PHYTOTHERAPY RESEARCH, VOL.

7, 190-193 (1993)

An Extract of the Leaves of the Breadfruit

Artocarpus aZtiZis (Parkinson) Fosberg Exerts a
Negative Inotropic Effect on Rat Myocardium

Ronald E. Young*
Physiology Department, University of the West Indies, Mona, Kingston 7, Jamaica, West Indies

Lawrence A. D. Williams
Scientific Research CounciVZoology Department, University of the West Indies, Mona, Kingston 7, Jamaica, West Indies

Michael T. Gardner and Cyril K. Fletcher

Department of Anatomy, University of the West Jndies, Mona, Kingston 7, Jamaica, West Indies

Ethyl acetate soluble extracts from the leaves of the breadfruit Artocurpus ultilis exerted a weak, negative
chronotropic effect @<0.05) and significantly reduced left ventricular pulse pressure @<0.001) in uiuo in the
rat. The electrocardiogram maintained a high amplitude QRS complex (ventricular excitation) even when the
ventricular pressure pulse was very depressed. On right ventricular myocardial strips, the same extracts
produced a significant negative inotropic effect @<0.0001). This indicates that the in uiuo effects might be due in
part, to a direct inotropic effect on the myocardium. An in uiuo side effect was extensive intravascular
haemolysis and consequent haemoglobinuria which could be caused by the vehicle, the extract, or a combined
effect of the two. The mechanism of action of the inotropic agent was not cholinergic, and may involve a
decoupling of excitation and contraction.

Keywords: Artocarpus altilis; breadfruit; ethylacetate extract; negative inotropic effects; rat myocardium.

breadfruit leaves. The present study reports acute

INTRODUCTION
effects on the mammalian (rat) heart, of an ethyl
acetate soluble subfraction of an ethanol extract.
An infusion made from the leaves of the breadfruit
Artocarpus altilis (Parkinson) is commonly believed in
some islands of the Caribbean to have antihypertensive
properties. Published reports of this observation (Feng MATERIALS AND METHODS
et al., 1962; Durand et al., 1962; Wong, 1976; Ayensu,
1981; Halberstein, 1983) suggest that active principles
Preparation of the ethyl acetate extract. Air-dried leaves
could include y-amino-butyric acid (GABA), phenols
(150 g) were crushed and extracted at 27-30 "C (room
such as camphorol, glycosides, the flavonoid quercetin,
temperature) for 120 h in 95% ethanol. The extract was
and/or various cardenolides. A search of the literature
decanted, treated with 1 g of activated charcoal per
revealed no laboratory studies designed to characterize
100 mL, and filtered through a Whatman No. 201 filter
the nature of the cardiodynamic effects, or to survey
paper. The filtrate was concentrated using a rotary
the effective fractions of extracts from Artocarpus.
evaporator under reduced pressure (4 kPa; 50 "C). This
Early reports of Feng et al. (1962) and Durand et al.
produced a dark brown, oily mixture, which was parti-
(1962) indicated that an aqueous extract of the leaves
tioned between hexane:methanol:water (10:9:1, v/v).
and small stems of Artocarpus incisa L. (=altilis
The hexane fraction was separated and put aside for
Parkinson) promoted contraction in guinea-pig ileum,
future study. Methanol was removed from the water
had no effect on rat uterus, increased blood flow in the
fraction under reduced pressure in a rotary evaporator.
hind limbs of the rat, was ineffective on the isolated
The remaining dark-brown residue was partit;oned
rabbit heart, and produced marked transient
depression of blood pressure in the pentobarbital between ethyl acetate:methanol:water (10:1:9; v/v).
Both fractions were separated and the water-methanol
anaesthetized dog. Durand et al. (1962) isolated the
fraction put aside. The ethyl acetate fraction was dried
inhibitory amino acid GABA, a known antihyperten-
and dissolved in a 50% dimethylsulphoxide-saline mix-
sive agent, from the extract. They did not determine
ture (50% DMSO) in concentrations of (w/v) 2.3%,
whether the extract was acting in the same manner as 8.2% and 30%.
GABA, nor did they investigate any nonaqueous
extracts. Since some of the putative active principles
suggested above are soluble in nonaqueous solvents, Physiological assay. In uiuo preparation. Adult Wistar rats
we set out to investigate more closely, the cardiovascu- weighing 3 5 W 5 0 g were anaesthetized using sodium
lar effects of some of the nonaqueous extracts of pentobarbital administered intraperitoneally at a
dosage of 35mg/kg body weight. The trachea was
* Author to whom correspondence should be addressed. cannulated and the animal placed on a respirator (2-

