Elimination Studies on a Preparation of Injectio Digitalis U. S. P. XII*

SCIENTIFIC EDITION

231

REFERENCES (1) Hoelscher, Helena, and Bacon, F. J., A m . Perjumer, 25 (1930), 249; also THISJOURNAL, 19 (1930), 624. Experimental data, however, did not appear here or in the M.A. thesis (unpublished) of Miss Hoelscher (“Histological Studies of Genus Menthu,” Western Reserve Univ., Cleveland, O., May, 1930). (2) Tunmann, 0..Ber. deut. phurm. Ges., 18 (1908), 491-540. (3) Anon., Pharm. Monutsh., 13 (1932). 27,28. (4) Tschirch, A., and Oesterle, Anut. Atlas, 1895-1900. (5) Tschirch, A., and Tunmann, 0.. Arch. Pkurm., 239 (1901), 7-16. (6) Tunmann, 0..Phurm. Ztg., 52 (1907), 353, 354.

(7) Pater, B., “Die Heilpflanzenversuchsanstalt der Landwirtschaftlichen Akademie in Kolozsvar,” 1914, Heft 1, 47 pp.; through Ann. Rept. Essential Oils, Synthetic Perjumes, etc., Schimmel & Co., 1914-1915, p. 65. (8) Tschirch, A. Handbuch der Pharmakognosie. lte. Aufl. Bd. 2. Teil 2. Leipzig: Verlag von Chr. Herm. Tauchnitz. 1917. (9) Hesse, F., “Ueber die Groessenverhaeltnisse und Inhaltsbestandteile der Haare einiger offizineller Manzen.” Diss. Wuerzburg: Druck Franz Standenraus, 1916, 61 pp. (10) Brandt, W., Proc. Int. Bot. Cong. (Fourth), Ithaca, N. Y . ,1926, vol. 11.. 1362-et seg. (11) Sievers, A. F., J . Agr. Research, 1 (1913). 129-146.

Elimination Studies on a Preparation of Injectio Digitalis u. s. P. XII* By Marvin L. Pabst and George F. Cartland

The Twelfth Revision of the United States were extracted with distilled water. The aqueous Pharmacopmia, which became official on extract was filtered and extracted vjith chloroform. The chloroform extract was concentrated to a small November 1, 1942, lists for the first time a volume and precipitated by the addition of petrosterile aqueous solution of digitalis for intra- leum ether. The amorphous precipitate containing venous use, “Injectio Digitalis.” This had the cardioactive glucosides was dissolved in alcohol, been under consideration for many years. diluted with water, clarified and assayed by the inI n 1929, Hatcher and Haag (1) described a travenous cat method (2). Phosphate buffer as recommended by Levy and Cullen (3) was added method of preparation which was recom- limiting the NapHPO, part so that the pH was bemended as a basis for further study should it tween 6.5 and 7.0. The solution was diluted so t h a t be decided to admit an injectable digitalis each cubic centimeter contained 0.33 U. S. P. Digito the U. S. P. The purpose of this paper is talis Unit, the alcohol adjusted to 10% and sterilized to report the results of studies on the rate of by Berkefeld filtration. The glucosidal complex assays approximatdy 1 U. 8.P. Digitalis Unit per elimination following intravenous adminis- 1.8 mg. Although it is possible t o prepare solutions tration to cats of a sterile solution of digitalis more highly concentrated than 0.33 U. S.P. Digiprepared using the method outlined by talis Unit per cubic centimeter, such solutions are likely to develop troublesome precipitates in the Hatcher and Haag. EXPERIMENTAL

Description of Digitalis Solutions.-The Injectio Digitalis’ used in these studies meets the U. S. P. specifications and was prepared as follows: The dried and powdered leaves of Digitalis purptreu *Received April 12, 1943, from the Research Laboratories, The Upjohn Company, Kalamazoo, Mich. Sterile Solution Digitalis-Upjohn lot 7984.

ampul. The 10% alcohol serves as an excellent preservative and also helps t o stabilize the solution against delayed precipitation. Digitalis purpureu, as compared to Digitalis lanab. has offered greater difficulties in attempts to study its chemical composition by isolation and crystallization of the individual glucosides (5). However, the work of Cloetta (6) on a glucosidal fraction comparable t o the one described here indicates that the chief active constituents are gitalin, bigitalin (gitoxin) and digitoxin.

