A Study of a Depressor Principle of Veratrum viride

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P U R D U E U N IV E R S IT Y

THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION

--------- Arthur-George Zupko-

ENTITUED_________ A

STUDY OF A

nRPRRSSOR PRINCIPLE

___________ OF VERATRIM VIRIDE____________

COMPLIES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES

AND IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS

FOR THE DEGREE OF

-Doctor-of Philosophy

Jt

a»^ «

P r o f e s s o r in C h a r g e o f T h e s is

H ead of S chool or D epartm ent

August 9_____ 19 49

TO THE LIBRARIAN:----IS Vid-t" THIS THESIS. IS NOT TO BE REGARDED AS CONFIDENTIAL.

FKOFBSSOR I N OHA

RHGISTRAH FOHM 10— 7-4 7— 134

A STUDY OF A DEPRESSOR PRINCIPLE OF VERATRUM VIRIDE

A Thesis Submitted to the Faculty of Purdue University by Arthur George Zupko In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy

August, 1949

ProQuest Number: 27712256

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 27712256 Published by ProQuest LLC (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

ACKMMJEDGBŒNTS

The writer is indebted to Prof. L. D. Edwards without whose encouragement> criticisms and guidance this work would not have been possible.

Thanks are also extended to the various members

of the staff of the School of Pharmacy and to Dr. L. P. Doyle of the Agricultural Experiment Station for aid in interpreting the histopathological sections. The writer also wishes to express his profound gratitude to the American Foundation for Pharmaceutical Education whose financial assistance aided greatly in the pursuance of this work.

TABLE OF CONTENTS Page ...............

INTRODUCTION EXPERIMENTAL PART I.

X

....

2

Isolation and Properties of the Depressor Principle

2

1.

Extraction procedure .........................

2

2.

Testing the residues ..........................

5

3.

Properties of the Depressor Principle

.........

Ô

PART II* Pharmacology of the Depressor Principle ............

IX

1. Effect on Blood Pressure and Respiration ........

11

A.

B.

C. D. E.

Dogs 1. Intravenous injection ............. 2. Intramuscular injection ...........

11 14

Cats 1. Intravenous injection ............. 2. After atropine .................. 3. After ergotoxine .............. 4. After tetraethylammonium bromide...

17 18 22 24

Rabbits 1. Intravenous injection .............

26

Rats 1.

29

Intravenous injection .............

Roosters 1. Intravenous injection

2. Effect on the Heart Rate .......................

31 31

A.

Intact dogs .............................

32

B.

Isolated frog heart .................. 1. Hypodynamie heart..

33 34

C.

Isolated turtle heart ....................

35

TABLE OF CONTENTS (Continued) Page 3*

U»

5#

Effect on Coronary Flow Rabbits

35

B*

Rats

36

Effect on Blood Vessels

36

A«

Hind leg of pithed frog

37

B#

Isolated hind leg of rabbit •••••••••♦•#♦

3B

Effect on Capillaries

39

Meso-appendix of rat «.•••••••••••••••»#«

Effect on Smooth Muscle ofIntestine••••••••«« A#

Guinea pig ileum *«••••••••«••••••••••••♦

39 AO AO

7.

Effect on Blood Sugar

A1

8»

Determination of the MinimumEffective Dose •••

42

9»

Acute Toxicity A. LD50 in rats, subcutaneously B. ID50 in rats, intravenously C. ID50 in mice, subcutaneously ••••••••«••••

A4 A4 47 48

10.

DISCUSSION

35

A,

A. 6.

....

Chronic Toxicity

.....

49

A. Histopathological study of rats after 60 doses intramuscularly

50

B. Histopathological study of rabbits after 30 doses intravenously

57

C* Histopathological study of rabbits after 45 doses intravenously .............

58

....

60

SUMMARY AND CONCLUSIONS..........

