Part 1

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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

Transcriber's Note: Underscores around words indicates italics while an underscore and curly brackets in an equation indicates a subscript.

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 Societe 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.

Ueber Mategerbstoff, Reuchlin (Dissertation) 1904.

SOME CONSTITUENTS OF THE POISON IVY PLANT.

(RHUS TOXICODENDRON)

INTRODUCTION.

Plants belonging to the natural order Anacardiaciae (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 Anacardiaciae, 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.

FOOTNOTES:

[1] Chesnut. Bull. No. 20, U. S. Dept. of Agr., Div. of Botany.

[2] Man. of Bot., p. 119.

[3] Man. of Bot., p. 119.

[4] Brook. Med. Jour., June, 1897.

[5] Rein, The Ind. of Jap., p. 338, et seq.

[6] H. Yoshida on Urushi Lacquer, Jour. Chem. Soc., 1883, p. 472.

[7] Ann. de Chem. et de Phys., Series VII, Vol. 12, p. 125, 1897.

[8] Amer. Jour. Pharm. 78, p. 53, Feb., 1906.

[9] An account of Pfaff's work will be found in another part of this paper.

[10] Amer. Jour. Med. Sci. 51 (1866), p. 560.

[11] Med. and Surg. Rep. 17, Nov., 1867.

[12] Jour. Exp. Med. 2 (1897), p. 181.

[13] Ibid.

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 fuer 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."

FOOTNOTES:

[14] A free translation of this paper is given in Amer. Jour. Pharm. for 1858, p. 542.

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.

FOOTNOTES:

[15] Proc. Amer. Pharm. Assn. 1865, p. 166, and Amer. Jour. Pharm. 1866, p. 4.

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 C_{21}H_{30}O_{4}Pb. 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 deg. 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 Anschuetze 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 deg. to 150 deg.

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.