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Since, in the preparation described above, yields are sacrificed to purity, it was considered desirable to make control elimination studies on the official Tincture of Digitalis U. S. P. XI1 which contains all of the active glucosides of dried digitalis leaves. This was prepared from official U. S. P. Digitalis Reference Standard, 1942. A s an additional control, similar elimination studies were carried out on Solution’ Digilanid, N. N. R . (4) which is a glucosidal TABLE I. PRELIMINARY ELIMINATION STUDIESO N THREE DIGITALIS PREPARATIONS Interval in Days Sterile Solution Digitalis Tincture Digitalis U. S. P. Digilanid N. N. R.

1

2

4

6

14

40(3)a 77(5)

76(3)

57 (2)

95 (3)

63 (2)

59 (2)

78 (2)

34 (2)

50 (2)

46 (3)

51 (2)

55 (2)

x(2)

106 (2)b

Per cent of initial dose eliminated during interval (number of cats in parentheses): Initial dose = 75% of average lethal dose. b A cat above average in resistance to the digitalis preparation is responsible for this apparent elimination of greater than 100yo of the initial dose.

complex of Digitalis lanata in an aqueous solution containing alcohol 28% and glycerine 10%. Procedure.-The average lethal dose per Kg. was first determined for each of the digitalis preparations using the U . S. P. XI1 cat method (2).

83F 84F 85M

0

1.96 2.02 1.95

2.62 2.70 2.61

14 14 14

1.97 2.00 2.08

1.97 2.01 2.02

starved during the final 24 hr. and again weighed. The average of the initial and final weights was used in calculating the final injection and the percentage elimination. At the end of the desired intervals the cats were killed by the intravenous injection of the digitalis solution allowing a period for the injection as near as possible to that specified by the u. P. Each of the major series (Tables 11, 111, IV) was completed within a period of 17 days during which the average lethal dose per Kg. was again determined on a parallel group of 6 cats. The amount of digitalis solution eliminated was calculated for each cat by subtracting the calculated fatal dose based on parallel assay and average weight from the total volume of solution actually injected at the beginning and end of the interval. Results.-Preliminary studies were carried out to estimate the percentage elimination of each of the preparations after intervals of 1,2, 4, 6 and 14 days. The results are given in Table I with the number of cats indicated in parentheses. From the results obtained, it was decided to study the major series at intervals of 2 , 6 and 14 days, the results of which are recorded in Tables 11, 111 and IV. The mortality a t the initial injection of 70% to 75y0 of the fatal dose was surprisingly low: 1 in 19 for Solution Digitalis lot 7984; 2 in 20 for Tincture Digitalis U. S. P.; 1 in 19 for Solution Digilanid N. N. R.

s.

2.89 2.04 2.95

3.61 3.68 3.70

5.51 4.74 5.56

1.90 1.06 1.86

73 39’ 71

Average 72% in 14 days

In parallel assay the L. D. (average of 6 cats) was found t o be 1.83 cc./Kg.

The cats were starved for 24 hr. and weighed. Under light ether anesthesia and using aseptic precautions 70% to 75y0 of the calculated fatal dose was injected into the femoral vein over a period of 2 to 3 min. The cats were kept in individual cages during the period allowed for digitalis elimination,

DISCUSSION

A comparison of Tables I1 and IV indicates a similar rate of elimination for the purified glucosidal fraction obtained from Digitalis purpureu (Solution Digitalis lot 7984) and that of Digitalis lanata

SCIENTIFIC EDITION

233

TABLE III.-ELIMINATION OF TINCTURE OF DIGITALISU. S P. XI1 (Tincture prepared from U. S. P. Digitalis Reference Standard, 1942)

Initial Injection Cat No. Weight, Dose, and Sex Kg. Cc. 143 F 2.44 1.11 144 F 2.34 1.06 145 F 2.26 1.03 2.02 0.92 156 M 147 F 2.50 1.14 148 F 1.82 0.83 137 M 1.98 0.90 138 F 2.16 0.98 139 F 0.85 1.86 2.42 1.10 140 F 141 F 3.00 1.36 142 M 2.90 1.32 131M 2.08 0.95 2.30 1.04 132 M 133 F 2.36 1.07 155 M 0.85 1.88 1.96 0.89 135 F 2.16 0.98 136M 0

c -

Interval, Days 2 2 2 2 2 2 6 6 6 6 6 6 14

14 14 14 14 14

Final InjectionAverage Lethal Weight, Dose, Kg. Cc. 0.92 2.30 2.24 0.95 1.08 2.16 0.70 1.93 0.72 2.39 0.66 1.75 1.98 1.11 1.98 2.19 0.64 2.22 0.68 1.91 1.96 2.41 0.83 2.40 0.99 2.72 2.86 2.86 1.42 2.82 2.09 1.11 2.10 0.83 2.28 2.29 0.80 2.34 2.35 0.83 2.12 2.00 0.67 1.98 1.97 1.04 2.12 2.14