62

BIBLIOGRAPHY

64

ABSTRACT

LIST CF TART.ES Table 1

2 3 4 5 6 7 8 9 10 11

Page Effect of Oral Administration of the Depressor Princi­ ple on the Blood Pressure of Normal and Hypertensive Intact Unanesthetized Rats

7

The Effect of Intravenous Injections of the Depressor Principle on the Blood Pressure of Cats

19

The Effect of an Intravenous Dose of the Depressor Principle on the Blood Pressure of a Rabbit .****...*#*

27

The Effect of the Depressor Principle Given Intra­ venously on the Blood Pressure of Anesthetized Rats .*#

30

The Effect of the Depressor Principle on the Perfusion Rate Through the Hind Leg of a Frog

38

The Effect of An Intraperitoneal Injection of the Depressor Principle on the Blood Sugar of Rats ••♦••••»

43

The Effect of a Subcutaneous Injection of the Depressor Principle on the Blood Pressure of Normal Rats ,•••••♦♦

45

The Effect of a Subcutaneous Injection of the Depressor Principle on the Blood Pressure of Hypertensive Rats.**

46

Results of Subcutaneous Injections of the Depressor Principle into Albino (Wistar strain) Rats ...*.**#..**

47

Results of Intravenous Injections of the Depressor Principle into Albino (Wistar strain) Rats

48

Results of Subcutaneous Injections of the Depressor Principle into Mice

49

LIST OF FIGURES Figure I I—Â II TIT IV V VI

Page The Effect of the Depressor Principle on the Blood Pressure of a Normal Dog ..*******»».»***.*** *»**#*#**

13

The Effect of An Intramuscular Dose of the Depressor Principle on the Blood Pressure of a Normal Dog ...***»

16

Effect of the Depressor Principle on the Blood Pressure of a Cat

18

The Effect of the Depressor Principle on the Blood Pressure of a Cat After Atropinisation ..**••••••••••••

21

The Effect of the Depressor Principle on the Blood Pressure of a Cat After Ergotoxine

23

The Effect of the Depressor Principle on the Blood Pressure of a Cat After Tetraethylammonium Bromide *****

25

The Effect of the Depressor Principle on the Blood Pressure of a Rabbit ********************************

28

LIST

ça?

PHOTOMICROGRAPHS

Photo­ micrograph 1

2 3

Page

Rat kidney showing granular degeneration of tubular epithelium and apparently a shrinkage of vascular tufts in the glomeruli ********************************

53

Rat kidney showing tubular epithelial degeneration in some tubules *************************************

54

Rat spleen showing paucicity of splenic pulp and dilatation of small blood vessels and sinusoidal spaces ********************************************

56

1 INTRODUCTION ESM The dried rhizome and roots of Veratrum viride (Green Hellebore, American Hellebore) have long been used as a cardiac depressant and hypotensive agent, especially in eclampsia and other conditions of high blood pressure.

The injection of a physiological dose of an aqueous

extract of Veratrum viride is followed by a characteristic lowering of the blood pressure of long duration, transient slowing or stoppage of the respiration and a slowing of the heart rate.

Up to the present

time the principles fran Veratrum viride responsible for its physio­ logical action have not been isolated, although much work has been done in this field since 1879 when Wright (l) reported the isolation of the alkaloids jervine, rubijervine and pseudojervine* Many workers have contributed to the present day knowledge of the alkaloids and principles present in Veratrum viride. Notable among these are Seiferle, Johns and Richardson (2) who were the first to isolate germine, and more recently, Jacobs and Craig (3# 4) who have isolated isorubijervine, veratrosine, veratramine and a new unnamed alkaloid.

However, these isolated constituents have been

shown to be relatively inactive or comparatively weak in activity, especially with respect to vasodepression of long duration. Previous workers have first isolated a principle, purified it, worked out its chemical configuration and then tested it pharmaco­ logically.

In an effort to minimize the problem of working with

relatively inactive principles, the present study was undertaken to isolate a principle which would show activity similar to that of the aqueous extract of the whole drug and thus merit further chemical study*

2 EXPERIMENTAL

Part I.

Isolation and Properties of the Depressor Principle

1. Extraction ZÎEfiSSâüEâ* A variety of coarse and powdered samples of crude Veratrum viride, generously supplied by Irwin, Neisler and Company, Decatur, Illinois, were used in this study*

A majority of the samples contained (>% or more

of acid-insoluble-ash* Extractions were done on a small scale, one to three kilograms of crude drug being macerated with 95% ethyl alcohol, shaken frequently over a twenty-four hour period, then filtered by suction*

This process

was repeated three times over a seventy-two hour period with each sample.

The combined filtrates were concentrated to a syrupy consis­

tency using a steam bath and reduced pressure.

