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TOXICITY OF BIKUKULLA FORMOSA (WESTERN BLEEDINGHEART) '

By O. F. BLACK, Senior Biochemisty W. W. EGGLESTON, Assistant Botanist, and J. W. KELLY, Junior Biochemist, Office of Drug and Related , Bureau of Industry, United States Department of Agriculture INTRODUCTION Three different native plants of the genus BikukuUa (also known as ) have previously been studied in this laboratory and reported on with respect to their poisonous properties and danger to grazing animals.^ One of these, B, cucullaria, was found to contain an intensely poisonous alkaloid and to be a grave danger to animals on ranges where it may occur. The other two, B. canadensis and B. eximia, although found to contain alkaloids in appreciable quantity, could be classed as harmless, since their basic contents were not toxic. Still another member of this group of plants, B, formosa, has been found growing wild in the West and is the subject of the present paper. BOTANICAL DESCRIPTION OF BIKUKULLA FORMOSA BikukuUa formosa (Andr.) Co ville. Western bleedingheart. (Fig. 1.) A smooth, succulent perennial with thick fleshy branching rootstock; stemless; flowering stalks several, 6 inches to 2 feet high; clusters twice branched, slightly taller than the ; leaves twice ternately dissected, long-stemmed; oroUa flattened, ovate-cordate at the base, the four 7 to 9 lines long, united up to the middle, withering persistent about the pod, crests of inner petals tubular, conspicuous; two, scalelike; in two sets of three each; stigma one, 2-lobed; seed pod 1-celled. Shady woods of British Columbia (where ñrst discovered by Menzies), south through the lower mountain woods of western and , the coast ranges of to Alameda County, and in the to Tulare County. More at home in the moist climate from British Columbia to Oregon. CHEMICAL EXAMINATION Although most of these plants have been the subject of more or less chemical study, little is known concerning the physiological properties of their constituents. Apparently all of them contain alkaloids, but in few instances have these been investigated physio- logically. The alkaloids of BikukuUa formosa were studied by Heyl,^ who, working with roots of plants obtained from one of the German botanical gardens, was able to isolate protopine and two other crystalHne alkaloids. Protopine is a well-known alkaloid foimd among the numerous constituents of opium and also in a variety of plants.

1 Received for publication Dec. 31,1929; issued May, 1930. 2 BLACK, O. F., EGGLESTON, W. W., KELLY, J, W., and TURNER, H. C. POISONOUS PROPERTIES OF BIKUKULLA CUCULLARIA (DUTCHMAN'S-BREECHES) AND B. CANADENSIS (SQUIRREL-CORN). Joui. Agr. Research 23: 69-78, illus. 1923. EGGLESTON, W. W., BLACK, O. F., and KELLY, J. W. A BOTANICAL AND CHEMICAL STUDY OF BIKUKULLA EXIMIA, WITH A KEY TO NORTH AMERICAN SPECIES OF BIKUKULLA. JouF. AgT. Research 39: 477-481, illus. 1929. 3 HEYL, G. UEBER DIE ALKALOIDE VON (ANDR.) D. C. Arch. Pharm. 241: 313-320. 1903.

Journal of Agricultural Research, Vol. 40, No. 10 W^hington, D. C. May 15, 1930 Key No. G-729 (917) 918 Journal of Agricultural Research Vol, 40, No. 10

FIGURE 1.— of Bikukulla formosa May 15,1930 Toxícüy of Bikukulla formosa 919

It is physiologically active, producing local anesthesia and, in mam- mals, narcosis and convulsions, but it has no extensive use in medicine. The other two alkaloids were not named by Heyl, and nothing is known concerning their physiological properties. In June, 1928, a quantity of Bikukulla jormosa was collected in the Cascade Range about Fish Lake ranger station, Santiam National Forest, Oreg. The tops were dried and sent to Washington, D. C, where they arrived in fair condition. Eight hundred grams of this material, ground to a moderate fineness, was packed in a percolator and moistened with 80 per cent alcohol weakly acidified with acetic acid. After standing 24 hours it was exhaustively percolated with alcohol of the same strength, and the solution thus obtained was concentrated by vacuum distillation to about 500 c. c. A moderate quantity of 10 per cent lead acetate solution was added, the precipitate filtered, and the excess of lead removed by dilute H2SO4. The resulting solution was partially neutralized with solid CaCOs, then concentrated to a volume of about 300 c. c. This extract was made strongly alkaline with Na2C03, resulting in a voluminous precipitate, which was removed by filtration and the filtrate shaken out twice with ether. Further treatment with ether and other solvents failed to remove any further basic material, showing that the extraction had been complete. The precipitated bases and residues from the ether extractions, which dried to a brown sirupy material, were united and treated with hot benzol, which left a slight quantity of black tarry substance undissolved. The crude basic material prepared by this procedure weighed 8.3 gm., or just over 1 per cent of the plant material. Heyl ^ stated that he obtained 3 per cent of crude alkaloid, but as he worked with the roots alone, the difference between his results and the writers' may well have been due to the presence of a much greater percentage of alkaloid in the roots than in the tops. The crude basic material, when freed from the solvent and dried, consisted of a light-brown amorphous gummy mass which grew darker in color on standing in the desiccator and responded to all the ordinary tests for alkaloids. It showed no tendency to crystallize, although Heyl reports that under the same conditions his material yielded crystals of protopine. The crude preparation described was sub- jected to a number of methods of purification, but neither through these nor by the use of any of the ordinary solvents or combinations of such solvents could any well-defined separation or crystallization be brought about. PHYSIOLOGICAL TEST OF THE EXTRACT Since the main object of this investigation was to gain a practical knowledge of the toxicity of the plant to animals, the crude basic material afforded a starting point. Two-tenths of a gram was dissolved in very dilute hydrochloric acid and the volume made up to 5 c. c. A white mouse weighing 21 gm. was given 0.25 c. c. of this solution by subcutaneous injection. The animal remained in a quiescent, semistupified condition for 15 minutes and was then seized with convulsions which lasted intermittently for 30 minutes,

* UEYL, G. Op. cit. 920 Journal of Agricultural Research voi. 40, No. 10 when it died. The lungs were collapsed, the extremities blue, the heart distended, and death was probably due to respiratory paralysis. By gradually diluting the solution, progressively weaker doses of the base were administered to mice in the same way until a dilution was reached that failed to prove fatal. In all cases the symptoms of poisoning were the same. From this procedure it was determined that the lethal dose of the crude alkaloids lies between 5 and 2.5 mgm. for a mouse of about 20 gm. in weight; or, calculated on the basis of percentage of alkaloids in the plant, it would be represented by from one-half to one-fourth gram of dry plant. Although the toxicity of the plant to mice does not prove that it is equally poisonous to other animals, it is evident that BikukuUa formosa should be placed in the category of poisonous plants and should be regarded as a potential danger to livestock wherever it is found.