Relaxant Effects of an Alkaloid-Rich Fraction from Aspidosperma Ulei Root Bark on Isolated Rabbit Corpus Cavernosum

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

1Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara´, Fortaleza, CE, Brazil; 2Department of Organic and Inorganic Chemistry, Federal University of Ceara´, Fortaleza, CE, Brazil and 3Institute of Biomedicine, Veterinary College, State University of Ceara´, Fortaleza, CE, Brazil

We described earlier that an alkaloid-rich fraction (F3–5)fromAspidosperma 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 þ À1 (1 lM)orK 60 mM.F3–5 (1–300 lgml ) relaxed the RbCC strips in a concentration-dependent and À1 reversible manner. The relaxant effect of F3–5 (100 lgml ) 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 þ contraction induced by K 60 mM as well as the maximal contraction elicited by increasing 2 þ external Ca 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 state is conversely maintained by the a-adrenergic neuroeffector system and by other vasoconstrictors, The erectile dysfunction is a common condition that such as endothelin-1. In recent years, the selective affects majority in the world causing considerable phosphodiesterase 5 (PDE5) inhibitors, sildenafil, distress, unhappiness and relationship problems. tadalafil and vardenafil have become the treatment Recent research on penile smooth muscle physiol- option for ED but their uses have contraindications, ogy has increased the number of drugs available for such as with concomitant nitrate administration or treating erectile dysfunction (ED).1 Penile erection a-adrenergic blockers.4 Besides, there were reports occurs when the lacunar spaces of the corpora of undesirable cardiovascular and visual distur- cavernosa expand with blood through relaxation of bances5,6 presumably due to differential expression smooth muscle and vasodilatation of the helicine of phosphodiesterase enzymes in various tissues arteries.2 This is triggered by nitric oxide (NO) and their selectivity. Therefore, alternative forms of release from cavernosal nerve terminals and en- medical treatment remain clinically interesting, dothelial cells, which activates intracellular guany- including plant extracts, which many patients prefer late cyclase to produce more cGMP.3 Penile flaccid as a treatment modality.7 Natural product research can often give substan- tial contribution to drug innovation by providing novel chemical structures and/or mechanisms of Correspondence: Professor VSN Rao, Department of action.8 Many plant extracts are traditionally em- Physiology and Pharmacology, Faculty of Medicine, ployed among different cultures in order to improve Federal University of Ceara, POB: 3157, Fortaleza, CE 9,10 60430-270, Brazil. sexual performances. Some plant-derived alka- E-mail: [email protected] loids such as papaverine, apomorphine, berberine Received 16 July 2007; revised 26 October 2007; accepted and yohimbine have some degree of evidence that 1 November 2007; published online 29 November 2007 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 H alkaloids and several of them exhibit important N N pharmacological properties that include antimalar- N ial, antileishmania, antidiabetic and anti-inflamma- N tory effects.15–18 The use of bark extract from H H Aspı´dosperma quebracho blanco has been a com- Uleine Nor-uleine mon traditional practice in many parts of South America to treat impotence, benign prostatic hyper- trophy and to obtain relief from cardiac- or asthma- 19 H related dyspnea. An a-adrenoceptor blocking N activity of it has been described in literature. The bark extract binds nonselectively to human penile N a1-anda2-adrenoceptors and cloned human a-adrenoceptor subtypes, and this effect was attrib- H uted to the bark’s yohimbine content, which has Tetrahydro-3,14,4,21-ellipticine moderate but well-documented effects on ED.20,21 Figure 1 Chemical structures of ulein, nor-ulein and tetrahydro- Aspidosperma ulei is yet another plant that largely 3,14,4,21-elipticin. 