International Journal of Impotence Research (2008) 20, 255–263
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ORIGINAL ARTICLE
Relaxant effects of an alkaloid-rich fraction from Aspidosperma
ulei root bark on isolated rabbit corpus cavernosum
AR Campos1, KMA Cunha1, FA Santos1, ER Silveira2, DEA Uchoa2, NRF Nascimento3 and VSN Rao1
1
Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, CE, Brazil;
Department of Organic and Inorganic Chemistry, Federal University of Ceará, Fortaleza, CE, Brazil and 3Institute of
Biomedicine, Veterinary College, State University of Ceará, Fortaleza, CE, Brazil
2
We described earlier that an alkaloid-rich fraction (F3–5) from Aspidosperma ulei (Markgr) induces
penile erection-like behavioral responses in mice. This study verified a possible relaxant effect of
this fraction on isolated rabbit corpus cavernosum (RbCC) strips precontracted by phenylephrine
(1 lM) or K þ 60 mM. F3–5 (1–300 lg ml1) relaxed the RbCC strips in a concentration-dependent and
reversible manner. The relaxant effect of F3–5 (100 lg ml1) on phenylephrine contraction was
unaffected in the presence of atropine, N-x-nitro-L-arginine methyl ester or 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one and by preincubation with tetrodotoxin, glibenclamide, apamine and
charybdotoxin suggesting that mechanisms other than cholinergic, nitrergic, sGC activation or
potassium channel opening are probably involved. However, the phasic component of the
contraction induced by K þ 60 mM as well as the maximal contraction elicited by increasing
external Ca2 þ concentrations in depolarized corpora cavernosa was inhibited by F3–5. We conclude
that F3–5 relaxes the RbCC smooth muscle, at least in part, through a blockade of calcium influx or
its function.
International Journal of Impotence Research (2008) 20, 255–263; doi:10.1038/sj.ijir.3901624;
published online 29 November 2007
Keywords: Aspidosperma ulei (Markgr); alkaloid fraction; rabbit corpus cavernosum; smooth
muscle relaxation
Introduction
The erectile dysfunction is a common condition that
affects majority in the world causing considerable
distress, unhappiness and relationship problems.
Recent research on penile smooth muscle physiology has increased the number of drugs available for
treating erectile dysfunction (ED).1 Penile erection
occurs when the lacunar spaces of the corpora
cavernosa expand with blood through relaxation of
smooth muscle and vasodilatation of the helicine
arteries.2 This is triggered by nitric oxide (NO)
release from cavernosal nerve terminals and endothelial cells, which activates intracellular guanylate cyclase to produce more cGMP.3 Penile flaccid
Correspondence: Professor VSN Rao, Department of
Physiology and Pharmacology, Faculty of Medicine,
Federal University of Ceara, POB: 3157, Fortaleza, CE
60430-270, Brazil.
E-mail: vietrao@ufc.br
Received 16 July 2007; revised 26 October 2007; accepted
1 November 2007; published online 29 November 2007
state is conversely maintained by the a-adrenergic
neuroeffector system and by other vasoconstrictors,
such as endothelin-1. In recent years, the selective
phosphodiesterase 5 (PDE5) inhibitors, sildenafil,
tadalafil and vardenafil have become the treatment
option for ED but their uses have contraindications,
such as with concomitant nitrate administration or
a-adrenergic blockers.4 Besides, there were reports
of undesirable cardiovascular and visual disturbances5,6 presumably due to differential expression
of phosphodiesterase enzymes in various tissues
and their selectivity. Therefore, alternative forms of
medical treatment remain clinically interesting,
including plant extracts, which many patients prefer
as a treatment modality.7
Natural product research can often give substantial contribution to drug innovation by providing
novel chemical structures and/or mechanisms of
action.8 Many plant extracts are traditionally employed among different cultures in order to improve
sexual performances.9,10 Some plant-derived alkaloids such as papaverine, apomorphine, berberine
and yohimbine have some degree of evidence that