The temperature of the

alcoholic filtrates during concentration ranged from 20-25° C,

The

syrupy alcoholic concentrate was air-dried until all the alcohol was removed. The dried extract was taken up in distilled water at room tem­ perature , agitated for two hours, then filtered. used did not exceed one liter. of the resinous material,

The volume of water

The filtration removed a large portion

Hie filtrate was concentrated over a steam

bath under reduced pressure in order to make the volume easier to handle. The concentrated aqueous filtrate was washed with ether several times to remove aromatic substances, mixed with freshly prepared lead hydroxide and filtered to remove the organic acids.

The fresh lead

hydroxide was prepared by mixing aqueous portions of lead acetate and

3 concentrated sodium, hydroxide, then -washing the precipitate several times with water*

The resultant filtrate was then made alkaline to a

pH of 7*2 with anhydrous sodium bicarbonate.

The alkaline solution

was next shaken out with 50 cc. portions of ether at least six times, each extraction consuming about thirty minutes.

The two distinctly

different solutions, ether (I) and aqueous (II), were then further extracted individually* The ether solution (I) was evaporated to dryness at room temper­ ature, taken up in anhydrous ether, filtered and vacuum desiccated immediately. and filtered.

The dried residue was next dissolved in distilled water The residue (Y) was saved for testing purposes.

The

filtrate was shaken out with ether 5 to 6 times, the aqueous portion (B) being air-dried and saved for testing.

The ether solution (X) was

evaporated to dryness, taken up with a small amount of anhydrous ether, and vacuum desiccated. The aqueous portion (II) was shaken out with a mixture of two parts chloroform to one part alcohol several times in a separatory funnel.

The chloroform—alcohol solution was separated from the aqueous

portion, evaporated at room temperature, and the residue (C) saved for testing.

The aqueous portion remaining was air-dried and this residue

(A) also was saved for testing. A total of five distinct residues was obtained from each sample and set aside for depressor activity tests. The extraction procedure may best be illustrated by the following flowsheet$

4 FLOWSHEET OF EXTRACTION Crude Drag (1-3 kilos) 4 Macerate with 95$ ethyl alcohol (at least three times) Filter (suction) Concentrate to thick svrup (steam bath and reduced pressure) ■

•

.

-

■

.

.

.

Dry in air (remove all alcohol) 'It... Take up in water (room temperature-not more than one liter) Fil ter (suction)- removes resinous materials 4r Concentrate (steam bath and reduced pressure) Ar Wash ^several times with ether (removes aromatic substances) MIy with fresh lead hydroxide and filter-removes organic acids, etc# '|r Make a^kfllnne to pH 7.2 with sodium bicarbonate 4f : ' Shake out with ether 5 to 6 times (thirty minutes for each) 4f ' ^ . . .

.

Aqueous solution (ll) Shake outi With 2:1 CHCl^-Alc. (several times

Ether solution (I) 4f Evaporate to drvness Take ^up in anhydrous ether. filter

Aqueous (A) rtfït) (ts

0Hgl3-£L^£l

Vacmim desiccate filtrate

Evaporate (test)

Take up in water at room temperature t 1 - 4 Residue (X) Soluble part 4f * , Shake out with ether 5 to 6 times Aqueous (B) (test)

v Ether 'If Evaporate to dnmasa "

Take up in anhydrous ether Vacuum desiccate (X) residue (test)

5 2.

Testing of the Residues» Using the apparatus described by Williams, Harrison and Grollman

(5) for measuring systolic blood pressure in the tail of the intact un­ anesthetized rat, all five residues were given a preliminary screening for vasodepressor activity• One group of ten normal Wistar strain Albino rats and a second group of ten Wistar strain Albino rats, made hyper­ tensive by bilateral constriction of the renal artery as described below, were used in testing an individual residue* The rats were made hypertensive in the following manner: Young rats of either sex weighing about 150 grams were selected and all food withheld from them for forty-eight hours.

On the day of the operation

the rats were anesthetized with 50 mg./kg. of sodium pentobarbital given intraperitoneally. After sufficient anesthesia was obtained each animal was operated on as follows: 1* The animal was fixed to an operating board which was equipped with a taut rubber band on each side for holding retractors* 2* The abdominal hair was removed from the xyphoid process to the pubic region. 3* A single midline incision was made through the skin and peritoneum* 4* The viscera were pressed away from the operation site and under the edge of the incision on the side opposite the operation site* 5* The edge of the incision on the operation side was then retracted. Using small cotton swabs, the renal artery was isolated from the surrounding fat and connective tissue. 6* A paper slip probe was passed under the artery and slight upward pressure was exerted to cut off the blood supply. A change in the color of the kidney from reddish brown to green served to confirm the isolation of the proper vessel. Afine silk thread was then passed under the arteiy by means of a fine suture needle. 7* The suture needle was removed and a 26-gauge platinum wire placed parallel to the artery. The far end of the platinum wire was held by a small artery clamp which was allowed to rest on the operating board; the wire was bent so that the free end wouldlie next to the vessel without having to be held.

