NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
ACTION OF GIGANTIN (ACTIVE PRINCIPLE OF MADAR
JUICE) ON THE AMPHIBIAN HEART
By V. RATHNASABAPATHY, B.V.Sc., J. C. DAVID, M.B., Ph.D., and V. ISWARIAH, B.A.,
M.B.B.S., M.R.C.P.
(From the Research Unit in Indigenous Drugs, Department of Pharmacology, Madras
Medical College)
(Received for publication on 3 April 1950)
(With Plates VII-XII)
M ADAR juice is a white milky liquid obtained from Calotropis gigantea, known
as erukku in Tamil, jelledu in Telugu and madar in Hindustani. It is common-
ly used as a criminal poison, for infanticide and producing abortion. Several thera-
peutic properties have been attributed to it. It is said to be an effiecient anti-
spasmodic, alterative and nervine tonic in doses of one grain to three grains. It is
claimed to be useful in cases of asthma, epilepsy and paralysis.
Although the emetic and toxic effects of the root bark and milk juice of Calo-
tropis were repeatedly observed in animals and men, it remained for Lewin [1913]
to show conclusively the digitalis like action of the milky juice of Calotropis procera.
Chemists efforts hence forth have been directed to the isolation of these active
principles. Wieland succeeded in isolating 'Calotropin' as a chloroform addition
compound. His work was followed by Jacobi on the same product. Both of them
withheld publication. Hesse et al [1939] in their investigation of African arrow
poisons, obtained from the Calotropis saps several poisonous principles, Uscharin,
Calotoxin and Calactin, of which Uscharin is of particular interest, for unlike most
cardiac glycosides it contained nitrogen and sulphur in its molecule. Chen [1942]
has also isolated Uscharin and other principles and studied their digitalis like action.
Sheshadri et al. [1943] state that they obtained from madar juice cardiac poisons
containing nitrogen and sulphur, similar to Uscharin but they did not isolate any
poisonous principle in a pure state.
Pichandi [1948] in his endeavour to isolate the poisonous principle of madar
juice in a pure state succeeded in his attempts and got it in the form of pure crystals
with a melting point of 232°C. with decomposition. The molecular weight as obtained
by Rast's method using camphor as solvent was found to be about 464. The formula
C 24 H 36 O 9 was therefore proposed as the tentative molecular formula and the
tentative name 'gigantin' was suggested by the same author.
Using the same method for isolation, the authors [1949] of this paper, obtained
the crystalline substance tentatively named 'gigantin' from the madar juice
and did some preliminary pharmacological studies, the results of which have been
presented in a previous publication. The present study is the outcome of the
95
96
ACTION OF GIGANTIN ON THE AMPHIBIAN HEART
[Vol. XXI, Part II,
detailed investigation being carried out with a view to explore the possibilities of
therapeutic usefulness of the substance, which resembles digitalis in many respects.
MATERIALS AND METHODS
Gigantin obtained as mentioned above [Pitchandi, 1948] was insoluble in water,
Therefore a stock solution of 1 in 1,000 was prepared in 90 per cent alcohol and
dilutions were made in distilled water according to the experimental needs. The
amount of alcohol present in a single dose of the drug after large dilutions, was
negligible and it did not interfere with the true action of the drug and this has been
proved by control experiments by administering similar dilutions of 90 per cent
alcohol alone.
Rana-hexa-dactyla, the species found in large numbers in and around Madras
was used in all the experiments. The weight of the frogs ranged from 180 g. to 250 g.
and the heart weight 500 mg. to 800 mg.
Methods
The action of the drug on the hearts in situ was studied by myocardiogram of
the exposed hearts, the drug being administered intra-hepatically through any one
of the lobes.
Perfusion of isolated hearts was done through the inferior venacava using
Clark's [1928] perfusion fluid. The perfusion apparatus consists of a Syme's canula
connected with two perfusion funnels through a two-way stop-cock. By this
arrangement fluid from any one funnel could be switched on to the canula with
great ease and with least disturbance to the heart. The perfusion canula designed
by Symes [1911] and [1918] was slightly modified by blowing a hole about 1 cm.
above the tip of the nozzle. This hole serves three purposes. (1) It keeps the
perfusion pressure constant at about 2 cm. on the ventricle and a little over 1 cm.
on the auricle. (2) When the perfusion fluid is changed the fluid remaining in the
connecting tubes is flushed out quickly through the side hole without any appre-
ciable change in the pressure. (3) A certain amount of the perfusion fluid escaping
through the exit keeps the outside of the heart moist.