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 Chemicals blanco, there were not many reports available on the Phentolamine hydrochloride, PHE, atropine, 1H- pharmacological activity of A. ulei alkaloids with [1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (ODQ), the exception of one study that describes the in vitro N-o-nitro-L-arginine methyl ester (L-NAME), tetrodo- relaxant property of the alkaloid containing extract toxin (TTX), charybdotoxin, apamin, glibenclamide, on vascular and nonvascular smooth muscles from nifedipine, ethyleneglycol-bis(b-aminoethylether)- rats, guinea-pigs and rabbits.22 In awake mice, very N,N0-tetraacetic acid, guanethidine and dimethyl recently we demonstrated a pro-erectile activity of sulfoxide (DMSO) were purchased from Sigma/ an alkaloid-rich fraction (F3–5) from A. ulei root bark Aldrich Chemical Co (St Louis, MO, USA). Drug that might have resulted from both central and solutions were prepared in saline always fresh on peripheral sites of action involving a-adrenergic, the day of experiment. F3–5 was dissolved in 3% dopaminergic and nitrergic receptor mechanisms.23 (v/v) DMSO. Controls were administered 3% DMSO To have a greater insight into the mechanism(s) of in saline (v/v) to serve as vehicle-treated controls. pro-erectile effect of F3–5, the present study was designed to investigate its effect on phenylephrine (PHE) or high potassium-precontracted rabbit RbCC smooth muscle strips in vitro corpus cavernosum (RbCC) in vitro, and further to The study protocols were approved by the Institu- elucidate the possible involvement of adrenergic, tional Ethics Committee of the Federal University of cholinergic and nitrergic neuroeffector systems and Ceara´ in accordance with the guidelines of National the role of potassium and calcium channel activa- Institute of Health on the use and care of animals for tion in its relaxant effect. 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, Materials and methods 35–40 mg kgÀ1, i.v.) and killed by exsanguination. The penis was removed at the level of attachment of Plant material, fractionation and identification of the corporal body to the ischium, immersed in cold alkaloids Krebs solution (pH 7.4). The corporal tissues were A. ulei (Markgr) was collected from the Garapa area carefully dissected free from the tunica albuginea, of Acarape, Ceara´, Brazil after its identification, and strips were prepared and mounted under 1 g resting a voucher specimen has been deposited in Herbar- tension in 5 ml organ baths filled with warmed ium Prisco Bezerra (no. 30823) of Federal University (37 1C) and oxygenated (95% O2 þ 5% CO2) Krebs 25 of Ceara´, Fortaleza. The fraction, F3–5 was obtained solution. Following an equilibration period of from the ethanolic extract of A. ulei root bark 60 min, tension was induced by the addition 22 according to a previously described procedure. of PHE (1 mM). At the plateau of contraction, 1 H NMR analysis of this fraction (F3–5) revealed the relaxation responses to cumulative concentrations À1 presence of three major indole-type alkaloids, which (1–300 mgml )ofF3–5 were registered on a desk were identified as uleine, nor-uleine and tetrahydro- model polygraph (DMP-4B, Narco Bio-Systems, 3,14,4,21-elipticin (Figure 1) based on spectral de- Houston, TX, USA), using a model FT-60 (Narco tails and in comparison with literature data.24 Bio-System) force displacement transducer.