6 ôe A double square knot was tied about both the artery and plat­ inum wire. 'Hie platinum wire was then removed and the site gently massaged until a return of redness to the kidney came about, indicating circulation had been restored. 9.

The operation was repeated on the other side in a similar manner#

10* The retractor was removed and the viscera redistributed to the noimal position. Fine silk was used to close both incisions with a simple continuous suture. Hypertension may be expected in 2 to 3 weeks with bilateral con­ striction. After an 18-24 hour period of fasting, the normal blood pressure of each rat was determined.

Varying dosages of the residue being tested

were administered orally and blood pressure readings taken every half hour. Residues A and B were only weakly active, C only slightly more active and Y showed a moderate vasodepressor activity.

The X residue

or final ether extract was the most active vasodepressant by far.

The

X residue, henceforth called the depressor principle, exhibited fairly consistent strong vasodepressor activity in both groups of rats as shown in Table 1. The depressor principle (X residue or final ether extract) was further tested on two cats and two rabbits and gave evidence of suf­ ficient vasodepressor activity to warrant further testing. The (Y) and (C) residues were tested for rodenticidal properties by mixing with various agents and feeding the mixtures to groups of rats. The residues were first tested by intimately mixing finely ground rat pellets with the residue in such proportions that 1 mg. of the residue tested was contained in each gram of ground food.

This mixture

K

1• §) CM in H § 5 a :S ■a 5 m 5 3 3 a 5 is

r

:S3 a e •Ha °

§ § .a m O« g o«

8^ Q$ ! u

s 8

m H

in H

i

3 à § m in H

g

3 S a c-e to » cn o O d d H cn -4 m >0 to O' S

$ $ inO' s ITi O' §

90 150 90 90

a >oo«

v>*" to 8 r-t

90

H

3 s 8

100

S h a g° H °to *

7

8 8 8 8 mo> | 8 to 8 8 1 d

a â

1 o2 3 8 8 §

§ a 3 | m vO -4 g O ■g O« O« d 1 i m o *1 H CM r ■

m m O'

i 3 to o> O d d H i to 3

Ô was fed to a group of six rats vhich were observed over a twenty-four hour period.

The rats ate freely of the mixture and a few showed signs

of some toxicity, i.e. salivation, depression, etc., but no deaths resulted.

Upon increasing the amount of residue per gram of ground food,

it was found that the rats no longer ate liberal portions but wisely separated the food from the residue.

The Y residue exhibited the

greater toxicity. The residues were next tested by incorporating large amounts of each residue in leaf lard and making the mixture into an ointment which was then smeared on rat pellets and fed to the rats.

The identical

procedure was used with white glucose but neither residue showed ideal rodenticidal properties although an occasional death occurred. Rats can apparently overcome a toxic dose of these residues when they are mixed with other materials if sufficient time is allowed for recovery since oral administration of these residues alone, at the same dosage levels, caused death in a majority of the rats. 3. Properties of the Depressor Principles Approximately one hundred samples from different lots of the crude drug were extracted by the procedure outlined.

It was found that one

kilogram of crude drug yielded an average of about 25 milligrams of the depressor principle.

Fresh lots of the drug yielded up to 50 milli­

grams per kilogram while old samples yielded as low as 15 milligrams per kilogram. The chemical nature of the principle has not, as yet, been fully determined.

On the basis of a heavy precipitate vdth Mayer’s reagent

and a positive nitrogen test the principle appears to be alkaloidal

9 in natures

However, aqueous solutions of the depressor principle respond

basic to phenol red but not to phenolphthalein*

Solubilities suggest

that it also may be glycosidal, even though tests vdth Benedict's and Barfoed's reagents were negative. The principle was found to be soluble in water, alcohol, dilute mineral acids, ether, chloroform, acetone, propylene glycol, methanol, propanol and butanol.

It was slightly soluble in benzene and concen­

trated alkalies but insoluble in carbon bisulfide.