they may be helpful for impotence and ED.11–14
A. ulei on rabbit corpus cavernosum
AR Campos et al
256
Aspidiosperma species commonly grown in tropical
America have proven to be a rich source of indole
alkaloids and several of them exhibit important
pharmacological properties that include antimalarial, antileishmania, antidiabetic and anti-inflammatory effects.15–18 The use of bark extract from
Aspı´dosperma quebracho blanco has been a common traditional practice in many parts of South
America to treat impotence, benign prostatic hypertrophy and to obtain relief from cardiac- or asthmarelated dyspnea.19 An a-adrenoceptor blocking
activity of it has been described in literature. The
bark extract binds nonselectively to human penile
a1- and a2-adrenoceptors and cloned human
a-adrenoceptor subtypes, and this effect was attributed to the bark’s yohimbine content, which has
moderate but well-documented effects on ED.20,21
Aspidosperma ulei is yet another plant that largely
grows in the Amazon region of Brazil and in many
other parts of South America that is found to be rich
in indole alkaloids. In contrast to A. quebracho
blanco, there were not many reports available on the
pharmacological activity of A. ulei alkaloids with
the exception of one study that describes the in vitro
relaxant property of the alkaloid containing extract
on vascular and nonvascular smooth muscles from
rats, guinea-pigs and rabbits.22 In awake mice, very
recently we demonstrated a pro-erectile activity of
an alkaloid-rich fraction (F3–5) from A. ulei root bark
that might have resulted from both central and
peripheral sites of action involving a-adrenergic,
dopaminergic and nitrergic receptor mechanisms.23
To have a greater insight into the mechanism(s) of
pro-erectile effect of F3–5, the present study was
designed to investigate its effect on phenylephrine
(PHE) or high potassium-precontracted rabbit
corpus cavernosum (RbCC) in vitro, and further to
elucidate the possible involvement of adrenergic,
cholinergic and nitrergic neuroeffector systems and
the role of potassium and calcium channel activation in its relaxant effect.
Materials and methods
Plant material, fractionation and identification of
alkaloids
A. ulei (Markgr) was collected from the Garapa area
of Acarape, Ceará, Brazil after its identification, and
a voucher specimen has been deposited in Herbarium Prisco Bezerra (no. 30823) of Federal University
of Ceará, Fortaleza. The fraction, F3–5 was obtained
from the ethanolic extract of A. ulei root bark
according to a previously described procedure.22
1
H NMR analysis of this fraction (F3–5) revealed the
presence of three major indole-type alkaloids, which
were identified as uleine, nor-uleine and tetrahydro3,14,4,21-elipticin (Figure 1) based on spectral details and in comparison with literature data.24
International Journal of Impotence Research
H
N
N
N
N
H
H
Uleine
Nor-uleine
H
N
N
H
Tetrahydro-3,14,4,21-ellipticine
Figure 1 Chemical structures of ulein, nor-ulein and tetrahydro3,14,4,21-elipticin.
Chemicals
Phentolamine hydrochloride, PHE, atropine, 1H[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (ODQ),
N-o-nitro-L-arginine methyl ester (L-NAME), tetrodotoxin (TTX), charybdotoxin, apamin, glibenclamide,
nifedipine, ethyleneglycol-bis(b-aminoethylether)N,N0 -tetraacetic acid, guanethidine and dimethyl
sulfoxide (DMSO) were purchased from Sigma/
Aldrich Chemical Co (St Louis, MO, USA). Drug
solutions were prepared in saline always fresh on
the day of experiment. F3–5 was dissolved in 3%
(v/v) DMSO. Controls were administered 3% DMSO
in saline (v/v) to serve as vehicle-treated controls.
RbCC smooth muscle strips in vitro
The study protocols were approved by the Institutional Ethics Committee of the Federal University of
Ceará in accordance with the guidelines of National
Institute of Health on the use and care of animals for
experimentation. Male New Zealand white rabbits
(6-month-olds; 2.5–3.0 kg) were used for the
study (n ¼ 15). For experiments, animals were
anesthetized with pentobarbital sodium (Hypnol,
35–40 mg kg1, i.v.) and killed by exsanguination.