Weight, Kg. 2.16 2.14 2.06 1.84 2.28 1.68

Fatal dose Calculated on Basis of Average Weight, Cc. 1.40' 1.37 1.32 1.18 1.46 1.07 1.21 1.34 1.17 1.47 1.74 1.74 1.27 1.40 1.43 1.22 1.20 1.31

Per Total InAmount Cent of jected, ElimiInitial nated Initial Dose and Over Final Interval Elimin Cc. in Cc. inated 2.03 0.63 57 60 0.64 2.01 0.79 77 2.11 0.44 48 1.62 0.40 35 1.86 0.42 51 1.49 2.01 0.80 89 1.62 0.28 29 0.36 42 1.53 0.46 42 1.93 0.61 45 2.35 1.00 76 2.74 2.06 0.79 83 0.47 45 1.87 0.44 41 1.87 0.46 54 1.68 0.36 40 1.56 0.71 72 2.02

Elimination during Period

Average 55% in 2 days

Average 54% in 6 days

Average 56% in 14 days

In parallel assay the L. D. (average of 6 cats) was found to be 0.61 cc./Kg.

(Solution Digilanid N. N. R.). Both are eliminated to the extent of approximately 75% over a period of 14 days. However, the tincture prepared from the U. S. P. Digitalis Reference Standard, 1942 (Table 111) is more persistent in its action since only slightly more than half of it is eliminated over a period of 14 days. From Table 11, the amount of Solution Digitalis lot 7984 eliminated is 63y0in 2 days, but only 4070 in 6 days. Irregularities such as this have also been observed by others (1, 7), and are considered t o

signify a cumulative digitalis effect but not necessarily an increase in the direct cardiac action. SUMMARY

A preparation of Injectio Digitalis U. S . P. XII is described and its rate of elimination after intravenous injection in cats has been studied. Parallel control studies are reported On a Tincture Of Digita1is prepared from the official u. s. P. XI1 Digitalis Ref-

TABLEIV.-ELIMINATIONOF SOLUTION DIGILANIDN. N. R.

Initial Injection Cat No. Weight, Dose, Cc. Kg. and Sex 1.65 118F 1.92 1.69 119F 1.96 1.77 121F 2.06 1.84 122F 2.14 2.15 123M 2.50 1.70 1.98 130F 1.74 112F 2.02 1.84 113F 2.14 114F 2.14 1.84 1.72 115M 2.00 1.94 1.67 116F 1.93 117F 2.24 1.57 1.82 106F 1.77 107M 2.06 1.81 108F 2.10 1.86 109F 2.16 1.55 110M 1.80 1.67 lllF 1.94 0

Val, Days 2 2 2 2 2 2 6 6 6 6 6 8 14 14 14 14 14 14

Fatal dose Calculated Final Injection-on Basis Average Lethal ofAverage Weight, Dose, Weight, Weight, Cc. Kg. Cc. Kg. 1.82O 0.99 1.72 1.82 1.87 1.87 1.14 1.78 1.62 1.93 1.80 1.93 2.05 2.05 1.00 1.96 1.32 2.41 2.32 2.41 1.90 1.90 1.43 1.82 1.05 1.94 1.86 1.94 1.16 2.11 2.08 2.11 1.85 2.14 2.14 2.14 1.63 1.96 1.92 1.96 1.87 1.87 1.00 1.80 1.27 2.25 2.25 2.26 1.81 1.75 1.80 1.81 2.08 2.08 1.40 2.10 1.53 2.00 1.90 2.00 2.18 2.18 1.76 2.20 1.87 1.87 1.52 1.94 1.77 1 95 1.95 1.96

In parallel assay the L. D. (average of 6 cats) was found to be 1.00 cc./Kg.