The excessive solu­

bility of the principle leads one to believe that sugar is present which makes the substance (basic fonn) soluble in water, a characteristic not usually present in alkaloids* A 6 mg, % aqueous solution of the depressor principle was tested for polarographic activity using an 0,1 M solution of tetraethylaamonium bromide as the electrolyte.

The polarogram showed no reducible

ions under the stipulated conditions and can therefore be considered as polarographically inactive. The principle gave only a few typical color reactions when tested with various reagents. With concentrated sulfuric acid a reddish brown color forms and with Frohde *s reagent a brown color foms which goes to a dark red and then to a violet color over a one-half hour period. A precipitate forms with bromine water but when the bromine volatil­ izes the precipitate disappears. following agents:

It failed to react with any of the

formaldehyde, Millon1s reagent, Schiff fs reagent,

ferric chloride, 5% phenol, silver nitrate, and sodium nitroprusside* A melting point deteimination was done on the depressor principle extracted from each sample.

An average melting point, via the capillary

method, of 93O"108° C, (uncorrected) was obtained*

10 Preliminary attempts at crystallizing the depressor principle by the use of hydrochloric acid gas, dried picric acid, salicylic acid, benzoic acid and quinine—sulfanilamide by the method of Bibbins et al (6) met vdth little success.

An anhydrous ether solution of dry

hydrochloric acid gas, when added drop by drop to a solution of the depressor principle in anhydrous ether, gave a fine amorphous precip­ itate.

The hydrochloride salt had a melting point range of 1780-165°

(uncorrected) vfcen determined by the capillary method.

It gave the

identical color reactions as did the crude depressor principle. When tested on rats, cats and rabbits the hydrochloride salt lowered the blood pressure but the duration of vasodepressor activ­ ity was considerably less than the crude depressor principle.

It is

possible that the acid caused a splitting off of a chemical linkage necessary for prolonged vasodepression. Aqueous solutions of the crude depressor principle were allowed to stand overnight and were then tested for vasodilator response on cats. It was found that the vasodepressor activity was considerably dimin­ ished, presumably due to a slow hydrolysis of the principle. When the depressor principle was extracted at a pH range of 7.8 to 9.4 using concentrated sodium hydroxide in lieu of anhydrous sodium bicarbonate, the vasodepressor activity was greatly lessened when tested on several rats and two cats. Strong alkalies apparently change the chemical configuration necessary for prolonged vasodepressor activity.

The best results were obtained at a pH of 7.2 using sodium

bicarbonate to alkalinize. In view of the fact that when salts were made of the crude depressor principle much of the vasodepressor activity was lost, it

11 was decided to investigate the pharmacology of the crude depressor principle in detail to ascertain whether or not it merited further ehemieal study»

Part II. Pharmacology of the Depressor Principle 1. Effect on Blood Pressure and Respiration. The outstanding effects of moderate doses of an aqueous extract of Veratrum, viride injected intravenously in animals are a fall in blood pressure of long duration and a transient slowing or stoppage of the respiration.

These same physiological responses were observed

with the depressor principle in experiments on dogs, cats, rabbits, rats and roosters. The duration of vasodepressor activity varied with the species of animal used, the rooster exhibiting only a comparatively transient fall in blood pressure lasting no more than fifteen minutes.

Via

intravenous injection, the depressor principle effectively lowered blood pressure for as much as five hours in the other species. When administered intramuscularly in dogs, the blood pressure was lowered for almost seven hours without toxic manifestations. A transient slowing of the respiration was observed in the dog, eat and rabbit after intravenous injection of the depressor principle. However, the respiration was not slowed appreciably in dogs injected intramuscularly» A. Dogs,: 1. Experiments on three dogs showed that intravenous injec­ tion of the depressor principle effectively lowered blood pressure

12 for more than two hours duration.

The animals were anesthetized with

35 mg./kg. of sodium pentobarbital administered intraperitoneally. The carotid artery was cammlated and the blood pressure recorded by means of a mercury manometer.

Respiration was recorded via a pneumo­

graph about the thoracic cage connected to a writing tambour.

The

femoral vein was isolated for injections. Dog

Male dog weighing 7.00 kg. with a normal blood

pressure of 150 mm. mercury.

After an 0.02 mg./kg. injection of the

depressor principle the blood pressure fell to 75 mm.