International Journal of Impotence Research A. ulei on rabbit corpus cavernosum AR Campos et al 257 Other experimental protocol consisted of indu- experiments and compared with the response cing frequency–response curves of relaxation (what obtained by isovolumetric addition of vehicle (3% are mainly from nitrergic origin) on 1 mM PHE DMSO).The concentration producing a 50% relaxa- precontracted RbCC with 10 s train of transmural tion of maximal response (EC50) was calculated by electrical field stimulation (EFS; 20 V; 0.5 ms; 2– sigmoidal curve-fitting analysis by using GRAPH 16 Hz). The frequency–response curves were per- PAD 3.0 software and expressed along with its 95% formed in the absence and presence of vehicle or CI in brackets. The statistical differences were À1 F3–5 (1, 3 or 10 mgml ). analyzed by one-way analysis of variance (ANOVA) In separate experiments, when once a stable with Tukey’s test as post hoc, with Po0.05 con- contraction to PHE (1 mM) was attained, F3–5 sidered to indicate statistical significance. Differ- (100 mgmlÀ1) was added to the organ bath in the ences among the relaxation obtained before and presence of vehicle or one of the following pharma- after the specific blockers were compared by paired cological blockers: 10 mM atropine, 100 mML-NAME, two-tailed Student’s t-test with significance set at 100 mM ODQ, 100 mM glibenclamide, 1 mM TTX or Po0.05. 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. Results In another set of experiments RbCC were pre- À1 treated during 30 min with 10 mM guanethidine and The cumulative addition of F3–5 (1–300 mgml ) 10 mM phentolamine (a1-anda2-adrenergic receptor concentration dependently inhibited the tension blockers) and thereafter a concentration–response induced by either PHE (1 mM)or60mM potassium curve for SF3–5 was performed in strips tonically in a nonadrenergic medium (that is, enriched with þ precontracted with K 60 mM. guanethidine and phentolamine; Figures 2a–c). The In the protocols described until this point the relaxant effect of F3–5 was reversible after repeated effects of F3–5 were studied using paired segments washings with calculated EC50 and 95% CI for PHE- mounted on different baths and cumulatively in- induced contraction being 26.3 mgmlÀ1 (15.1–45.9) þ À1 creasing concentrations of F3–5 in one bath while and for K 60 mM being 94.8 mgml (19.5–459). adding vehicle, isovolumetrically, to the other. The The EFS-induced relaxation was not modified by differences between baseline of the control and test F3–5 in any frequency studied. For example, the segment were used to express the relaxation in- maximal relaxation induced by 2 Hz (10 s train; 20 V; duced by F3–5. The relaxation (negative deflections) 0.5 ms) stimulation was 29.7±1.4% in the presence were expressed as percentage of the maximal tonic of vehicle and 33.4±1.9; 33.3±5.6 or 40.4±6.8% in À1 contraction (positive deflection) induced by PHE or the presence of 1, 3 or 10 mgml F3–5, respectively. þ K 60 mM. In the same way, the maximal relaxation attained In another set of experiments, F3–5 (30, 100 and after 16 Hz stimulation was 68.8±9.1% in the 300 mgmlÀ1) was added to the preparation 5 min control group compared with 59.2±1.7, 56.1±2.8 before the induction of phasic contractions with K þ or 68.1±8.1% obtained in the presence of 1, 3 or À1 60 mM. The phasic component was considered at the 10 mgml F3–5, respectively (Figure 3). The con- peak upward deflection after 10 s exposure to the centration–response curve to F3–5 was not shifted by high-potassium solution. The response obtained in atropine (10 mM), L-NAME (100 mM) or ODQ (100 mM). the presence of each concentration of F3–5 was Nevertheless, the relaxation obtained by the lowest expressed as percentage of the K þ -induced phasic concentration (that is, 1 and 3 mgmlÀ1) was reduced contraction in the absence of F3–5 and compared in amplitude by L-NAME or ODQ but not by with the effect of vehicle added isovolumetrically. atropine. The relaxation induced by F3–5 (1 and Finally, concentration–response curves obtained 3 mgmlÀ1) in control conditions were 15.7±4.5 and by increasing concentrations of calcium chloride 26.2±5.1%, respectively and were 2.9±1.5 and (CaCl2; 1–100 mM) were performed in RbCC 10.4±2.6%, respectively, in the presence of previously depolarized with Krebs–Henseleit with L-NAME (Po0.05, Student’s t-test vs control). The þ K -60 mM and containing zero nominal calcium. This same pattern occurred in the presence of ODQ with whole procedure was repeated in the presence of F3–5 relaxation of 3.7±2.5 and 7.7±2.7% attained with 1 À1 À1 (100 mgml ) or vehicle in separate experiments. or 10 mgml 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 Statistical analysis by F3–5 (Figures 5a and b). There was also no The magnitude of relaxant RbCC responses to F3–5 is statistical difference with regard to the relaxant given as the percentage of the precontraction responses of the cavernosum to F3–5 in the presence induced by PHE, or high potassium. The results of KATP channel blocker, glibenclamide (100 mM) are expressed as the mean±s.e.m. of the number of or apamin (1 mM) plus charybdotoxin (100 nM),