Total Per InAmount Cent jected, Elimiof Initial nated Initial and Over Dose Final Interval Elimiin Cc. in Cc. nated 0.82 50 2.64 2.83 0.96 57 3.39 1.46 82 2.84 0.79 43 3.47 1.06 49 3.13 1.23 72 0.85 49 2.79 3.00 0.89 48 3.69 1.55 84 3.35 1.39 81 0.80 48 2.67 3.20 0.95 49 96 1.51 3.32 62 3.17 1.09 1.34 74 3.34 1.44 77 3.62 3.07 1.20 77 89 3.44 1.49

Elimination during Period

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erence Standard, 1942, and also on Solution Digilanid N. N. R. After the administration of approximately 75y0of the lethal dose, the average elimination in 14 days, expressed in per cent of the initial dose, was 72y0 for Injectio Digitalis U. S. P. XII; 79y0 for Solu-

tion Digilanid N. N. R.; and 56y0 for the Tincture of Digitalis U. S. P. XII. Thus, the purified glucosidal preparations from Digitalis purpureu and lunatu have similar rates of elimination but differ from the crude tincture which exerts a more prolonged effect.

REFERENCES (1) Hatcher, R. A., and Haag, H. B., THIS (4) “New and Non-official Remedies,” 1942, JOURXAL, 18 (1929), 670. p. 270. (2) “Pharmacopoeia of the United States,” ( 5 ) Stoll, A., THISJOURNAL, 27 (1938), 761. Twelfth revision, p. 510. (6) Cloetta, M.. Arch. exptl. Path. Pharmakol., (3) Levy, R. L., and Cullen, G. E., J . ExptZ. 112 (1926), 261. (7) Gold, H., Arch. Internal Med., 32 (1923), 779. Med., 31 (1920),267.

A Preliminary Study of

Piper marginaturn

Jacq. *

B y Emilia Hoyo de Ndiiez and Carl H . Johnsont

’

Piper marginaturn Jacq. (Fam. Piperacme) is a Puerto Rican plant to which a number of medicinal properties have been attributed. de Grosourdy (1)stated that the leaves of this plant act as a hemostatic in cases of traumatic hemorrhages, lesions, epistaxis, hemoptisis and in uterine hemorrhages. He also claimed that in diarrheas and in the chronic dysenteries so common in the tropics an infusion of the leaves is very eff ective. The present investigation was carried out to determine the active constituents present in the leaves, especially those responsible for the medicinal value of the plant. A review of the literature revealed that no phytochemical work has been reported on this species, all the work recorded being of a botanical nature. The leaves’used in this investigation were collected in Puerto Rico during October and December of 1940. They were air-dried on screens in a well-ventilated room and then were ground to a No. 60 powder. The ground material was of a dark green color with a very aromatic odor and, on rubbing * This paper is based on a thesis presented to the Graduate Council of the University of Florida, Gainesville, Fla., in partial fulfillment of the requirements for the degree of Master of Science in Pharmacy. Received Sept. 4, 1942. t Assistant Professor of Pharmacognosy and Pharmacology, University of Florida.

between the fingers, left a greasy film suggesting the presence of a fatty substance. EXPERIMENTAL GENERAL INVESTIGATION

In a general study using the methods of the United States Pharmacopaeia, Eleventh Revision, the following results were obtained: Total ash.. . . . . . . . . . . . . . . . . . . . . . . . . .10.690/, Acid-insoluble ash.. . . . . . . 2.91y0 . .14.66% Moisture, oven method.. Moisture, toluene method. . . . . . . . . . . . 8. 55y0 By treatment of a 20-Gm. sample in a Soxhlet apparatus with the solvents listed in succession the followingamounts of extractives were obtained: Petroleum benzin. . . . . . . . . . . . . . . . . . . .14.99% Ether. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.16y0

Total extractives.. . . . . . . . . . . . . . . . . ..48.63yo An aqueous extract of a small amount of the material gave a positive reaction for tannin with ferric chloride T. S. and for reducing sugars with Fehling’s solution. An acidulated water extract reacted positively with the alkaloidal reagents, iodine T. S . and mercuric potassium iodide T. S. Tests for starch and saponins in the solid material gave negative results. DETAILED INVESTIGATION

The Stas-Otto Process.-Fifty grams of the material was heated with alcohol acidified with tartaric acid, filtered, evaporated, dissolved and again evaporated. When this residue was dissolved in acid water and shaken with ether no alkaloids I,