The action of the drug on isolated hearts was studied by two different methods.
(1) By perfusing the heart for a short while with ringer solution containing the drug
and subsequently followed by normal ringer. (2) By perfusing the heart conti-
nuously with ringer containing the drug in a known concentratoin, to study the
effect of the drug on the heart muscle.
EXPERIMENTAL DATA
Action on the rate and amplitude of the heart in situ
The results of nine experiments conducted in this series showed that 0.0025 mg.
of gigantin given to frogs intra-hepatically produced either an increase or no change
in the rate of the heart beat. The amplitude was decreased initially and within two
minutes gradually returned to normal (Plate VII, fig. la.)
Higher doses from 0.01 to 0.05 mg. caused an immediate slowing of the rate
and diminution of the amplitude, recovery to normal taking place in about 5 to 10
Indian J. vet. Sci., Vol. XXI, Part II]
[ Plate VII
FIG. 1a. A record of the movements of the frog's ventricle in situ. Note
that 0.0025 mg. of gigantin does not alter the rate, but diminishes
the amplitude. Systolic level is higher, diastole deficient. (Time 6
seconds. Upstrokes are systole.)
FIG. lb. Same after
2 minutes.
FIG. 2. Tracings of the auricle (upper) and ventricle (lower) of
an isolated frog's heart, perfused with 1 in 1,00,000
gigantin in ringer. Note the auricles beat even after
the arrest of ventricle. The ventricle responds to
mechanical stimuli, applied at ↑ point. (Time 6
seconds ↑ Systole.)
Plate VIII
.]
[Indian
J.
vet.Sci.,
vol. XXI, Part II
A record of the movements of the ventricle of an isolated frog's heart perfused with 1 c.c. of 1 in 10,000 solution of gigantin in the ringe. (Time 6 seconds.
Upstrokes are systole.)
(a). Note the acceleration, diminution of amplitude Normal systolic level
and deficient diastole.
(b) Same after 1
minute.
(c). Same after 2 minutes. Note the contractuie and systolic
arrest.
Indian J. vet. Sci., Vol. XXI Part II]
[
plate IX
A record of the movements of the ventricle of an isolated frog's heart perfused with 1 in 5 million gigantin in the ringer. (Time 6 seconds.
Upstrokes are systole.)
(a). Note there is no change in
rate, the amplitude is increased.
(b). After 5 minutes.
Rate is slowed.
(c). After 10 minutes.
Systolic level is increased.
(d). After 20 minutes.
(e). After 60 minutes.
Plate X]
[ Indian J. vet. Sci., Vol. XXI Part II
A record of the movements of the ventricle of an isolated frog's heart perfused with ringer containing 1 in 1 million of gigantin. (Time 6 seconds. Upstrokes are systole.)
(a). Note the gradual slowing and diminution of amplitude during the
first 3 minutes. The amplitude is increased afterwards.
(b). Same after 8 minutes. Note the
extra systoles.
(c). Same after 15 minutes. Note the intermissions
of the ventricle and the diastolic arrest.
Indian .J. vet. Sci., Vol, XXI Part II ]
[Plate
XI
Particular movements of an isolated frog's heart perfused with ringer containing 1 in 1,00,000 of gigantin. (Time 6 seconds. Upstrokes are systole.)
(a)there is no initial change.
(b). Same after 3 minutes. Note
the marked slowing and in-
creased amplitude.
(c). After 5 minutes extra
systoles.
(d). After 10 minutes. Note the intermissions of the ventricle,
complete block and arrest in partial systole.
Indian J. vet. Sci., Vol. XX[ Part II ]
[ Plate XI
A record of the ventricular movements of an isolated frog's heart perfused with ringer containing 1 in 1,00,000 of gigantin.
(Time 6 seconds. Upstrokes are systole.)
(a). Note there is no initial change.
