Brazilian Journal of Medical and Biological Research (2005) 38: 111-118 111

Inotropic effect of C. sinensis on the guinea pig atrium

ISSN 0100-879X

Inotropic effect of Citrus sinensis (L.)

Osbeck leaf extracts on the guinea
pig atrium
E.D. Oliveira1,2, 1Laboratório de Biofísica do Coração, Departamento de Fisiologia,
T.S. Leite1, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Sergipe,
B.A. Silva2 and Aracaju, SE, Brasil
E.A. Conde-Garcia1 2Laboratorio de Tecnologia Farmacêutica, Universidade Federal da Paraíba,

João Pessoa, PB, Brasil

Abstract

Correspondence The objective of the present investigation was to determine the Key words
E.A. Conde-Garcia contractile effect of crude and acetone leaf extracts of Citrus sinensis • Citrus sinensis
Laboratório de Biofísica do Coração (L.) Osb. on mammalian myocardium. Crude leaf extracts have been • Contractility
Departamento de Fisiologia
used in folk medicine to treat neurological disorders. Some flavonoids • Heart
CCBS, UFSE • Myocardium
49100-000 Aracaju, SE
isolated from this plant presented a positive inotropic effect on
• Woodworth phenomenon
Brasil myocardium. This motivated us to test the extracts on the atria of
Fax: +55-79-246-3377 guinea pigs of both sexes (300-500 g) and surprisingly we observed
E-mail: egarcia@ufs.br inotropic depression instead of an increase in force. The maximum
effect of the crude extract was 79.4 ± 8.1% of the control force
Research supported by Centrais amplitude (N = 5 hearts, 10 trials, 27 ± 0.1ºC, stimulus: 2 Hz, 400 V,
Elétricas Brasileiras (ELETROBRAS,
0.5 ms). The EC50 for crude, ethanol, acetic, aqueous, and acetone
No. 23113.009351/03-67), Fundação
extracts was 300, 300, 600, 1000, and 140 µg/ml, respectively, with a
de Amparo à Pesquisa do Estado de
Sergipe (FAP-SE/FUNTEC FNS/Process
Hill constant of 1.8, 2.0, 2.5, 2.0, and 1.4, respectively. Blockade of
No. 01/2003) and CNPq. cholinergic, beta-adrenergic, or opioid membrane receptors with 1.5
µM atropine sulfate, 1 µM propranolol, and 10 µM naloxone, respec-
tively, did not change the effect of the crude extract. The acetone
extract abolished the Bowditch positive staircase phenomenon (N = 5
Received March 3, 2004
hearts, 10 trials, 27 ± 0.1ºC), suggesting a possible reduction of the
Accepted October 6, 2004
calcium inward current, and also promoted the so-called Woodworth
phenomenon. The effect was concentration-dependent and indicated
the existence of another inhibitory contractile mechanism such as the
simultaneous activation of some of the membrane potassium channels
reducing the myocardial action potential duration and further decreas-
ing the cellular calcium entry.

Introduction of great economical importance. Different

extracts obtained from the leaves and the
Citrus sinensis (L.) Osb., hereafter re- peel of the fruit have been used to kill mos-
ferred to as C. sinensis, is a plant of the quito larvae and mites (4,5), as antimicrobial
Rutaceae family native to Asia and India (1- agents (6), and as antifungal agents (7). Leaf
3), which is widely cultivated all over the extracts have been used in folk medicine to
world. Its fruit, rich in vitamin C, is con- treat neurological disorders and to facilitate
sumed fresh or industrialized as juice and is the digestion of food (8).