The penis was removed at the level of attachment of
the corporal body to the ischium, immersed in cold
Krebs solution (pH 7.4). The corporal tissues were
carefully dissected free from the tunica albuginea,
strips were prepared and mounted under 1 g resting
tension in 5 ml organ baths filled with warmed
(37 1C) and oxygenated (95% O2 þ 5% CO2) Krebs
solution.25 Following an equilibration period of
60 min, tension was induced by the addition
of PHE (1 mM). At the plateau of contraction,
relaxation responses to cumulative concentrations
(1–300 mg ml1) of F3–5 were registered on a desk
model polygraph (DMP-4B, Narco Bio-Systems,
Houston, TX, USA), using a model FT-60 (Narco
Bio-System) force displacement transducer.
A. ulei on rabbit corpus cavernosum
AR Campos et al
Other experimental protocol consisted of inducing frequency–response curves of relaxation (what
are mainly from nitrergic origin) on 1 mM PHE
precontracted RbCC with 10 s train of transmural
electrical field stimulation (EFS; 20 V; 0.5 ms; 2–
16 Hz). The frequency–response curves were performed in the absence and presence of vehicle or
F3–5 (1, 3 or 10 mg ml1).
In separate experiments, when once a stable
contraction to PHE (1 mM) was attained, F3–5
(100 mg ml1) was added to the organ bath in the
presence of vehicle or one of the following pharmacological blockers: 10 mM atropine, 100 mM L-NAME,
100 mM ODQ, 100 mM glibenclamide, 1 mM TTX or
10 mM apamin plus 100 nM charibdotoxin. The
cavernosal strips were preincubated with these
pharmacological agents in the bath chamber for a
30 min period before the addition of F3–5.
In another set of experiments RbCC were pretreated during 30 min with 10 mM guanethidine and
10 mM phentolamine (a1- and a2-adrenergic receptor
blockers) and thereafter a concentration–response
curve for SF3–5 was performed in strips tonically
precontracted with K þ 60 mM.
In the protocols described until this point the
effects of F3–5 were studied using paired segments
mounted on different baths and cumulatively increasing concentrations of F3–5 in one bath while
adding vehicle, isovolumetrically, to the other. The
differences between baseline of the control and test
segment were used to express the relaxation induced by F3–5. The relaxation (negative deflections)
were expressed as percentage of the maximal tonic
contraction (positive deflection) induced by PHE or
K þ 60 mM.
In another set of experiments, F3–5 (30, 100 and
300 mg ml1) was added to the preparation 5 min
before the induction of phasic contractions with K þ
60 mM. The phasic component was considered at the
peak upward deflection after 10 s exposure to the
high-potassium solution. The response obtained in
the presence of each concentration of F3–5 was
expressed as percentage of the K þ -induced phasic
contraction in the absence of F3–5 and compared
with the effect of vehicle added isovolumetrically.
Finally, concentration–response curves obtained
by increasing concentrations of calcium chloride
(CaCl2; 1–100 mM) were performed in RbCC
previously depolarized with Krebs–Henseleit with
K þ -60 mM and containing zero nominal calcium. This
whole procedure was repeated in the presence of F3–5
(100 mg ml1) or vehicle in separate experiments.
Statistical analysis
The magnitude of relaxant RbCC responses to F3–5 is
given as the percentage of the precontraction
induced by PHE, or high potassium. The results
are expressed as the mean±s.e.m. of the number of
experiments and compared with the response
obtained by isovolumetric addition of vehicle (3%
DMSO).The concentration producing a 50% relaxation of maximal response (EC50) was calculated by
sigmoidal curve-fitting analysis by using GRAPH
PAD 3.0 software and expressed along with its 95%
CI in brackets. The statistical differences were
analyzed by one-way analysis of variance (ANOVA)
with Tukey’s test as post hoc, with Po0.05 considered to indicate statistical significance. Differences among the relaxation obtained before and
after the specific blockers were compared by paired
two-tailed Student’s t-test with significance set at
Po0.05.
257
Results
The cumulative addition of F3–5 (1–300 mg ml1)
concentration dependently inhibited the tension
induced by either PHE (1 mM) or 60 mM potassium
in a nonadrenergic medium (that is, enriched with
guanethidine and phentolamine; Figures 2a–c). The
relaxant effect of F3–5 was reversible after repeated
washings with calculated EC50 and 95% CI for PHEinduced contraction being 26.3 mg ml1 (15.1–45.9)
and for K þ 60 mM being 94.8 mg ml1 (19.5–459).
