International Journal of Impotence Research (2008) 20, 255–263 & 2008 Nature Publishing Group All rights reserved 0955-9930/08 $30.00 www.nature.com/ijir 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]oxadia- zole[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