Braz J Med Biol Res 38(1) 2005

112 E.D. Oliveira et al.

Many substances have been isolated from is another taxonomic way of referring to C.
C. sinensis leaves: glycosides (apigenin and sinensis L. Osb. (14). A voucher specimen
diosmetin), ruteosides (luteolin), caffeine, (No. 36,773) was deposited in the Herbarium
hydroxyproline, flavonoids (natsudaidaine, of the Federal University of Pernambuco,
HEPTA (3,5,6,7,8,3',4'-hexamethoxyfla- Recife, PE, Brazil.
vone), hesperidin, and diosmin), and the
triterpene linomin (9-12). Hesperidin and Drugs
diosmin have been shown to have anti-in-
flammatory, antihypertensive, diuretic, an- The following drugs, diluted in water,
algesic, and hypolipidemic properties (10). were used: 1.5 µM atropine sulfate, 0.7 µM
Natsudaidaine and HEPTA showed a posi- propranolol, 0.3 µM acetylcholine chloride,
tive inotropic effect on the guinea pig right 1 µM epinephrine bitartrate, and 10 µM
ventricle (13). This cardiac effect motivated naloxone. All drugs were purchased from
us to study the contractile action of C. sinensis Sigma-Aldrich (St. Louis, MO, USA) except
extracts on the mammalian heart. However, for naloxone, which was from Rhodia Farma
when the crude water-alcohol extract was (São Paulo, SP, Brazil).
tested on the guinea pig atria, a marked
depressant effect was observed instead of Extract preparation
the positive one reported in the literature
(13). This surprising result motivated us to A crude water-alcohol extract was ob-
investigate the effect of C. sinensis on myo- tained by macerating dry leaves in an ethanol
cardial contractility. In addition, it is well and water solution (6:4, v/v) maintained at
known that substances that depress the heart 27 ± 3ºC for 10 days. The extract was con-
are commonly used as antiarrhythmic agents centrated in a rotary evaporator (Buchi RE
(i.e., verapamil). Thus, the present study can 111; Buchi Laboratoriums-Tecnick AG,
contribute to the elucidation of some charac- Flawil, Switzerland).
teristics of new active compounds that might The hexane, chloroform, acetone, etha-
be of importance in the future as pharmaco- nol, methanol, and acetic acid extracts were
logical tools for the treatment of cardiac prepared with a Soxhlet extractor using sol-
disorders. In spite of the wide use of C. vents of analytical grade purchased from
sinensis, very little was found regarding its VETEC (Rio de Janeiro, RJ, Brazil).
effects on the mammalian heart. After removal of the solvent, the extracts
The aim of the present study was to char- were stored at 27 ± 3ºC in a dry atmosphere
acterize and report the negative inotropic without any light protection and their Na+
effect produced by some of the C. sinensis and K+ contents were determined by flame
extracts on the guinea pig left atrium. photometry using an Analyzer Model 900
(Analyser Comércio e Indústria Ltda., São
Material and Methods Paulo, SP, Brazil).

Plant material Experimental procedures

C. sinensis leaves were collected in the The experimental procedures were pre-
town of Boquim (Sergipe, Brazil, 11º 15' 15" viously approved by the Research Ethics
S latitude, 37º 33' 45" W longitude) from Committee of the Federal University of
agrotoxic-free trees during the winter sea- Sergipe. The animals were handled accord-
son. A botanical specimen was classified as ing to usual procedures extensively described
Citrus aurantium L. var. sinensis Gall., which in the specialized literature, following the