The EFS-induced relaxation was not modified by
F3–5 in any frequency studied. For example, the
maximal relaxation induced by 2 Hz (10 s train; 20 V;
0.5 ms) stimulation was 29.7±1.4% in the presence
of vehicle and 33.4±1.9; 33.3±5.6 or 40.4±6.8% in
the presence of 1, 3 or 10 mg ml1 F3–5, respectively.
In the same way, the maximal relaxation attained
after 16 Hz stimulation was 68.8±9.1% in the
control group compared with 59.2±1.7, 56.1±2.8
or 68.1±8.1% obtained in the presence of 1, 3 or
10 mg ml1 F3–5, respectively (Figure 3). The concentration–response curve to F3–5 was not shifted by
atropine (10 mM), L-NAME (100 mM) or ODQ (100 mM).
Nevertheless, the relaxation obtained by the lowest
concentration (that is, 1 and 3 mg ml1) was reduced
in amplitude by L-NAME or ODQ but not by
atropine. The relaxation induced by F3–5 (1 and
3 mg ml1) in control conditions were 15.7±4.5 and
26.2±5.1%, respectively and were 2.9±1.5 and
10.4±2.6%, respectively, in the presence of
L-NAME (Po0.05, Student’s t-test vs control). The
same pattern occurred in the presence of ODQ with
relaxation of 3.7±2.5 and 7.7±2.7% attained with 1
or 10 mg ml1 F3–5, respectively (Po0.05, Student’s
t-test vs control; Figure 4).
On the other hand, TTX a neuronal sodium
channel blocker did not affect the relaxation elicited
by F3–5 (Figures 5a and b). There was also no
statistical difference with regard to the relaxant
responses of the cavernosum to F3–5 in the presence
of KATP channel blocker, glibenclamide (100 mM)
or apamin (1 mM) plus charybdotoxin (100 nM),
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AR Campos et al
258
Figure 2 Concentration–response curves to F3–5 (1–300 mg ml1) on isolated RbCC precontracted by phenylephrine (PHE) 10 mM or K þ
60 mM in a medium containing 10 mM phentolamine and guanethidine is shown in (a). Data are expressed as mean±s.e.m. (n ¼ 8–10).
(b and c) Representative physiographic recordings showing the relaxant effects of F3–5 (1–300 mg ml1) on isolated RbCC precontracted
by PHE 10 mM (b) or K þ 60 mM in tissues pretreated with phentolamine and guanethidine (c).
the potent inhibitors of small and medium and
large conductance KCa channels, respectively
(Figures 6a–c).
In experiments that were designed to verify the
calcium antagonism, the phasic contraction elicited
by K þ 60 mM was blocked by F3–5 in a concentration-related way with maximal inhibition attained at
300 mg ml1 (96.7±4.3% inhibition; Figures 7a and
b). Similarly, F3–5 (100 mg ml1) significantly inhibited (53.6%) the maximal contraction elicited by
cumulative additions of calcium chloride (1–
100 mM) to the Krebs medium in corpora cavernosa
previously depolarized by K þ 60 mM in a ‘Ca2 þ free’ medium (Figures 7c and d). The curve was only
International Journal of Impotence Research
shifted downwards in the concentration range
studied.
Discussion and conclusions
The isolated RbCC is a common experimental model
used to assess the erectile activity of compounds
and the relaxation of the cavernosum is considered
a positive result for the test substance.17–19 This
in vitro system was utilized for the present study
to verify the underlying mechanism in the proerectile activity of A. ulei fraction (F3–5) observed
A. ulei on rabbit corpus cavernosum
AR Campos et al
259
Figure 3 Frequency–response curves to transmural electrical
field stimulation (EFS; 20 V; 0.5 ms; 2–16 Hz) in the presence of
vehicle (control) or 1, 3 or 10 mg ml1 F3–5 on isolated RbCC
precontracted by phenylephrine (PHE) 1 mM. The data are
expressed as mean±s.e.m. (n ¼ 6–8).