Over a 45 minute

period the blood pressure showed a slow gradual rise to 105 mm*

In one

and one-half hours the blood pressure had gone to 125 mm. and in two hours and four minutes the animal had a pressure of 142 mm. The respiration was slowed for a few minutes and returned to normal with great rapidity. The animal had three bowel movements during the test period, perhaps due to a stimulation of gut activity. Dog; #2: Male dog weighing 9.65 kg. with a normal blood pressure of 125 mm. After an 0.02 mg./kg. injection of the depressor principle the blood pressure fell to 60 mm.

In one-half hour the

pressure gradually rose to 95 mm. and in one hour and fifteen minutes had gone to 110 mm.

After a two hour period the blood pressure was

within 5 to 10 mm. of normal. There was a transient slowing of the respiration with a return to normalcy within a few minutes. The dog defecated three times during the course of the experiment.

The effect of the depressor principle on the blood pressure of a normal dog» A shows the noimal blood pressure and the result of an intravenous injection of 0*015 mg*/kg, B, G, D and E show the grad* ual rise in blood pressure over a two hour and ten minute period. Time interval » one minute*

Figure

13

u Dog #3t Female dog weighing 8*29 kg. with a normal blood pressure of 165 mm*

After an intravenous dose of 0*015 mg*/kg* dose of

the depressor principle the blood pressure fell to 65 mm*

In one-half

hour the pressure showed a gradual rise to 85 mm., and in one hour was U 0 mm*

At the end of a two hour and ten minute period the blood pres­

sure was 155 mm. and slowly approaching normalcy.

Figure I illustrates

these changes in blood pressure. As with the other dogs there was a transient slowing of the respiration.

The dog defecated but once during the course of the

experiment. It is apparent that the depressor principle elicits a vasodilator response in dogs under the experimental conditions described# The duration of activity was somewhat over two hours in *11 eases* 2*

Two dogs were given intramuscular injections of the

depressor principle.

The fall in blood pressure was gradual, in one

ease consuming one hour before the maximum lowering was reached.

The

duration of activity with 0*01 mg./kg# of the depressor principle was almost 7 hours and with 0.05 mg./kg* it was well over 9 hours but with some toxic manifestations in the fora of eraesis and cardiac irregu­ larities# The respiration was not appreciably slowed in either dog after the intramuscular injection* Dog

Male dog weighing 6*60 kg. was anesthetized with

35 mg./kg. of sodium pentobarbital administered intraperitoneally* The normal blood pressure of the dog was l60 mm*

After an intramuscular

injection of 0*5 mg*/kg. of the depressor principle there was a slow

15 fall in pressure over a 20 minute period to 75 mm.

Over the next three

hours the dog emesed several times and developed cardiac irregularities* At the end of four hours the hlood pressure was 85 mm.

The dog emesed

again at the end of the fifth hour but the blood pressure remained at a level between 85 to 90 mm*

There was no change in the blood pres­

sure for the next four hours and, at the end of 9 hours, the pressure remained 85 to 90 mm., at which time the experiment was terminated. Dog #5; Female dog weighing 8*65 kg. was anesthetized with 35 mg./kg. of sodium pentobarbital administered intraperitoneally. The noimal blood pressure of the dog was 135 mm. After an 0.1 mg./kg* intramuscular dose of the depressor principle the blood pressure fell very slowly to 80 mm. over a period of one hour.

At the end of two

hours the pressure climbed only 5 to 10 mm. and at the end of four hours was approximately 100 mm.

At the end of six hours the blood

pressure was about 120-125 ma. and slowly approaching normalcy.

After

6 hours and 45 minutes the experiment was terminated and the blood pressure had still not reached the noraal level. Figure I-A shows the changes occurring during the course of the experiment. It is apparent that intramuscular administration of the depressor principle is far superior to any other method of adminis­ tration as evidenced by the gradual fall in blood pressure, little or no respiratory embarrassment and greater duration of activity due to slower absorption*

The effect of an intramuscular dose of the depressor principle on the blood pressure of a noraal dog. A shows the normal blood pressure; B shows the gradual lowering twenty minutes later; C shows the maximum lowering reached over a one hour period; D and B show the gradual rise in blood pressure three and six hours later, respectively. Time interval ■ one minute#

Figure I-À

16

17 B*

Gat%#

X# Experiments on seven cats showed that an intravenous injection of the depressor principle effectively lowered blood pres­ sure for varying periods of time depending upon the physiological state of the animal*

The animals were anesthetized with either sodium phéno­

barbital (170 mg./kg.) or a 1 cc./kg. mixture of 0.03 Qm. sodium pento­ barbital in 1 cc. 25% urethane.

