Journal of Ethnopharmacology 104 (2006) 240–244 Pro-erectile effects of an alkaloidal rich fraction from Aspidosperma ulei root bark in mice Adriana R. Campos a , Roberto C.P. Lima Jr. a , Daniel E.A. Uchoa b , Edilberto R. Silveira b , Flavia A. Santos a , Vietla S.N. Rao a,∗ a Department of Physiology and Pharmacology, Federal University of Ceará, Rua Cel. Nunes de Melo, 1127, C.P. 3157, Porangabussu, 60430-270 Fortaleza, CE, Brazil b Department of Organic and Inorganic Chemistry, Federal University of Ceará, Rua Cel. Nunes de Melo, 1127, C.P. 3157, Porangabussu, 60430-270 Fortaleza, CE, Brazil Received 24 February 2005; received in revised form 9 August 2005; accepted 6 September 2005 Available online 17 October 2005 Abstract In recent years, there has been a renewed interest in the search for novel natural substances active against erectile dysfunction. Plants that belong to the genus Aspidosperma (Apocyanaceae) are known to be very rich in indole alkaloids and have an ethnomedical history of use as traditional remedies for erectile dysfunction. This study examined whether the indole alkaloidal rich fraction (F3–5 ) from Aspidosperma ulei Markgr. root bark could manifest penile erection-related behavioral responses (penile erection, erection-like and genital grooming) in mice. Intraperitoneal injection of F3–5 (25 and 50 mg/kg) elicited all the three different behavioral responses in a manner similar to yohimbine (2 mg/kg, i.p.), a known indole alkaloid. Seventy-five percent of mice treated with yohimbine or F3–5 showed penile erections, which were completely blocked by clonidine, an alpha-2-adrenoceptor agonist and haloperidol, a dopaminergic antagonist and as well as by l-NAME, a nitric oxide synthase inhibitor. These results point out that F3–5 facilitates penile erection in mice possibly through the activation of central dopamine and blockade of presynaptic alpha-2 adrenoceptors with a subsequent enhancement in nitric oxide release from the penile nerves and arteries. This study further supports the traditional use of extracts from Aspidosperma species in erectile dysfunction. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Penile erection; Aspidosperma ulei Markgr.; Root bark; Alkaloidal fraction; Pro-erectile effect; Yohimbine; Male mice 1. Introduction Erectile dysfunction (ED) has been described in medical literature since ancient times (Van Driel et al., 1994). Its prevalence rate may vary from 11 to 33.9% in male population (Shabsigh and Anastasiadis, 2003). Traditional herbs may be a potential source of natural drugs for therapy against ED (Adimoelja, 2000; Drewes et al., 2003; Ghadiri and Gorji, 2004). Pyranoisoflavones from the roots of Eriosema kruassianum; forskolin, a diterpene from Coleus forskohlii; several alkaloids like berberine, which occurs in Berberis plants, papaverine from Papaver somniferum and yohimbine from Corynanthe yohimbe bark are few examples that have enjoyed reputation as traditional remedies for the treatment of ED (Mullhall et al., 1997; Chiou et ∗ Corresponding author. Tel.: +55 85 4009 8341; fax: +55 85 4009 8333. E-mail address: vietrao@ufc.br (V.S.N. Rao). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.09.009 al., 1998; Zaher, 1998). Yohimbine is an indole alkaloid with ␣2 -adrenergic-blocking activity that has been used for over a century in the treatment of erectile dysfunction (Morales, 2000; Guay et al., 2002). Although it improves sexual performance (Ernst and Pittler, 1998), its use has been reported to be associated with undesirable side-effects, such as hypertension, anxiety and manic symptoms (Riley, 1994). The genus Aspidosperma (Apocyanaceae) is known to be very rich in indole alkaloids like aspidospermine and quebrachamine (Deutsch et al., 1994). In some countries, Aspidosperma quebracho blanco extract is in use as a prescription drug to treat ED and the beneficial effect could be largely due to its yohimbine content. Unlike yohimbine, the extract seems to bind non-selectively to penile ␣1 - and ␣2 -adrenoceptors (Sperling et al., 2002). Aspidosperma ulei Markgr. (Apocyanaceae) is a large tree that grows widely in the regions of North and North-Eastern Brazil. Chemical extraction and fractionation study on its root bark revealed the presence of indole alkaloids in fraction F3–5 . A.R. Campos et al. / Journal of Ethnopharmacology 104 (2006) 240–244 In the search for novel indole alkaloids effective against erectile dysfunction and free from yohimbine’s side-effects, the present study assessed the F3–5 fraction from Aspidosperma ulei for penile erection-related behavioral activity in mice. 2. Material and methods 2.1. Plant material, extraction and fractionation The root bark of Aspidosperma ulei Markgr. was collected from Garapa (Acarape Municipality), Ceará State (Brazil), after its identification by Botanist Prof. Edson de Paula Nunes. A voucher specimen (#30823) was deposited in the Herbário Prisco Correia of Federal University of Ceará, Fortaleza. Ground root bark (4.0 kg) of Aspidosperma ulei was macerated with EtOH (10 L, 3×) to yield, after solvent evaporation, a brown viscous extract designated as Aspidosperma ulei crude ethanolic extract (AuCE, 144.6 g). AuCE (51.0 g) was then suspended in H2 O (200.0 mL) and partitioned with EtOAc (200 mL, 4×) to yield AuCE-Aq (40.6 g) from the water soluble and AuCE-EA (8.6 g) from the organic soluble phase, after lyophilization and rotaevaporation, respectively. When AuCE, AuCE-Aq and AuCE-EA extracts were subjected to behavioral tests in mice, only AUCE and AuCE-EA showed the penile erection-related behavioral activity. AuCEEA (1.2 g) was subsequently dissolved in MeOH and submitted to exclusion chromatography over Sephadex LH-20 (45.0 g) using MeOH as eluent. Eight fractions (200.0 mL) were collected to yield three pooled fractions: F1–2 (587.4 mg), F3–5 (162.4 mg) and F6–8 (371.9 mg). In behavioral tests, F3–5 was found to be most active, and therefore, the procedure was repeated several times in order to obtain enough material for in vivo testing. 1 H NMR analysis of this fraction (F3–5 ) revealed the presence of two major indole-type alkaloids (Staerk et al., 2001). 241 2.4. Penile erection-related behaviors The method described by Rampin et al. (2003) was followed. Mice were habituated to the testing room for at least a 2 h period and to the test chamber (a glass aquarium, 25 cm × 20 cm × 20 cm) for 5 min, before the start of experimentation. In the first series of experiments, four groups of mice (n = 8 per group), respectively, received either a single intraperitoneal injection of the plant extract F3–5 (25 and 50 mg/kg), the vehicle (3% DMSO, 10 mL/kg) that was used to suspend the F3–5 or the reference drug yohimbine (2 mg/kg) (Clark et al., 1985). The dose selection of 25 and 50 mg/kg F3–5 was based on our acute toxicity study with mice (24 h intraperitoneal DL50 in mice = 400 ± 40 mg/kg) and at the doses employed in this study, no clinical toxicity was apparent in treated animals. Personnel unaware of the treatments of the mice participated in the behavioral evaluation. Observations were commenced 30 min after the plant extract injection by placing each male in the test chamber. Three behavioral responses were counted over a period of 30 min: erection (when the male stood on its hind limbs, bent its body forward, bent its head down to reach the genital area, licked its penis and displayed hip movements); erection-like response (the male stood on its hind limbs, bent its body forward, bent its head down, licked the abdomen but not the penis and displayed hip movements; the penis was not erect); and genital grooming (the male sat on its hindquarters, bent his head down and performed grooming of the genital area). The latency for the first erection was also measured. In a second series of experiments, the influence of adrenergic, dopaminergic and nitrergic systems on the penile erectionrelated behaviors was analyzed in groups of mice (n = 8 per group) pre-treated with clonidine (100 ␮g/kg, i.p.) or haloperidol (2 mg/kg, i.p.) or l-NAME 10 mg/kg (s.c.), 15 min before F3–5 injection. 2.5. Pentobarbital-induced sleeping time 2.2. Animals Male Swiss mice (25–30 g) obtained from the central animal house of Federal University of Ceará were used. They were housed in polypropylene cages at 23 ± 2 ◦ C before experimentation under standard environmental conditions (12-light/12-h dark cycle; 55–60% relative humidity) and had free access to pellet diet (Purina chow) and tap water. The Institutional Committee on the Care and Use of Animals for Experimentation approved the experimental protocols, which were in accordance with the guidelines of National Institute of Health, Bethseda, USA. 2.3. Drugs and reagents Clonidine (Sigma, USA), haloperidol (Cristália, Brazil), NG -nitro-l-arginine-methyl ester (l-NAME) (Sigma, USA), dimethyl sulfoxide (DMSO) (Vetec, Brazil), Diazepam (DZP) (Sigma Pharma, Brazil) were used. All the reagents used were of analytical grade. For experimentation, the drugs were dissolved in 0.9% saline. Sleeping times induced by pentobarbital (50 mg/kg, i.p.) were established in groups of mice (n = 8), 30 min following the treatments with F3–5 (25 and 50 mg/kg, i.p.), vehicle (DMSO 3%, 10 mL/kg, i.p.) or DZP (1 mg/kg, i.p.). The sleeping times were measured in minutes by observing the loss and the recovery of righting reflex (Carlini et al., 1986). 