Braz J Med Biol Res 38(1) 2005

Inotropic effect of C. sinensis on the guinea pig atrium 113

ethical rules disposed by the Brazilian Col- for about 1 h. The preparation was consid-
lege for Animal Experimentation (COBEA, ered to be well adapted when its force ampli-
Colégio Brasileiro de Experimentação Ani- tude and resting tension remained unchanged.
mal). The extracts were added cumulatively to
The experiments were carried out on the the organ bath and the experiments were
guinea pig left atria removed from animals carried out using the following concentra-
of both sexes (300-500 g) killed by a blow to tion range: crude extract (100-2000 µg/ml),
the skull base. The chest was opened and the ethanol extract (30-2500 µg/ml), acetone
heart promptly excised and transferred to an extract (10-1200 µg/ml), aqueous extract (30-
organ bath. The left atrium was immersed in 4000 µg/ml), and acetic acid extract (30-
a modified Tyrode solution consisting of 2500 µg/ml).
137 mM NaCl, 5.0 mM KCl, 0.5 mM MgCl2,
12.0 mM NaHCO3, 1.8 mM CaCl2, 6.0 mM Phytochemical screening
glucose, and 1.8 mM NaH2PO4, salts of ana-
lytical grade manufactured by and purchased Phytochemical screening was performed
from Merck S.A. Indústrias Químicas, Rio on the C. sinensis acetone extract according
de Janeiro, RJ, Brazil. The bath was oxygen- to the techniques proposed by Domínguez
ated and buffered with a carbogen mixture (15).
(95% O2 + 5% CO2 purchased from either
Aga S.A., São Paulo, SP, Brazil or White Acute toxicity testing
Martins Gases Industriais S.A., São Paulo,
SP, Brazil). The bath temperature was main- Twenty Mus musculus mice (10 males
tained at 27 ± 0.1ºC by means of a thermo- and 10 females weighing 23 to 33 g) were
statically controlled water jacket. studied after 7 days of acclimation. The ani-
The atria were stretched to 10 mN (1 gf = mals were housed in groups of 5 in wide
9.807 mN) as a control diastolic tension. cages, with free access to water and com-
Suprathreshold rectangular electrical pulses mercial laboratory chow. They remained at
of 2 Hz, 400 V, and 0.5 ms, provided by a room temperature (27 ± 2ºC) under a natural
Programmer (4030) and a Stimulator (3072) 12-h light-dark cycle (from 5:30 to 17:30 h,
manufactured by Digitimer Limited, Welwyn approximately). Ten mice were randomly
Garden City, Hertfordshire, England, were assigned to test groups of 5 males (23 to 26
delivered by a pair of Ag/AgCl electrodes g) and 5 females (28 to 33 g). The control
placed inside the organ bath to pace the group consisted of 5 males (24 to 27 g) and 5
preparations. These electrodes were arranged females (26 to 32 g). Acute toxicity testing
along the atrium in order to provide a simul- was performed by injecting intraperitoneally
taneous excitation of the entire preparation (ip) the male and female control group with
(field stimulation). The myocardial force was 0.25 ± 0.01 and 0.30 ± 0.02 ml of saline,
recorded isometrically with a force trans- respectively, whereas the test groups received
ducer (HP FTA 10-1 Sunborn, HP 8805B, ip 2 g/kg body weight of the C. sinensis
Chicago, IL, USA) with the aid of a thermal extract. The injected volume was 0.25 ± 0.01
paper polygraph (HP 8805B, HP 7754A, HP ml for males and 0.30 ± 0.02 ml for females.
7754B). Data were also stored in a computer The animals remained under observation for
(512 samples/s; A/D converter DI-400, 7 days after extract administration.
WINDAQ Proacquisition, DATAQ Instru-
ments, Akron, OH, USA) for off-line pro- Data processing
cessing. Before starting the experimental pro-
cedures, the atrium was allowed to stabilize Stored data were processed with the

Braz J Med Biol Res 38(1) 2005

114 E.D. Oliveira et al.

EG1v.1 software (Patent deposit No. sinensis extracts (1200 µg/ml) on the mem-
00051104, Instituto Nacional de Propriedade brane calcium inward current. The experi-
Industrial, INPI, Ministério do Desenvolvi- ments were carried out on animals previ-
mento, Indústria e Comércio Exterior/ ously treated with 5 mg/kg reserpine (Gross
Brasília, DF, Brazil, also deposited in the Laboratory, Rio de Janeiro, RJ, Brazil) in-
Cartório do 10º Ofício, Títulos e Documentos jected into the peritoneal cavity 24 h before
e Pessoas Jurídicas, Aracaju, SE, Brazil). To the experiment. Reserpine was used to de-
determine the effect of the extract on the plete the myocardial catecholamines and then
guinea pig atrium contractility, the follow- to minimize extrasystole occurrence. The
ing variables were measured: a) atrial force experimental protocol consisted of measur-
maximum amplitude, b) atrial contraction ing the atrial force at different stimulation
time, c) atrial relaxation time, and d) atrial rates: 1) low frequency (0.2 Hz, control rate),
force derivatives related to the contraction 2) high frequency (0.33, 0.5, 0.66, 1.0, 1.33,
and relaxation phases. Contraction time was 1.66, or 2.0 Hz, 40 s), 3) silent period (no
measured at three different levels, i.e., 20, stimulation was applied for 40 s), followed
50, and 80% of the atrial force amplitude. A by 4) low frequency (0.2 Hz). This cycle was
similar procedure was performed to deter- performed when normal Tyrode (control so-
mine the relaxation time. To obtain the mean lution) was used or after adding a C. sinensis
value of each of these variables, 50 succes- extract to the organ bath (test solution). The
sive contractions were processed. These Nayler and Merrillees (19) protocol is based
measurements were made during the follow- on the Bowditch phenomenon (positive stair-
ing experimental phases: 1) control, 2) after case) that appears when the stimulation rate
adding the extract to the organ bath, and 3) is suddenly increased. In this paper, ‘force
after the washout recovery. overshoot’ stands for the amount of force
To determine the extract concentration over the force amplitude observed during the
required to produce 50% of the maximum control phase.
extract effect (EC50), the experimental data
were fitted by curves calculated by the Hill- Statistical analysis
Langmuir equation (16-18):
The Student t-test for independent
samples was used to determine differences
between means. Results were considered to
be statistically different when P was less
than 0.05 (Statistica for Windows). Data are
reported as means ± SD.