The carotid artery was eannulated and

the blood pressure recorded by means of a mercury manometer. Respir­ ation was recorded by a pneumograph about the thoracic cage connected to a writing tambour.

The femoral vein was isolated for injections.

All the animals exhibited a lowering of blood pressure and a transient slowing of the respiration after an intravenous injection of the depressor principle.

The results are tabulated in Table 2.

Figure II illustrates a typical lowering of blood pressure with slow­ ing of the respiration after intravenous administration of 0*02 mg./kg. of the depressor principle. over three hours.

The duration of activity in this case was

18

Figure II

S

CD

o. e

19 Table 2 The Effect of Intravenous Injections of the Depressor Principle on the Blood Pressure of Cats

Gat Sex Weight Dose Normal No. _ kg* mg*/kg* B.P*

Blood Pressure in mm* Mercury Time in hours è 1 1& 2 2& 3

1

M

2*176

0*02

135

66

90

105

115 125

*•

2

M

1*960

0.025

143

76

96

112

123 136

—

3

F

4*000

0.025

120

66

62

100

105 114

4

M

3*125

0*02

164

76

92

105

115 132

158

5

M

2*640

0,02

156

62

96

118

124 136

144

6

F

2*350

0.03

116

63

80

96

7

M

2*645

0.01

146

90 108

115

116 134 145

2* Effect of Atropine» Previous workers (?) have shown that the fall in blood pressure following the intravenous adminis­ tration of Veratrum viride was not abolished by atropine*

Experiments

were conducted on three cats to determine the role played by the vagus in the vasodilator response to the depressor principle* 2fce cats were anesthetized with a 1 cc*/kg. mixture of 0*03 Gsu sodium pentobarbital in 1 ec, of 25% urethane.

The carotid artery was

eannulated and the blood pressure recorded by means of a mercury manometer* Respiration was recorded via a pneumograph connected to a writing tambour*

Atropine sulfate (0*1 cc*/kg, of a 1% solution) was

injected via the femoral vein and five minutes later acetylcholine (0*1 ce*/kg. of an 0.01# solution) was injected to test for complete

20 vagal paralysis#

After vagal paralysis was assured, the normal blood

pressure was obtained and a dose of the depressor principle was administered# In two of the cats the blood pressure was lowered considerably but the duration of activity was only about one and one-half hours* The remaining cat died as a result of paroxysmal tachycardia#

Transient

slowing of the respiration was seen in all the cats# Cat #1 had a normal blood pressure of 165 mm# after atropin­ isation# Following an intravenous dose of 0.02 mg#/kg# of the depressor principle the blood pressure fell to 95 mm* and rose to 135 mm* over a one hour period.

The blood pressure approached normalcy after one

and one-half hours#

This change is illustrated in Figure III*

Cat #2 had a normal blood pressure of 136 mm* after atropin­ isation*

Following an intravenous dose of 0.02 mg./kg. of the

depressor principle the blood pressure fell to 84 mm* but within a few minutes a state of paroxysmal tachycardia occurred and the animal . expired within one-half hour, presumably due to the poor physiological state of the animal* Cat #3 had a normal blood pressure of 144 mm. after atropin­ isation. Following an intravenous dose of 0.02 mg./kg* of the depressor principle, the blood pressure fell to 88 ram* and rose to 125 mm. within one hour*

The normal level of pressure was reached in one hour and

forty minutes* Apparently paralysis of the vagus with atropine does not abolish the vasodilator response to the depressor principle but does affect the duration of activity*

This indicates that the parasympa-

21

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thetie nerve fibers play only a minor role in the mechanism of the vasodilator action of the depressor principle* 3* Effect of Ergotoxine* Two experiments were conducted on eats to ascertain if the vasodilator response of the depressor prin­ ciple was abolished by paralysis of the sympathetic augmentory fibers* Each cat was anesthetized with a 1 ee*/kg« of a mixture of 0,03 Gm. sodium pentobarbital in 1 cc. of 25% urethane.

The carotid artery was

eannulated and the blood pressure and respiration recorded as previously described,

A dose of 3 mg./kg. of ergotoxine was administered via the

femoral vein.

After the blood pressure returned to normal the depressor

principle was administered. In both cats the blood pressure fell considerably after ergo— toxine was administered.