(b). Same after 3 ninrates. Note
the marked slowing and in-
creased amplitude.
(c). After 5 minutes extra
systoles.
(d). After 10 minutes, Note the intermissions of the ventricle,
complete block and arrest in partial systole.
98
ACTION OFGIGANTIN ON THE AMPHIBIAN HEART [Vol. XXI, Part II,
TABLE I
Gigantin on the rate and amplitude of isolated frog's heart
Concentration of
the drug in the
perfusion fluid
Number of experiments
Time
Bofore drug
1 minute after drug
2 minutes after drug
3
do.
5
do.
10
do.
15
do.
20
do.
25
do.
30
do.
60
do.
Time in minutes for the
arrest of the heart
A
1 in 5 million
1
Rate
80
80
70
60
45
40
..
40
..
35
35
Ampli-
tude
17
23
..
28
35
32
. .
29
..
26
21
Alive
B
1 in 1 million
4
Average
Rate
46±20
44±18
..
. .
23±14
16±7
13±7
8±5
8±4
3
..
Amplitude
26±13
..
..
23±10
24±4
27±1
. .
. .
32±8
..
28±1
C
1 in 100 thousand
3
Average
Rate
66±9
59±16
..
28±8
22±5
7
..
..
..
..
..
Amplitude
19 ±6
20±7
..
21±7
21±4
22
..
..
..
..
..
11±1
Rate: Rate per minute. Amp.: Amplitude in mm.
Relation of nervous mechanism—The right vagus nerve of a pithed frog attached
to the terminals of an induction coil was stimulated by a moderate strength of faradic
current and a record of the normal inhibition of the heart was taken. Now 0.01 mg.
of gigantin was given intra-hepatically and this was found to produce the slowing
of the heart. Stimulation of the vagus, when the heart was under the influence of the
drug, produced a further inhibition of the heart beat.
The heart was allowed to recover from the influence of the drug. A few drops
of a 0.1 per cent. atropine solution, which blocks the peripheral nerve endings of the
post gangionic fibres of vagus, was applied to the heart and ascertained by vagal
stimulation which failed to produce the normal inhibition. A dose of gigantin, when
repeated, produced the characteristic slowing of the heart as before atropinisation.
100
ACTION OF GIGANTIN ON THE AMPHIBIAN HEART
[Vol. XXI, Part II,
systole of the ventricle was determined in distance of the primary from the secondary
coil, both before and after the administration of the drug.
Studied according to this method it was found that the irritability of the cardiac
muscle was increased by gigantin. The minimal effective current for stimulating
the heart of frog was found to be considerably decreased as shown below (Table II).
TABLE II
Distance of secondary from the primary coil in centi metres
Serial number.
1
2
Before the drug
9.5
12
Dose of drug
0.0025 mg.
0.0025 mg.
After the drug
10.5
13
Latent period—The effect of the drug on the latent period in frog's heart has
been studied by the authors [1949] previously and it was found to decrease the
latent period considerably. Further experiments carried out using the same tech-
nique confirmed the previous findings.
Refractory period—The influence of the drug on the refractory period of the
frog's heart was studied using the same technique mentioned by the authors [1949].
The experiments done then and a number of them repeated later showed that this
substance decreased the refractory period of the heart muscle.
Contraction and relaxation—Under a physiological concentration of gigantin
in the perfusion fluid (1 in 5 million) the ventricle of the frog's heart was found to
contract more completely than before. This was represented by the increase in the
systolic level of the individual beats of the heart. There was no change in the extent
of relaxation as seen by the normal diastolic level (Plate IX). Close observations
by the inspection of exposed hearts and study of the individual beats showed
that the speed of contraction was quickened by the increased strength of contraction.
In tracing the normal beat the lever raised slowly from the diastolic position and
attained the highest point only after an appreciable interval. Under gigantin this
interval was shortened, the lever rose more rapidly as well as to a higher point.
The diastole was weakened and the relaxation time was considerably increased,
the lever coming down more slowly to the base line (Plate IXc).
After some time the extent of systolic contraction often became less, the lever
no longer rising to the full systolic position as earlier in the experiment. The total
excursion of the lever however was persistently above normal.