where: E = effect of the extract on the contrac- Results

tile force amplitude; Emax = maximum extract
effect on the contractile force amplitude; C = A sodium and potassium content equal to
extract concentration in the organ bath; EC50 = 0.013 mM and 1.2 mM was obtained by
extract concentration producing 50% of the flame photometry for 1 g/l of C. sinensis
maximum effect, and n = Hill constant. crude extract. The yields of solids for each of
the C. sinensis extracts were: 16% (160 g/kg
Study of the inward calcium current dry leaves) for the crude water-alcohol ex-
tract and 3.4% (34 g/kg dry leaves) for the
The protocol of Nayler and Merrillees hexane extract, 2.05% (20.5 g/kg dry leaves)
(19) was employed to study the effect of C. for the chloroform extract, 1.6% (16 g/kg dry

Braz J Med Biol Res 38(1) 2005

Inotropic effect of C. sinensis on the guinea pig atrium 115

leaves) for the acetone extract, 1.2% (12 g/ tors participated (Figure 3), as shown by the
kg dry leaves) for the ethanol extract, 3.4% fact that none of the following receptor an-
(34 g/kg dry leaves) for the methanol extract, tagonists modified the myocardial effect of
3.7% (37 g/kg dry leaves) for the aqueous the crude extract: 10 µM naloxone, 1 µM
extract, and 4.3% (43 g/kg dry leaves) for the propranolol, or 1.5 µM atropine sulfate when
acetic acid extract. added to the bath.
The phytochemical screening of the ac- The C. sinensis effect on the calcium
etone extract of C. sinensis revealed the inward current was also studied by the Nayler
following types of compounds: 1) saponin and Merrillees protocol (19). This protocol
[foam (-)]; 2) alkaloid [Bouchardat (-), Mayer is based on the development of the Bowditch
(-), Dragendorff (-), tungstosilicic acid (-)]; positive staircase phenomenon that is a con-
3) steroid [acetic anhydride plus sulfuric sequence of an increased calcium entry pro-
acid (+)]; 4) tannin [gelatin 0.5% (++), FeCl3 moted by a sudden increase of the stimula-
(++)]; 5) flavonoid [magnesium tape (+++),
fluorescence (+++)].
Figure 1 shows the negative inotropic
effect of the crude water-alcohol extract of
5 mN
C. sinensis with a concentration-dependent
reduction in atrial force. Despite its relevant Control 100 1000 3000 5000 5 min

magnitude (79%), this effect promptly dis- Figure 1. Negative inotropic effect promoted by the crude extract of Citrus sinensis leaves
appeared when the extract was removed from on the guinea pig left atrium. The extract was cumulatively added to the organ bath after a
control period and induced a concentration-dependent effect. The atrium was paced at 2
the organ bath. Similar results were obtained Hz with pulses of 400 V, and 0.5 ms (27 ± 0.1ºC). Similar results were observed in 10 trials
in 10 trials carried out on 5 atria. carried out on 5 atria. 5 mN = 0.51 gf.
Experiments carried out to determine
which solvent extract of C. sinensis had the
Negative inotropic effect (% of the control force amplitude)