2.6. Open-field test Mice (n = 8) were observed for locomotion by placing them in the open-field arena and the locomotion frequency (the number of floor units the animal entered with all its limbs) was counted for a period of 4 min, following 30 min after the intraperitoneal administration of F3–5 25 and 50 mg/kg, DMSO 3%, 10 mL/kg or DZP 1 mg/kg (Archer, 1973). 2.7. Rota-rod test The motor coordination and performance of each male mouse was evaluated in a rota-rod apparatus, 30 min after the intraperi- 242 A.R. Campos et al. / Journal of Ethnopharmacology 104 (2006) 240–244 toneal treatment with F3–5 (25–50 mg/kg), vehicle (DMSO 3%, 10 mL/kg) or DZP (1 mg/kg). This apparatus has a 2.5 cm diameter bar, divided into six parts, and it is placed at a height of 25 cm, rotating at 12 rpm. Latency to fall from the rotating bar during a 2 min period was registered (Dunham and Miya, 1957). 2.8. Hole-board test A possible anxiogenic effect was evaluated in a hole-board apparatus (35 cm × 35 cm × 15 cm), 30 min after the intraperitoneal treatment of mice with F3–5 (25 and 50 mg/kg), vehicle (DMSO 3%, 10 mL/kg) or DZP (1 mg/kg). Each animal was placed on the central square of the arena and the number of holes poked (hole-dips) were recorded for 5 min. A decrease of the hole-poking response was considered a positive anxiogeniclike effect (File and Pellow, 1985). 2.9. Forced swimming test This test was performed according to the method described by Porsolt et al. (1977). Mice were individually forced to swim in a transparent glass vessel filled with water at 24–26 ◦ C, 30 min after the intraperitoneal administration of F3–5 (25 and 50 mg/kg), vehicle (DMSO 3%, 10 mL/kg) or clonidine (100 ␮g/kg). The total duration of immobility (s) was measured during a 6 min period. Mice were considered immobile, when they made no further attempts to escape except the movements necessary to keep their heads above the water. Each experimental group consisted of eight animals. 2.10. Statistical analysis Comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by Dunnet test. Values are expressed as mean ± S.E.M. from eight animals. Differences were considered statistically significant, when p < 0.05. 3. Results F3–5 elicited all the three distinct behaviors in Swiss male mice: erection, erection-like responses and genital grooming (Fig. 1). During erection, the erect penis was visible, erection was always accompanied by grooming of the penis and Fig. 1. Histogram displaying the number of erections, erection-like responses and genital groomings during the 30 min observation period in mice that received i.p. injections of vehicle (control), F3–5 (25 and 50 mg/kg) or yohimbine (2 mg/kg). Data are presented as mean ± S.E.M. from eight animals. * p < 0.05, ** p < 0.01 and *** p < 0.001 significantly different from control (ANOVA, Dunnet test). the mouse held the penis with forepaws. During erection-like responses, this penile grooming was not observed. In contrast, the mouse groomed the abdomen. During erection and erection-like responses, the animal bends on the extremity of its hind limbs. Genital grooming was the third behavioral response observed. It could not be confused with the other two behaviors, namely erection and erection-like responses, as it was recorded, when the mouse sat on its hindquarters. One-way ANOVA revealed the statistical significance (p < 0.001) of treatment effect on the number of mice that displayed erections as well as on the mean number of erections. F3–5 (25 and 50 mg/kg) elicited erections in 11 of the 16 mice (68%) during the 30 min period of observation. In contrast, the reference drug yohimbine treated groups showed erections in six of eight animals (75%). There was no statistical significance between these two treatments. The animals of the control group that received the vehicle showed no erection. The mean number of erections in F3–5 (25 and 50 mg/kg) and yohimbine (2 mg/kg) were in the order of 1.25 ± 0.36, 1.00 ± 0.37 and 1.12 ± 0.29, respectively. The latency to first erection with F3–5 (25 and 50 mg/kg) was not significantly different from control group of animals that received yohimbine (556.62 ± 168.11, 596.62 ± 242.42 and 698.62 ± 198.23 s, respectively). Clonidine (100 ␮g/kg), the ␣2 -adrenoceptor agonist, haloperidol (2 mg/kg), the dopamine D2-receptor antagonist or the nitric oxide synthase inhibitor, l-NAME (10 mg/kg) failed to Table 1 Effects of Aspidiosperma ulei F3–5 on pentobarbitone sleeping time and in open-field, rota-rod, hole-board and forced swimming tests in mice Behavioral tests Control Sleeping time Open-field Rota-rod Hole-board Forced swimming 64.14 34.28 120.00 33.14 158.71 F3–5 (25 mg/kg) ± ± ± ± ± 4.24 3.22 0.00 3.15 13.80 42.85 56.57 115.72 25.71 148.28 ± ± ± ± ± 6.42** 4.80** 4.28 3.33 12.57 F3–5 (50 mg/kg) 38.14 56.50 102.50 15.57 84.00 ± ± ± ± ± 2.96** 4.58** 11.45 2.41** 6.25** DZP (1 