Figure 4 Concentration–response curves to F3–5 (1–300 mg ml1)
on isolated RbCC precontracted by phenylephrine (PHE) 1 mM in
tissues pretreated with N-o-nitro-L-arginine methyl ester(LNAME; 100 mM), ODQ (100 mM) or atropine (10 mM). The data are
expressed as mean±s.e.m. (n ¼ 6–8). *Po0.05, Student’s t-test vs
control.
in vivo.23 The initial objective of our study was to
verify a possible relaxant effect of F3–5 on PHE or
high potassium-precontracted RbCC and then to
elucidate the likely involvement of adrenergic,
cholinergic and nitrergic neuroeffector systems as
well as the role of potassium and calcium channel
activation in its relaxant mechanism.
F3–5 showed a concentration-dependent and reversible relaxing effect on RbCC strips precontracted
by PHE; 10 mM) or high potassium (60 mM). Since
F3–5 relaxed the RbCC precontracted by K þ 60 mM
in a nonadrenergic medium (that is, enriched with
guanethidine and phentolamine), a postsynaptic
blockade of a-adrenergic receptor by F3–5 is unlikely
to explain its relaxant activity.
The relaxation evoked by transmural EFS, which
is mainly dependent on neuronal NO release and
increased intracellular cGMP concentration,26 was
not modified by F3–5 and this argues against a
possible PDE5-inhibitory activity of this compound.
Figure 5 (a) Concentration–response curves to F3–5
(1–300 mg ml1) on isolated RbCC precontracted by phenylephrine
(PHE) 1 mM in the absence or presence of TTX (10 mM).
(b) Representative physiographic recording showing the effect of
F3–5 (1–300 mg ml1) in tissues pretreated with tetrodotoxin (TTX)
is presented. The data are expressed as mean±s.e.m. (n ¼ 6–8).
Studies of Williams et al.27 suggest that NO and
cGMP act synergistically to reduce Ca2 þ release
from intracellular stores and thereby the relaxation
of the corpus cavernosum, leading to erection.
Nevertheless the main component of the relaxant
effect of F3–5 seems to be neither related to a direct
stimulating action on muscarinic cholinergic receptors releasing endothelial NO, nor to an increased
eNOS or nNOS activity or due to NO activation of
soluble guanylate cyclase with subsequent increase
in cGMP generation. The concentration–response
curve to F3–5 was unaffected by the muscarinic
blocker atropine, the NO-synthase inhibitor
L-NAME or by the soluble guanylate cyclase inhibitor
ODQ, suggesting a different mechanism. Nevertheless, the decrease in magnitude of the relaxation
induced by low concentrations of F3–5 (1 and
3 mg ml1) might be associated to the inhibition of
basal NO–GC activity by L-NAME and ODQ, which
per se increases RbCC tonus. Alternatively, this may
reflect a first component of the relaxation that is
dependent on NO release, but the concentration–
response curve, EC50 values and maximal response
International Journal of Impotence Research
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260
Figure 6 (a) Concentration–response curves to F3–5 (1–300 mg ml1) on isolated RbCC precontracted by phenylephrine (PHE) 1 mM in the
absence or presence of glibenclamide (GLIB 100 mM) or apamin (APAM 1 mM) plus charybdotoxin (CHARBD, 100 nM). The data are
expressed as mean±s.e.m. (n ¼ 6–8). Representative recordings are depicted in (b) (APA þ CHAR) and (c) (GLIB).
are not significantly modified and this component
(if it exists) is of minor contribution to the overall
relaxant activity.
A direct nitrergic activation, as demonstrated to
Tityus serrulatus scorpion venom toxin28 is also
unlikely since the relaxation was insensitive to TTX.
The potassium channels such as BKCa, Kv and
KATP are physiological regulators of the membrane
electric potential and transmembrane calcium flux,
and therefore they may have a key role in the
regulation of smooth muscle tone including corpus
cavernosum.29–31 However, in the present investigation, the blockade of KCa channels (both high MaxiK and low-conductance KCa), with charybdotoxin
and apamin, respectively, and the KATP channels
by glibenclamide did not modify significantly the
International Journal of Impotence Research
F3–5-induced relaxation. These findings suggest that
the relaxation effect of F3–5 is unrelated to Ca2 þ activated, ATP-sensitive and voltage-dependent
K þ channels.