International Journal of Impotence Research A. ulei on rabbit corpus cavernosum AR Campos et al 258

À1 þ Figure 2 Concentration–response curves to F3–5 (1–300 mgml ) on isolated RbCC precontracted by phenylephrine (PHE) 10 mM or K 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). À1 (b and c) Representative physiographic recordings showing the relaxant effects of F3–5 (1–300 mgml ) on isolated RbCC precontracted þ by PHE 10 mM (b)orK 60 mM in tissues pretreated with phentolamine and guanethidine (c).

the potent inhibitors of small and medium and shifted downwards in the concentration range large conductance KCa channels, respectively studied. (Figures 6a–c). In experiments that were designed to verify the calcium antagonism, the phasic contraction elicited þ by K 60 mM was blocked by F3–5 in a concentra- Discussion and conclusions tion-related way with maximal inhibition attained at 300 mgmlÀ1 (96.7±4.3% inhibition; Figures 7a and The isolated RbCC is a common experimental model À1 b). Similarly, F3–5 (100 mgml ) significantly inhib- used to assess the erectile activity of compounds ited (53.6%) the maximal contraction elicited by and the relaxation of the cavernosum is considered cumulative additions of calcium chloride (1– a positive result for the test substance.17–19 This 100 mM) to the Krebs medium in corpora cavernosa in vitro system was utilized for the present study þ 2 þ previously depolarized by K 60 mM in a ‘Ca - to verify the underlying mechanism in the pro- free’ medium (Figures 7c and d). The curve was only erectile activity of A. ulei fraction (F3–5) observed

International Journal of Impotence Research 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 À1 vehicle (control) or 1, 3 or 10 mgml 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 5 (a) Concentration–response curves to F3–5 (1–300 mgmlÀ1) 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 À1 Figure 4 Concentration–response curves to F (1–300 mgmlÀ1) F3–5 (1–300 mgml ) in tissues pretreated with tetrodotoxin (TTX) 3–5 ± on isolated RbCC precontracted by phenylephrine (PHE) 1 mM in is presented. The data are expressed as mean s.e.m. (n ¼ 6–8). tissues pretreated with N-o-nitro-L-arginine methyl ester(L- NAME; 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 Studies of Williams et al.27 suggest that NO and control. cGMP act synergistically to reduce Ca2 þ release from intracellular stores and thereby the relaxation in vivo.23 The initial objective of our study was to of the corpus cavernosum, leading to erection. verify a possible relaxant effect of F3–5 on PHE or Nevertheless the main component of the relaxant high potassium-precontracted RbCC and then to effect of F3–5 seems to be neither related to a direct elucidate the likely involvement of adrenergic, stimulating action on muscarinic cholinergic recep- cholinergic and nitrergic neuroeffector systems as tors releasing endothelial NO, nor to an increased well as the role of potassium and calcium channel eNOS or nNOS activity or due to NO activation of activation in its relaxant mechanism. soluble guanylate cyclase with subsequent increase F3–5 showed a concentration-dependent and re- in cGMP generation. The concentration–response versible relaxing effect on RbCC strips precontracted curve to F3–5 was unaffected by the muscarinic by PHE; 10 mM) or high potassium (60 mM). Since blocker atropine, the NO-synthase inhibitor þ F3–5 relaxed the RbCC precontracted by K 60 mM L-NAME or by the soluble guanylate cyclase inhibitor in a nonadrenergic medium (that is, enriched with ODQ, suggesting a different mechanism. Neverthe- guanethidine and phentolamine), a postsynaptic less, the decrease in magnitude of the relaxation blockade of a-adrenergic receptor by F3–5 is unlikely induced by low concentrations of F3–5 (1 and to explain its relaxant activity. 3 mgmlÀ1) might be associated to the inhibition of The relaxation evoked by transmural EFS, which basal NO–GC activity by L-NAME and ODQ, which is mainly dependent on neuronal NO release and per se increases RbCC tonus. Alternatively, this may increased intracellular cGMP concentration,26 was reflect a first component of the relaxation that is not modified by F3–5 and this argues against a dependent on NO release, but the concentration– possible PDE5-inhibitory activity of this compound. response curve, EC50 values and maximal response

International Journal of Impotence Research A. ulei on rabbit corpus cavernosum AR Campos et al 260

À1 Figure 6 (a) Concentration–response curves to F3–5 (1–300 mgml ) 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 F3–5-induced relaxation. These findings suggest that 2 þ (if it exists) is of minor contribution to the overall the relaxation effect of F3–5 is unrelated to Ca - relaxant activity. activated, ATP-sensitive and voltage-dependent A direct nitrergic activation, as demonstrated to K þ channels. Tityus serrulatus scorpion venom toxin28 is also It has been shown that in RbCC smooth muscle unlikely since the relaxation was insensitive to TTX. PHE induces contraction not only by increasing 2 þ The potassium channels such as BKCa,Kv and intracellular Ca concentration, but also by in- 2 þ KATP are physiological regulators of the membrane creasing Ca sensitivity of the contractile appara- electric potential and transmembrane calcium flux, tus.32 Evidence also exists for the role of Ca2 þ entry and therefore they may have a key role in the in the tonic contraction of the corpus cavernosum regulation of smooth muscle tone including corpus smooth muscle cells and this tonic contraction can cavernosum.29–31 However, in the present investiga- be abolished by voltage-dependent Ca2 þ channel tion, the blockade of KCa channels (both high Maxi- blockers, such as nifedipine or by removal of 2 þ 33 K and low-conductance KCa), with charybdotoxin extracellular Ca . A possible calcium channel and apamin, respectively, and the KATP channels blockade by F3–5 cannot be ruled out from the pre- þ by glibenclamide did not modify significantly the sent study since it could block the K 60 mM-induced