One in one million concentration of: gigantin in the perfusion fluid manifested
similar changes except for a short period in the beginning. The contractions were
more powerful and were accompanied by the quickening of the speed of contraction,
whereas the relaxations were slower than normal and the lever took a longer time to
reach the base line than before (Plate X).
Higher concentrations, as 1 in 500,000 to 1 in 100,000 of the drug in the perfusion
fluid, increased the strength of contractions from the beginning as shown by the
June, 1951]
V.
R.,
J.
C.
D.
AND
V.
I.
101
systolic level which was persistently above normal. The diastole on the other
hand was progressively deficient from the increasing slowness of relaxation. The
diastolic level was also reduced. The ventricle remained in a condition of semicon-
tracture after the active movement towards the end of systole and contractions began
even before the heart returned to its normal position of rest in diastole (Plate XI,
b and c). Due to the setting in of auriculo-ventricular block by this time,
diastole became more complete, because more time was given for relaxation before
impulses arrived. The total excurtion of the lever was more than normal till the
arrest of the heart in diastole by the heart block.
In a very toxic concentration (1 in 10,000) the heart was arrested in complete
systole in three minutes by the progressively deficient diastole. At every successive
beat the contractions were normal while the relaxations were imperfect and greatly
reduced, probably due to the predominance of contracture.
Thus in addition to changes in rhythm, individual beats undergo modifications
under gigantin. Under physiological concentrations the phase of increased contrac-
tility may be prolonged until the drug is excreated or destroyed. The relaxations
may be slower but complete. After larger doses (lethal dose) this phase accompanied
by deficient diastole is prolonged until it is interrupted by the intermissions of the
ventricle. In a highly toxic dose this phase may be of very short duration, mainly
indicated by deficient diastole and systolic arrest of the ventricle. The 'contracture'
which culminates in the arrest in systole, is essentially in its beginning an imperfect
relaxation.
The work of the heart—In physiological doses the rate of the frog's heart is
slowed, but the total amplitude and the strength of the beat is increased. The
ventricle relaxes to the normal level slowly and allows a better and complete filling
of the ventricle. This is followed by a systole which is more powerful and perfect
than normal. Thus the output per beat may be said to be increased. The
slowing of the rate is compensated by the increased pulse volume (output per beat)
and the minute volume might not be altered considerably by the drug.
It has been observed during the experiments that a number of irregular hearts
became regular after the administration of gigantin. In such cases it has necessarily
increased the efficiency; further in diseased conditions of the heart more rest to the
heart is ensured by the slowing of the heart without affecting the efficiency and the
minute volume.
Cumulation and fixation in the heart—When 0.01 mg. of gigantin was injected
intra-hepatically, slowing of the rate and a slight diminution of the amplitude
were produced in the frog's heart. It returned to normal in about three minutes.
But when the same dose was repeated after the heart has returned to apparent
normality, in addition to the above changes heart block was produced. This
phase was however of a short duration and the heart recovered from the
block and attained normal rhythm after about five minutes.
Plate XII ]
[ Indian J. vet. Sci., Vol. XXI Part I
A record of the movements of the ventricle of an isolated frog's heart. (Time 6 seconds. Upstrokes are systole.)
R.= Normal ringer: Rē no ea = ringor with no calcium : R—C+G=ringei with gigantin as substitute for calcium :R—CA =
ringer with no calcium.
(a). Note that linger with no calcium causes diastolic
arrest of the ventricle substitution of gigantin
after the arrest has no effect.
(b). Suhstitution of gigantin before the complete arrest keeps the heart going
on for about 10 minutes.
104
ACTION ON GIGANTIN ON THE AMPHIBIAN HEART
[Vol. XXI, Part II,
seen under veratrine, in tetanus, fatigue and similar conditions. It may be an
exaggeration of muscle tone in unstriated muscle and the heart itself, though this
hardly brings enlightenment, for tone is still an obscure quality.
Cushny [1935] after his experiments with digitalis series of drugs, forwards
that the contracture is due to some change in the muscle fibres which shorten them
without significantly changing their elasticity.