0951-418X/93/020190-04 $07.00
01993 by John Wiley & Sons, Ltd. Accepted 10 July 1992
 A MYOCARDIAL INHIBITOR FROM ARTOCARPUS

0 -
al
-0
3
.-
-I-
- -20 -
Q

6
E
.-K -40 -
I 1
T
W
0
Z -
Q -60
I
0
R
-
-80 1 1

-100 1 I I
I I I
0 20 40 60

TIME ( s )
B. 10

0
x
V
c Figure 2. (A) The cardiodepressive effect of 30% extract in
e,
3 -10
0-
Y
LL
.-K -20
W frequently seen at the highest doses of extract, perhaps as a
0 result of the circulatory compromise. Electrical activity of the
2 -30
I contractile strength falls. The pattern of spread of excitation,
0
R however, is probably altered, giving rise to the altered QRS
-40
I I I I
-50
0 20 40 60
TIME (s)
Figure 1. Changes over time in (A) inotropic and (6) chronotro-
pic responses to 50% DMSO-saline (vehicle) and to increasing
doses of extract. Responses are expressed as percent change
relative to the pretreatment level. At a dose of 149 mglkg there
was significant depression in frequency (p<0.05) and of left
ventricular pulse pressure (pSO.001)relative to the vehicle. The
results were obtained from seven experiments ( n = 7 ) . Vertical
bars indicate fl SE. 050% DMSO (3.17 mu/kg); 0 11.8 mg/kg;
V 42.2 mg/kg; V 149 mg/kg of extract i.v., n = 7 .
3 mL tidal volume) as soon as the thorax was opened.
A cannula filled with heparinized saline was inserted
into the left ventricle near the apex, and attached to a
Statham Spectramed P23XC pressure transducer
(Grass Instrument Co., Quincy, MA, USA). A Lead I1
standard electrocardiogram (ECG) was also recorded
after the cut edges of thc thoracic wall were re-apposed
and covered with gauze soaked in physiological saline.
Signals were amplified and recorded using a Grass
Model 7E polygraph (Grass Instrument Co., Quincy,
MA,WSA). Because the thorax was opened, the ECG
signals were non-standard in form, but provided an
index of electrical activity of the heart.
The femoral vein was catheterized to allow intrave-
vehicle (50% DMSO). Top trace, ECG; bottom, left ventricular
pressure. t is the point of injection of extract. There is a 30 s
gap between each segment of record. The very large excursions
on the ECG trace are movement artifacts produced when the
animal was showing signs of respiratory distress. This was
ventricular myocardium persists quite strongly although the
waveforms. These 'normalize' as the myocardium recovers. The
conducting tissues then, might be reversibly impaired by the
treatment. (6)In vitro responses of a right ventricular strip to
increasing doses of extract in 50% DMSO. t time of addition of
drug; 4 wash. The volume of drug added was always 0.07 mL
and the bath volume was 7.0 mL. The concentration of vehicle
therefore was the same in all cases. The strip was stimulated at
0.2 Hz with 5 ms suprarnaxirnal, square pulses. At all doses the
muscle recovered after washing. See text for details.
nous administration of the extracts. For each trial, a
volume of 0.18 mL of the different concentrations of
extract, or of vehicle, was injected. This ensured that
other factors such as dose of the carrier, remained
constant, while only the concentration of extract var-
ied.
Immediately after completion of the drug testing
procedure, animals were given a booster dose of anaes-
thetic. Samples of various tissues were fixed in formoi-
saline followed by routine embedding in Paraplast,
sectioned at 7 pm, and stained in haematoxylin-eosin
for histological examination.
In uitro preparations. Rats were killed quickly by cervical
dislocation. The heart was removed and a strip of
myocardium (10 x 3 mm) excised from the wall of the
right ventricle. The strips were held at 37 "C in a 7 mL
bath of Krebs-Henseleit saline, oxygenated with 95%
0 2 / 5 % COz. Resting tension was set at 5 mN.
Supra-maximal stimuli (5 ms; 100 V) were administered
at a rate of 0.2 Hz, using chlorided silver wire elec-
192 R. E. YOUNG E T A L .