International Journal of Impotence Research A. ulei on rabbit corpus cavernosum AR Campos et al 261

À1 Figure 7 Representative physiograph recordings (n ¼ 5) of the effect of F3–5 (30, 100 and 300 mgml ) on isolated RbCC on phasic þ contractions elicited by rapid exposition (10 s) to K 60 mM is shown in (a) and (b), the respective graph with data expressed as À1 mean±s.e.m. (c) Representative physiographic recording of effect of a single concentration F3–5 (100 mgml ) on contractions elicited by þ cumulative additions of calcium chloride to the external medium (CaCl2; 1–100 mM) of previously depolarized by K 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 alkaloid extract by rat cortical synaptosomes further calcium influx through voltage-dependent support this notion (unpublished observations). calcium channels.34 This is reinforced by the fact Nevertheless, the antagonism to calcium signaling that this alkaloid fraction strongly impaired con- as a second messenger as well as an interference on tractions of depolarized RbCC, in a zero nominal- the Ca2 þ sensitivity of the contractile machinery of calcium medium, elicited by increasing calcium RbCC smooth muscle were not investigated and may concentrations in the medium. An impaired calcium not be excluded as components of the relaxation influx through the membrane and consequently the induced by F3–5. calcium-induced calcium release from internal We have previously described that A. ulei fraction stores is likely to account for the observed relaxant (F3–5) in vivo induces pro-erectile-like behavioural effect of F3–5 on RbCC smooth muscle. The inhibi- activity in male mice that was effectively blocked by tion of calcium uptake observed in the presence of pretreatment with L-NAME, an inhibitor of NOS,

International Journal of Impotence Research A. ulei on rabbit corpus cavernosum AR Campos et al

262 23 suggesting an NO-mediated mechanism, whereas 8 Harvey AL. Medicines from nature: are natural products still in the current study, the same fraction produced a relevant to drug discovery? Trends Pharmacol Sci 1999; 20: profound relaxant effect on corpus cavernosum 196–198. 9Cıćero AF, Bandieri E, Arletti R. Lepidium meyenii Walp. smooth muscle in vitro, by an NO-independent improves sexual behaviour in male rats independently from mechanism, since the relaxation was unaffected by its action on spontaneous locomotor activity. J Ethnopharma- L-NAME or ODQ, the soluble guanylate cylase col 2001; 75: 225–229. inhibitor. The apparently paradoxical mechanisms 10 Drewes SE, George J, Khan F. Recent findings on natural products with erectile-dysfunction activity. Phytochemistry that we observed in vivo and in vitro are probably 2003; 62: 1019–1025. due to the fact that the fraction is a mixture 11 Zaher TF. Papaverine plus PGE, versus PG E1 alone for containing three major alkaloids. It is conceivable intracorporeal injection therapy. Intl Urol Nephrol 1998; 30: that this fraction may have both central and 193–196. peripheral sites of action, which are possibly related 12 Matsumoto K, Yoshida M, Andersson KE, Hedlund P. Effects in vitro and in vivo by apomorphine in the rat corpus to one or even more number of these alkaloids. cavernosum. Br J Pharmacol 2005; 146: 259–267. In conclusion, our data demonstrate for the first 13 Chiou WF, Chen J, Chen CF. Relaxation of corpus cavernosum time that the fraction F3–5 from A. ulei bark extract and raised intracavernous pressure by berberine in rabbit. Br J exerts a relaxant effect on RbCC smooth muscle Pharmacol 1998; 125: 1677–1684. 14 Dinsmore WW. Available and future treatments for erectile in vitro by a mechanism independent on NO–GC dysfunction. Clin Cornerstone 2005; 7: 37–45. pathway and probably related to a calcium antagon- 15 Deutsch HF, Evenson MA, Drescher P, Sparwasser C, Madsen ism. 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