Approximately one-half hour later the blood

pressure approached normalcy at which time an intravenous dose of 0.02 mg./kg. of the depressor principle was given.

The blood pressure

fell immediately and gradually returned to normalcy over a period of one and one-half hours.

The respiration showed a transient slowing as

usual. Gat #1 had a blood pressure of 150 mm. one-half hour after the injection of ergotoxine. The blood pressure fell to 70 mm. within * three minutes after an intravenous dose of 0.02 mg./kg* of the de­ pressor principle.

There was a gradual rise to 140 mm, after one hour

and complete normalcy within one hour and fifteen minutes.

Figure HT

illustrates the changes described above. Cat #2 had a normal blood pressure of 135 mm. one-half hour after the ergotoxine injection.

The blood pressure fell to 80 ma.

23

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24 within ©ne minute after an intravenous dose of 0.02 mg./kg. of the depressor principle. Within one hour the pressure rose to 125 mnw and within one and one-half hours it was back to nomal. The augmentory sympathetic nerve fibers do not seem to play a major role in the mechanism of the vasodilator response of the depressor principle since the fall in blood pressure was not abolished, although the duration of activity was considerably lessened. 4. Effect of Tetraethvlaimoniaia Bromide.

Since paralysis of

the parasympathetic and the sympathetic augmentory nerve fibers do not abolish the fall in blood pressure caused by the depressor princi­ ple, it seemed desirable to investigate the possibility of the auto­ nomie ganglia playing a major role in abolishing the vasodilator response. Experiments were conducted on two cats using tetraethylammonium bromide'as the autonomic blocking agent.

The animals were anesthetized

and set up for blood pressure and respiration recordings as described in the atropine experiments.

A dose of tetraethylammonium bromide

was injected via the femoral vein and after the blood pressure approached normalcy an intravenous dose of 0.02 mg./kg* of the depressor principle was administered#

The blood pressure lowering was pronounced

and the respiration was slowed in both cats. In Cat #1 20 mg./kg. of tetraethylammonium bromide immediately lowered the blood pressure from 160 to 80 mm. with a gradual rise to 144 mm. after forty—five minutes*

The depressor principle was injected

at this point and the blood pressure fell to 85 mm. with a gradual rise te 130 mm. in one and one-half hours as shown in Figure V. Cat #2 had a noraal blood pressure of 140 mm. which fell to

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26 78 ma. after a dose of 30 mg./kg. of tetraethylammonium. bromide.

The

blood pressure rose slowly to 105 mm. and remained at this level for one and one-half hours.

After an intravenous dose of 0.02 mg./kg. of

the depressor-principle the blood pressure fell to 45 mm. but the animal expired shortly thereafter.

It seemed that a dose of 30 mg./kg. of the

autonomie blocking agent was too great for the eat. The blockage of the autonomie ganglia does not abolish the fall in blood pressure but it does lessen the duration of activity somewhat.

Since paralysis of the parasympathetic and sympathetic

augmentory fibers or blocking the autonomic ganglia did not abolish the fall in blood'pressure, it would seem that the autonomic nervous system plays but a minor role in influencing the vasodilator response of the depressor principle. C. Rabbits. 1.

Six rabbits were used to demonstrate the vasodilator

property of the depressor principle.

Each animal was anesthetized and

set up for blood pressure and respiration recordings in the same manner as described for cats. The blood pressure of each animal, showed a pronounced fall after intravenous injection of the depressor principle as shown in Table 3#

A dose of 0.04 mg./kg. killed one rabbit, but it is question­

able as to whether or not this was due to the drug since the animal was in a poor physiological state. Figure VI shows a typical response of the rabbit to the depressor principle.

27

Table 3 The Effect of An Intravenous Dose of the Depressor Principle on the Blood Pressure of a Rabbit Rab- - . bit Sex Weight Dose Normal Ho. fcg. mg./kg. B.P.

Blood Pressure in mm. Mercury Time in hours £ 1 li 2 2h 3-

1

M

2.129

0 .0 2

155

80

95

no

n5

125

2

H

3*170

0 .0 2

130

75

90

100

n5

125

3

M

2.905

0.025

125

70

85

95

105

no

4

M

1.955

0.025

130

80

95

no

120

125

5

M

1.740

0 .0 4

85

40

Died

6

M

2.650

0 .03

120

70

90

95

105

no

140

ns

n5

28

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e

B e

B 0> bO 01