Figure 3. Haematoxylin-eosin stained sections of kidney and urinary bladder, fixed in formol-saline. Note the distribution of
eosinophilic material (arrows) in the tubules of the renal cortex (a), renal medulla (b), and in the urinary bladder (c) in a pattern
indicative of intravascular haemolysis. In (a) and (b), scale bar=25 pm; in (c), 50 pm. See text for details.

trodes located above and below the strip. Contraction RESULTS

force was measured using a Grass FT03C force trans-
ducer. Prior to testing, strips were allowed to stabilize
for at least 1h. Extract in 50% DMSO was added to the
bath to produce a 100-fold dilution of the original
sample.
Analytical methods. Basal pulse pressure in uiuo and
contractile force of the myocardial strips in uitro varied
depending upon size of the animal and size of the
muscle strips respectively. To facilitate pooling of the
results, values were expressed as per cent change rela-
tive to the pre-treatment level, and analysed using a
generalized linear model and repeated measures
ANOVA, to discern differences between treatment
levels. Post-hoc evaluation of the levels showing signifi-
cant differences was carried out using the Fisher’s LSD
Test (Number Cruncher Statistical Systems Ver. 5.0).
0 Henle and collecting ducts of the kidney, and in the
e,
Y unremarkable. Because of this finding, a single prelimi-
0
L
-10
.-S
w Strips (Home Diagnostics Inc., Behringwerke AG,
$ -20
a
I
0
\ 1
In the in uiuo preparation, both extract and vehicle
produced a negative chronotropic effect, and a fall in
ventricular pressure pulse (Fig. 1). At 149mg/kg the
extract produced a significantly greater response than
the vehicle alone (ANOVA: p<0.05 for chronotropic;
p<O.Ool for inotropic effects). A strong QRS complex
(ventricular excitation) in the ECG even after the
ventricular pressure pulse had been severely depressed
(Fig. 2A) suggests excitation-contraction decoupling as
a possible mechanism of action.
Post mortem examination of the urinary bladders
revealed dark-red urine, containing no erythrocytes,
indicating severe haemoglobinuria. This effect could be
mimicked by high doses of carrier (10 X normal doses),
but was not produced by the normal doses of vehicle
used throughout the experiment. Light microscopic
examination of the histological sections revealed eosi-
nophilic material in the convoluted tubules, loops of
urinary bladder (Fig. 3) suggesting massive intravascu-
lar haemolysis. Liver, lung and heart sections were
nary test was conducted on a known user of the bread-
fruit leaf infusion, using Diascan Self-Monitoring Test
Germany). The urine tested before use was negative
for haemoglobin, but was positive 2 h after ingestion,