It has been shown that in RbCC smooth muscle
PHE induces contraction not only by increasing
intracellular Ca2 þ concentration, but also by increasing Ca2 þ sensitivity of the contractile apparatus.32 Evidence also exists for the role of Ca2 þ entry
in the tonic contraction of the corpus cavernosum
smooth muscle cells and this tonic contraction can
be abolished by voltage-dependent Ca2 þ channel
blockers, such as nifedipine or by removal of
extracellular Ca2 þ .33 A possible calcium channel
blockade by F3–5 cannot be ruled out from the present study since it could block the K þ 60 mM-induced
A. ulei on rabbit corpus cavernosum
AR Campos et al
261
Figure 7 Representative physiograph recordings (n ¼ 5) of the effect of F3–5 (30, 100 and 300 mg ml1) on isolated RbCC on phasic
contractions elicited by rapid exposition (10 s) to K þ 60 mM is shown in (a) and (b), the respective graph with data expressed as
mean±s.e.m. (c) Representative physiographic recording of effect of a single concentration F3–5 (100 mg ml1) on contractions elicited by
cumulative additions of calcium chloride to the external medium (CaCl2; 1–100 mM) of previously depolarized by K þ 60 mM corpora
cavernosa is depicted and finally in (d) the graph representing data expressed as mean±s.e.m. (n ¼ 6–10). *Po0.01 vs vehicle (ANOVA
followed by Tukey in (b), and Student’s t-test for paired data in (d)).
phasic contraction, which largely depends on
calcium
influx
through
voltage-dependent
calcium channels.34 This is reinforced by the fact
that this alkaloid fraction strongly impaired contractions of depolarized RbCC, in a zero nominalcalcium medium, elicited by increasing calcium
concentrations in the medium. An impaired calcium
influx through the membrane and consequently the
calcium-induced calcium release from internal
stores is likely to account for the observed relaxant
effect of F3–5 on RbCC smooth muscle. The inhibition of calcium uptake observed in the presence of
alkaloid extract by rat cortical synaptosomes further
support this notion (unpublished observations).
Nevertheless, the antagonism to calcium signaling
as a second messenger as well as an interference on
the Ca2 þ sensitivity of the contractile machinery of
RbCC smooth muscle were not investigated and may
not be excluded as components of the relaxation
induced by F3–5.
We have previously described that A. ulei fraction
(F3–5) in vivo induces pro-erectile-like behavioural
activity in male mice that was effectively blocked by
pretreatment with L-NAME, an inhibitor of NOS,
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262
suggesting an NO-mediated mechanism,23 whereas
in the current study, the same fraction produced a
profound relaxant effect on corpus cavernosum
smooth muscle in vitro, by an NO-independent
mechanism, since the relaxation was unaffected by
L-NAME or ODQ, the soluble guanylate cylase
inhibitor. The apparently paradoxical mechanisms
that we observed in vivo and in vitro are probably
due to the fact that the fraction is a mixture
containing three major alkaloids. It is conceivable
that this fraction may have both central and
peripheral sites of action, which are possibly related
to one or even more number of these alkaloids.
In conclusion, our data demonstrate for the first
time that the fraction F3–5 from A. ulei bark extract
exerts a relaxant effect on RbCC smooth muscle
in vitro by a mechanism independent on NO–GC
pathway and probably related to a calcium antagonism. Taken together these results with our earlier
in vivo finding of its ability to induce penile
erections-like behavioral responses suggest that
F3–5, may have a clinical perspective in patients
with ED. However, it remains to be verified in future
studies whether all the three alkaloids or only
one among these contribute to the observed effect
of F3–5 and further elucidation on the precise
mechanism of its relaxant action on corpus cavernosum is necessary.
Acknowledgments
This study was supported by grants from Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq, proc. no. 472717/2003-0), and Fundação
Cearense de Pesquisa e Cultura (FUNCAP, proc. no.
30/2002).
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