mg/kg) 79.48 24.30 32.40 46.10 ± ± ± ± – 5.66* 7.65* 7.00** 1.50** Clonidine (100 ␮g/kg) – – – – 23.70 ± 4.30** Data are presented as mean ± S.E.M. from eight animals. Observations were made 30 min following the intraperitoneal injections of vehicle (control), F3–5 , diazepam (DZP) or clonidine. Sleeping time (min); open-field (counts/4 min); rota-rod (permanence in s); hole-board (head-dips/5 min); forced swimming (immobility time in s). * p < 0.05 from control (ANOVA, Dunnet test). ** p < 0.01 from control (ANOVA, Dunnet test). A.R. Campos et al. / Journal of Ethnopharmacology 104 (2006) 240–244 produce any per se pro-erectile effect. However, pre-treatment of mice with these agents abrogated the pro-erectile effects of 25 mg/kg F3–5 (data not shown). In general behavioral tests, the alkaloidal fraction F3–5 , at either dose induced a significant increase in spontaneous locomotor activity in open-field test but caused no motor impairment in rota-rod test (Table 1). In contrast, DZP, the reference compound, significantly depressed the locomotor frequency and rota-rod performance. Mice pre-treated with F3–5 and DZP showed differing effects on pentobarbital-sleeping times. While F3–5 decreased the sleeping time, DZP enhanced it. DZP, as expected, markedly enhanced the number of head-dips in hole-board apparatus, and thus, showed an anxiolytic effect, whereas F3–5 was an anxiogenic effect as evidenced from a decreased number of head dips. In forced swimming test, both F3–5 and clonidine were able to decrease the immobility time. 4. Discussion In this work, we have demonstrated that the fraction F3–5 from Aspidosperma ulei root bark elicits penile erection-related behaviors in male mice. Presence of indole alkaloids closely related to yohimbine in this fraction and previous studies that show the clinical efficacy of an yohimbine rich extract from the bark of Aspidosperma quebracho blanco (Sperling et al., 2002) prompted us to undertake this study. Indole alkaloids can exert potent central and peripheral pharmacological effects by influencing various neurotransmitter systems. The results show that F3–5 facilitates penile erections in mice and at the dose of 25 mg/kg, it displayed a better efficacy as indicated by the number of responders and the number of erections. F3–5 -induced erections in mice were abolished by clonidine, an ␣2 -adrenoceptor agonist, suggesting that it may function as an ␣2 -adrenoceptor blocker. It implies that F3–5 has a yohimbinelike effect. In this context, Clark et al. (1985) provided evidence for the modulation of sexual behavior by ␣2 -adrenoceptors and suggested that agonists like clonidine suppress, whereas antagonists, such as yohimbine, cause sexual arousal. Despite some controversy, controlled clinical studies have shown the efficacy of yohimbine alone or its combination with other agents that increase NO bioavailability, in erectile dysfunction regardless of its etiology (Riley, 1994; Lebret et al., 2002; Meston and Worcel, 2002; Kernohan et al., 2005). To establish the therapeutic effect may require 3–8 weeks of therapy and most of the clinical trials of yohimbine have been criticized for having methodological problems and inconsistent data (Guirguis, 1998). Haloperidol, a non-selective dopaminergic antagonist also abrogated the F3–5 -induced erections. Both animal and human studies demonstrate that male sexual behavior is partly regulated by dopamine mechanism and that penile erection can be induced by apomorphine, a dopamine receptor agonist (Andersson and Wagner, 1995; Paredes and Agmo, 2004). Both dopamine agonists and presynaptic ␣2 -adrenergic blockers seem to increase NO synthase activity and cause enhanced NO release, important in erectile function (Simonsen et al., 1997; Heaton, 2000). In the present work, the nitric oxide synthase inhibitor, l-NAME also 243 blocked the erectile responses evoked by F3–5 . These results point out the involvement of noradrenergic, dopaminergic and nitrergic mechanisms in F3–5 -induced penile erection. Indole alkaloids can exert potent effects on the central nervous system (CNS). Therefore, the present study analyzed the effects of F3–5 on pentobarbital-sleeping time and general behaviors in open-field, rota-rod, hole-board and in forced swimming tests. The results obtained indicate a general stimulant action of F3–5 on CNS. The finding that F3–5 decreases pentobarbital-sleeping time is consistent with the finding of Kushikata et al. (2002) observed with yohmbine, a known indole alkaloid. The fraction F3–5 , at the doses tested (25 and 50 mg/kg), did not evoke any signs of toxicity in treated animals and the intraperitoneal DL50 in mice was 400 mg/kg (unpublished observations). 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