suggesting the presence of a haemolytic agent in the

7.45rng/n
infusion.
6\” -30
(conc. o f In t-he in uitro preparations all doses of the extract,
extract in
bath.)
0.23 mg/mL and above, produced a significant
(p<O.OOol), maintained depression of contractility,
-40 relative to 50% DMSO alone (Fig. 4). At the highest
0 1 2 3 4 5 dose (7.45 mg/mL) the extract did not solubilize well,
TIME(min) P o s t - T r e a t m e n t and tended to have variable and irreversible effects.
This dose therefore was not used routinely.
Figure 4. Mean inotropic responses of the in vitro right ventri-
cular strip to increasing doses of extract, at 1 and 4 min after
treatment. All doses of extract produced a significant negative Chemistry. Thin layer chromatography (TLC) on the
inotropic effect relative to the vehicle (p<O.OOOl). The highest extract produced two bands with Rf at 0.38 (PPT2)
dose level (7.45 mglkg) is a single observation; for all others
n = 4 preparations, with three replications per dose, per prep- (75% of loading) and at 0.88 (PPT3) (25% of loading).
aration. Vertical bars represent f l SE. See text for details. PPT3 was found to have the major action on the heart.
 A MYOCARDIAL INHIBITOR FROM ARTOCARPUS
Further TLC separation of PFT3 revealed the presence
of six compounds. These are presently being chemically
characterized.
DISCUSSION
An ethyl acetate soluble fraction of an ethanol extract
of the breadfruit leaf has been shown to have cardiode-
pressive properties consistent with the folklore use of
the leaves of this plant as an antihypertensive agent.
The fall in ventricular pulse pressure induced in the
whole animal preparation by the 30% solution of
extract in 50% DMSO is quite marked. This could
result from diminished afterload (e.g. peripheral vaso-
dilation) or from a negative inotropic effect on the
ventricular myocardium.
The in uitro results indicate that a direct effect on the
myocardium is a contributing factor, without comple-
tely ruling out contributory effects in uiuo, due to
changes in peripheral resistance or to autonomic action
on the heart itself. The pattern of the in uiuo response
is not consistent with a cholinergic type of effect, while
the observations relating to changes in the ECG suggest
an alteration of the pattern of spread of excitation
through the myocardium (Fig. 2A) and a decoupling of
the excitationxontraction linkage.
The extract plus vehicle produces marked haemoglo-
binuria resulting from massive intravascular haemoly-
sis. This side effect might possibly be produced by
surface active contaminants such as saponins, which
could be present in the extract, along with the cardioac-
tive principles. It could also be induced by the vehicle
REFERENCES
Ayensu, E. S. (1981). Medicinal plants of the West Indies.
Reference Publications, Algonac, MI, USA.
Durand, E., Ellington, E. V., Feng, P. C., Haynes, L. J., Magnus, K.
E., and Philip, N. (1962). Simple hypotensive and hyperten-
sive principles from some West lndies medicinal plants. J.
Pharmac. Pharmacol. 14,562-566.
Feng, P. C., Haynes, L. J., Magnus, K. E., Plimmer, J. R., and
Sherratt, H. S. A. (1962). Pharmacological screening of some
West Indian medicinal plants. J. Pharm. fharmacol. 14,556-
561.
Halberstein, R. A. (1983). The use of medicinal plants to control
high blood pressure in the Caribbean. The Florida
193
(50% DMSO) which is known to produce haemolysis at
sublethal doses (Smith ef a f . , 1967). At doses of 5.2 g/kg
in the rat, DMSO can be lethal (Rubin, 1975) while
doses of 2.5 g/kg/day have been given over 2 weeks with
no observed system toxicity (Rosenkrantz et af.,1963).
The dose given per test in our experiments was
3.17 m d k g or 0.22 g/kg. Since haemolysis has been
observed even after a single test, it is unlikely that the
DMSO on its own is the agent responsible. The haemo-
lysis could be due to components in the extract, acting
either independently or by potentiating the haemolytic
action of DMSO. The single test conducted on a habi-
tual user of the folk remedy, supports the suggestion
that components of extracts from the leaf may cause
haemolysis, and indicates that components in the etha-
nol extract may well be acting in the aqueous infusion.
It should therefore be of interest to isolate the cardio-
active principle described here, and to determine its
mode of action and structure. A more comprehensive
study of the incidence of haemolysis in users of the
breadfruit leaf infusion might also be in order. For the
present, however, we can state that we have found
clear evidence of a cardioinhibitory activity, which is in
keeping with the traditional belief that infusions from
the breadfruit, Artocarpus altilis may have useful anti-
hypertensive properties.
Acknowledgements
We thank Professor M. West, Department of Pharmacology,
University of the West Indies, for his comments on the manuscript,
and Mr Thomas Denton, Department of Anatomy, for help in the
preparation of histological slides.
Anthropologist 36, 167-176.
Rosenkrantz, H., Hadidian, Z., Seay, H., and Mason, M. M. (1963).
Dimethyl sulfoxide. Its steroid solubility and endocrinologic
and pharmacologic-toxicologic characteristics. Cancer
Chemother. Rep. 31, 7-24.
Rubin, L. F. (1975). Toxicity of dimethyl sulfoxide alone and in
combination. Ann. NYAcad. Sci. 243, 98-103.
Smith, E. R., Hadidian, Z., and Mason, M. M. (1967). The single
and repeated-dose toxicity of dimethyl sulfoxide. Ann. NY
Acad. Sci. 141, 96-109.
Wong, W. (1976). Some folk medicinal plants from Trinidad.
€con. 60t. 30, 103-142.