The efficiency of the heart seems to be increased under the physiological doses
of gigantin, by the powerful systolic contractions and slow but complete relaxations,
when there is a better filling and emptying of the ventricle. Thus the pulse
volume may be said to be increased.
The work done in a unit of time is the product of the rate of beat and the amount
of blood expelled by each contraction. When the rate is slowed under gigantin,
the influence of the drug upon the output per beat is the chief point at issue. If
contractions become more complete and the relaxations remain unchanged the
output per beat is naturally greater. Though the output was not measured in these
experiments, but only the muscular contraction, it may be inferred that the pulse
volume increased in correspondence. But the work done per minute might not
be increased to a great extent since the increased pulse volume is compensated for
by the slowing.
It may be said that the efficiency of the frog's heart is increased especially
when it is diseased or irregular before the administration of the drug. The improve-
ment in the work is obvious when a previous irregular rhythm becomes regular
under the drug.
In a toxic dose there is no question of efficiency, for as contracture develops
it is obvious that each beat expells less blood from the ventricle.
It is of special interest to note the relation of gigantin to calcium salts since
the latter tend to increase the strength of the beat and promote contracture in the
same way as gigantin and digitalis. The weak beats which are characteristic of
the heart beating with insufficient lime are generally strengthened by the addition
of gigantin. When the heart has ceased to beat from want of calcium it can be
induced to contract for some time by the addition of gigantin sufficiently early
before the diastolic arrest. But these contractions under gigantin are merely evi-
dence that it increases spontanity of the heart muscle and may be compared to the
effect of electrical stimuli rather than to the action of calcium.
Gigantin and digitalis.
The action of gigantin on the frog's heart resembles in many respects that of
digitalis. The slowing, the contracture and the intermissions of the ventricle are
all characteristics of the digitalis series of drugs. Experiments show that the drug
gets fixed in the heart muscle and can be washed from it only with great difficulty,
thus giving rise to cumulative toxicity as digitalis. Whether the drug which is in
combination in the heart muscle, is simply deposited there in an inactive form or
whether the actual combination is the cause of the changes in the heart is still a
matter for study.
June, 1951]
V,
R.,
J.
C.
D.
AND
V.
I.
105
Gigantin differs from digitalis in that it increases the exitability of the heart
muscle and shortens the refractory period whereas the latter acts in the opposite
manner.
SUMMARY
The pharmacology of gigantin on the amphibian heart has been studied in detail.
By a direct action on the heart muscle, it is found to increase the efficiency of
the heart in physiological doses.
Though it produces a synergesic action with calcium, it cannot replace it in the
perfusion fluid.
The action on the heart is analogous to digitalis in many respects.
ACKNOWLEDGMENTS
The authors' thanks are due to the Indian Council of Agricultural Research
under whose auspices this work was carried out, and to the Director of Animal
Husbandry, Madras, for his keen interest evinced in this work. They are also
deeply indebted to Dr C. Vareed, M.B.B.S., M.Sc., Professor of Physiology, Madras
Medical College and Lieut.-Colonel R. Krishnaswamy, M.B.,B.S., Civil Assistant
Surgeon, Research Unit, and Sri K. Lakshman Rao, B.Sc., (Pharm.), Research
Assistant for their valuable technical help whenever needed.
REFERENCES
Arthur Cushny, (1935). The actions and uses in Medicine of digitalis, 63
Chen. K. K., Bliss. C. I., and Brown Robbins, (1942). J. Phar. Exptl. Ther. 74, 223
Clark, (1928). Exptl. Pharmacology, By Sollmann & Hanzlik, 302
Hesse, Reicheneder and Eysenbach, (1939). Annalen, 537, 67-86
Lewin, (1913). Arch. Exper. Path. U. Pharmakol, 71, 142
Pitchandi, N., (1948). J. Proc. Inst., Chem., 20, 34
Rathnasabapathy, V., Lakshman Rao, K., Krishnaswamy. A., David. J. C., (1949). Indian
J. med. Res., 37
Sheshadri and Bhaskara Ramamoorthy, (1943). The Proc. Ind. Acad. Sci, XVIII. 31, Sec. A.
Symes, W. L., (1911). J. Physiol, 48, 25
Symes. W. L., (1928). Ibid. 52, 46