most potent depressant effect are shown in

100
Figure 2. Hill-Langmuir curves used to de-
termine the EC50 of different extracts indi-
cated EC50 values equal to 300, 300, 600, 80
1000, and 140 µg/ml with Hill constants
equal to 1.8, 2.0, 2.5, 2.0, and 1.4 for the 60
crude, ethanol, acetic acid, aqueous, and
acetone extracts, respectively. The acetone 40
extract was the most potent and its EC50 was
statistically different (Student t-test) from
20
the crude, ethanol, acetic acid, and aqueous
extracts (P < 0.001). However, the toxicity
of the acetone extract was low, as indicated 0
1 10 100 1000 10000
by the fact that only one (a female) of 10
Citrus sinensis (µg/ml)
mice (5 males and 5 females) died 24 h after
being injected with the high dose of the
Figure 2. Hill-Langmuir plots constructed to determine the potency of the following Citrus
acetone extract (2 g/kg).
sinesis extracts: acetone (filled squares, EC50 = 140 µg/ml, Hill constant = 1.4), ethanol
The involvement of the ß-adrenergic, (open squares, EC50 = 300 µg/ml, Hill constant = 2), crude (triangles, EC50 = 300 µg/ml, Hill
cholinergic or opioid membrane receptors in constant = 1.8), acetic acid (circles, EC50 = 600 µg/ml, Hill constant = 2.5), and aqueous
the mechanism of action of the C. sinensis (lozenges, EC50 = 1000 µg/ml, Hill constant = 2). The EC50 of the acetone extract was
significantly different from that of all other extracts (P < 0.001). The experiments were
crude extract was also studied (N = 3 atria, 6 carried out on electrically paced guinea pig left atria (2 Hz, 400 V, 0.5 ms, 27 ± 0.1ºC, N = 6
trials). None of the above mentioned recep- hearts, resting tension: 10 mN = 1.02 gf).

Braz J Med Biol Res 38(1) 2005

116 E.D. Oliveira et al.

Figure 3. The receptor antago- tion rate. Experiments performed at 27 ±

(% of the control force amplitude)

100 Control
nists propranolol (1 µM), atro-
0.1ºC showed that the force overshoot ob-

Negative inotropic effect

pine sulfate (1.5 µM), and Propranolol
80
naloxone (10 µM) do not Atropine served during the positive staircase (Bowditch
Naloxone
modify the depressor effect
60 phenomenon) disappeared when the C.
produced by the Citrus
sinensis acetone extract (1200 µg/ml) was
sinensis crude extract, sug-
gesting that none of them par-
40 added to the organ bath (Figure 4). More-
ticipates in the mechanism of
20
over, the extract was able to induce a fre-
action of this extract on the
quency-dependent negative staircase phe-
guinea pig atrium (N = 3 atria
and 6 trials for each type of 0 nomenon (Figure 5). Similar results were
1.5 2.0 2.5 3.0 3.5
receptor studied, 27 ± 0.1ºC, observed in 6 hearts (10 trials, stimulus: 400
Log [Citrus sinensis (µg/ml)]
stimuli: 2 Hz, 400 V, 0.5 ms). V, 0.5 ms).

Discussion
0.33 Hz 0.66 Hz 1.33 Hz
In the present study, we investigated the
0.2 Hz inotropic effects of some C. sinensis leaf
extracts on the mammalian myocardium. In
the guinea pig atria, these extracts interfered
with the atrial contraction mechanism by
5 mN

40 s
depressing the myocardial force in a concen-
tration-dependent manner. The crude water-
alcohol extract reduced the atrial contractile
force with an EC50 equal to 300 µg/ml. In the
effort to isolate the active crude extract com-
pound, the following water-soluble leaf ex-
Figure 4. Membrane calcium inward current of isolated atria. The upper panels show the tracts were obtained: the acetone extract was
Bowditch positive staircase obtained with the control solution by pacing the atrium at the the most potent (EC50 = 140 µg/ml) when
following rates: 0.33, 0.66, and 1.33 Hz. The data in the bottom panels were obtained with
the same stimulation rates but in the presence of the Citrus sinensis acetone extract (1200
compared to the ethanol, acetic acid, ac-
µg/ml). In this case, an inverse Bowditch phenomenon occurred (negative force overshoot). etone, and aqueous extracts. The acetone,
Similar results were obtained with 6 other preparations (27 ± 0.1ºC, control rate: 0.2 Hz, 5 ethanol, acetic acid, aqueous, and crude ex-
mN = 0.51 gf).
tracts exert their myocardial effects by means
of a cooperative interaction with the effector
Figure 5. Effect of stimulation
site (Hill constant higher than 1). This Hill
frequency on the force over- constant presumably indicates the number
shoot of the guinea pig left of active molecules needed to interact with
atrium. The overshoot is the
amount of force that exceeds
each binding site in order to promote the
the control force (control rate: biological effect observed (11-13,20).
0.2 Hz). Control curve: note 150 Control Neither the cholinergic, nor the opioid or
that the increase of the stim- Citrus
ß-adrenergic membrane receptors appear to
(% of the control force)

