO^-f KgO = EljaOg-f 2KCii-f-S0 Permanganate was used as a remedy in seme cases m.ixed with dilute sulphuric acid, and in others, with zinc sulphate; also with lime water. It was found to be satisfactory whether used alone or v;ith any of the substances m.entioned, provided it was well rubbed into the skin. The concentration of the •55- solution used was varied according to the location and con- dition of the eruption. Where tlie skin v/as thin or already "broken, dilute solutioiB (one per cent) were used. In one case, the eruption appeared in the palm of the hand where the skin was so thick that it was necessary to open it hefcre the remedies could reach the poison. The difficulty of getting the remedy in contact with the poison in the skin is the reason T/hy the eruption is hard to cure. The remedy most conmonly used for this eruption is an alcoholic solution of lead acetate. This rem.edy is unsatis- f^actory for the reason that its action consists in depositing an unstable lead compound of the poison in the skin where the conditions of moisture and temperature are favorable for its decomposition, liberating the poison vifith all its irritant properties. Moreover, alcoholic preparations should not be used because the alcohol dissolves the poison and on evapora- tion leaves it spread over a larger surface like a varnish. Potassium perm^anganate , howver, oxidizes the poison completely. The only objection to the use of perinanganate of which the writer is aware is that it stains the skin. The stain can be removed by vigorous scrubbing with soap, or it will v/ear off gradually in a few days. It can be rem^oved at once by certain acids, but these should not be used by persons not famaliar with their action. With the knov/ledge of the facts mentioned, many solutions «56- were tested for poison by applying tiiem to the skin, and when an eruption appeared, it was cured q^uickly am permanently by rubbing in a permanganate solution, usually mixed with dilute sulphuric acid. 3UIJMARY. Leaves and flowers of the poison ivy plant were extracted with ether and the ether was removed by evaporation. In the residue, the following substances were found and studied: gallic acid, fisetin, the sugar rhamnose, and a poisonous tar, gum or wax. The lead compound of the poison was soluble in ether; this fact gave a means of separating the poisonous substance from the non-poisonous i;jatter in one operation. The poison was not volatile with vapor of acetic acid, or T.'ith vapor of alcohol* The poisonous tar or wax was decomposed by acids and yielded gallic acid, fisetin, and rhamnose, showing the probable source of these compounds in the plants, and indicating that the poison is a complex substance of a glucoside nature. It was found that a portion of the poisonous substance oould be precipitated by lead acetate from a solution of the tar in 50'/o alcohol. -57- All cases of poisoning developed on the writer were easily cured with potassium permanganate. The following method is suggested for olataiaaag the poisonous substance from the plant; Extract the plant v/ith alcohol, filter, and precipitate at once v/ith lead acetate, Y/ash the precipitate, dry, and extract with ether in Soxhlet extractors (loosely filled). Combine the ether extracts, mix with water, and pass in hydrogen sulphide. Separate the water and the ether solution, and filter the latter, Y/ash the ether solution thoroughly by shaking with water, and then evapotate at a low temperature, BIOGEaPKY. U, A, Syme was born in Raleigh, IT. C. , on July 11, 1879, He was prepared for college at the Raleigh Kale Academy, entered the Horth Carolina College of Agriculture and Mechanic Arts in 1895, and v/as graduated in 1899 v/ith the degree £, S, He was an Instructor in Chemistry at the same College from January 1st, 1900, until June, 1903, v/hen he received the degree U, S, for graduate work. In October following, he entered Johns Hojikins University as a graduate student in Chemistry, ana was awarded one of the Horth Carolina Scholar- ships. His m.inor subjects are Physical Chemistry and Biology. -58- This manuscript is the property of the Johns Hopkins University, but the literary rights of the author must be respected in any use made of it. 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