ulation rate led to an increase 100

be involved in the mechanism of action of
Force overshoot

in force overshoot. When the

Citrus sinensis extract (1200 50 the crude extracts of C. sinensis because
µg/ml) was added to the bath,
0 atropine sulfate (21), naloxone (22) and pro-
the opposite effect was ob-
served. In this case, when the pranolol (23) did not change the inotropic
drive frequency was in- -50 response induced by leaf extract.
creased, the force overshoot
-100 The disappearance of the Bowditch posi-
decreased (N = 6 hearts, 10
trials, stimulus: 400 V and 0.5
0.0 0.5 1.0 1.5 2.0 2.5 tive staircase promoted by the acetone ex-
ms, 27 ± 0.1ºC). Stimulation frequency (Hz) tract of C. sinensis leaves suggests that the

Braz J Med Biol Res 38(1) 2005

Inotropic effect of C. sinensis on the guinea pig atrium 117

extract inhibits membrane calcium inward abolished the force overshoot (amount of the
current. Since the acetone extract of C. force over the force control value), a phe-
sinensis induced a negative Bowditch stair- nomenon that can be observed when the
case, another mechanism may be acting in stimulus rate is suddenly increased (Bowditch
parallel to calcium channel inhibition, such phenomenon) for atria maintained in Tyrode
as activation of the membrane potassium solution. This effect indirectly suggests a
channels leading to shortening of the action reduction of the calcium inward current, per-
potential. haps by blocking the L-type calcium mem-
C. sinensis leaves are known to be rich in brane channels. We observed an inverse re-
flavonoids such as HEPTA, luteolin, apige- lationship between the acetone extract con-
nin, nobiletin, hesperidin, quercetin, natsu- centration and the force overshoot. Thus, the
daidain, and diosmin (10). It has been re- faster the frequency of stimulation, the more
ported that citrus flavonoids can affect car- depressed is the force overshoot.
diac muscle. Quercetin and 3-methylquercitin However, it is important to emphasize
are substances with a positive chronotropic that since the acetone extract is a mixture of
effect on the right guinea pig atrium (24). several unknown substances, it cannot be
Penta-O-ethylquercetin induces a rapid and ruled out that we are observing the result of
transitory fall in the rat arterial blood pres- several simultaneous activities. To under-
sure mainly due to the inhibition of the car- stand the mechanism underlying these re-
diac cyclic AMP phosphodiesterase (8). sults, we could hypothesize that the extract
HEPTA flavonoid has a positive inotropic could also be acting on the membrane potas-
effect on the guinea pig papillary muscle (8). sium conductance. If this is the case, the
Hesperidin, quercetin, HEPTA, and apige- extract may be opening some potassium chan-
nin - important bioactive substances present nels, contributing to the reduction of the
in C. sinensis leaves - do not seem to be atrial action potential plateau and leading to
directly involved in the depression of myo- a decrease of the time interval during which
cardial contractility because their inotropic the L-type calcium channels remain open.
effect actually corresponded to an increase In conclusion, we showed that the C.
in the contraction force (25). In contrast, sinensis acetone extracts exert a negative
quercetin antagonizes the rate of the sponta- inotropic effect and we speculate that they
neously cytoplasmic calcium oscillation (26), do this by reducing the calcium inward cur-
a phenomenon that is known to be related to rent and by opening some potassium mem-
the calcium-induced calcium release mech- brane channels. These effects may be clini-
anism. cally relevant because anti-arrhythmic agents
The acetone extract of C. sinensis leaves usually are myocardial depressant substances.

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