Life Sciences 112 (2014) 74–81

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Effects of the essential oil of Croton zehntneri and its major components, anethole and estragole, on the rat corpora cavernosa Pedro Henrique Bezerra Cabral a, Rafael de Morais Campos a, Manassés Claudino Fonteles a, Cláudia Ferreira Santos a, José Henrique Leal Cardoso b, Nilberto Robson Falcão do Nascimento a,⁎ a b

Ceará State University, Superior Institute of Biomedicine, Laboratory of Renal and Cardiovascular Pharmacology, Fortaleza, Ceará, Brazil Superior Institute of Biomedicine and Laboratory of Electrophysiology, Fortaleza, Ceará, Brazil

a r t i c l e

i n f o

Article history: Received 10 February 2014 Accepted 15 July 2014 Available online 30 July 2014 Keywords: Essential oil Anethole Estragole Corpora cavernosa

a b s t r a c t Aims: The effects of the essential oil of Croton zehntneri (EOCz) and its major components anethole, estragole and methyl eugenol were evaluated in phenylephrine precontracted rat corpora cavernosa (RCC). Main methods: RCC strips were mounted in 5 ml organ baths for isometric recordings of tension, precontracted with 10 μM phenylephrine and exposed to test drugs. Key findings: All major compounds relaxed RCC. The order of potency was estragole N anethole N methyl eugenol. The maximal relaxation to EOCz and methyl eugenol was 62.67% (IC50 of 1.67 μM) and 45.8% (IC50 of 1.7 μM), respectively. Estragole relaxed RCC with an IC50 of 0.6 μM (maximal relaxation—76.6%). The maximal relaxation to estragole was significantly reduced by L-NAME (43.46%—IC50 of 1.4 μM), ODQ (53.11%—IC50 of 0.83 μM) and indomethacin (24.41%—IC50 of 1.3 μM). On the other hand, anethole relaxed RCC by 66.73% (IC50 of 0.96 μM) and this relaxation was blunted by indomethacin (35.65%—IC50 of 1.6 μM). Both estragole and anethole increased the relaxation achieved upon electrical stimulation. Both compounds increased the levels of cAMP (estragole by 3fold and anethole by 2-fold when compared to controls). Estragole also increased the levels of cGMP (0.5-fold). Significance: The higher potency of these compounds to relax corpora cavernosa smooth muscle may form the pharmacological basis for the use of such substances as leading compounds in the search of alternative treatments of erectile dysfunction. © 2014 Elsevier Inc. All rights reserved.

Introduction Croton zehntneri Pax. et Hoffm. is an Euphorbiaceae, popularly known as “canela de cunhã``. This aromatic plant is widely distributed in the Northeastern region of Brazil and its leaves have an essential oil, mainly composed of terpenoids (Craveiro et al., 1978), which corresponds to 2–3% of its dry weight. Its leaves are used as flavoring food and extracts of its bark and leaves are employed, as decoctions or infusions, in the folk medicine in Northeast Brazil for the treatment of anxiety, anorexy or for the relief of gastrointestinal disturbances (Leal-Cardoso and Fonteles, 1999; Batatinha et al., 1995). The major terpenoid compounds present in the essential oil of C. zehntneri (EOCz) are estragole and anethole. Anethole (1-methoxy-4-(1-propenyl)benzene) is an incolor liquid with anis-like flavor and more than 10 times sweeter than common sugar. Estragole (1-allyl-4-methoxybenzene) is a phenylpropene and an isomer of anethole that shares its anis-like flavor, but it lacks the sweet taste ⁎ Corresponding author at: Universidade estadual do Ceará, Instituto Superior de Biomedicina, Laboratório de Farmacologia Renal e Cardiovascular,Av. Paranjana, 1700, Itaperi, 60740-903 Fortaleza, CE, Brazil. Tel.: +55 85 3101 9836; fax: +55 85 3101 9810. E-mail address: [email protected] (N.R.F. Nascimento).

http://dx.doi.org/10.1016/j.lfs.2014.07.022 0024-3205/© 2014 Elsevier Inc. All rights reserved.

(Raubenheimer, 1912) and therefore is less used than anethole in the food industry. Previous pharmacological studies have shown that the EOCz and its major components, estragole and anethole, were both relaxant and antispasmodic in intestinal smooth muscle (Coelho-de-Souza et al., 1997, 1998). Anethole and estragole were shown to relax aortic rings in vitro (Soares et al., 2007; de Siqueira et al., 2006a). It has been documented that anethole and estragole have potent anti-inflammatory effect (Ponte et al., 2012) and EOCz and anethole present cicatricial activity (Cavalcanti et al., 2012). Estragole induces, in concentrations reaching the millimolar range, a myorelaxant effect on skeletal muscle (Albuquerque et al., 1995) and blocks the compound action potential of the sciatic nerve (Leal-Cardoso et al., 2004). This compound, at the milimolar concentration, is also able to block the excitability of primary afferent axons of the dorsal root ganglia by a local anesthetic mechanism (Silva-Alves et al., 2013). The relaxation of the corpora cavernosa is a crucial step in the penile erection mechanism (Andersson and Wagner, 1995). Nitric oxide is recognized as the main mediator of corpora cavernosa relaxation, leading to penile erection (Andersson, 2011). Although phosphodiesterase 5 inhibitors are effective pharmacological treatment for most patients with penile erection, there are still some groups

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of subjects that do not benefit from this treatment (Andersson, 2011) and alternative pharmacological treatments are currently under investigation. Since the detumescence of the corpora cavernosa is maintained by both neurogenic, i.e., noradrenergic, and intrinsic myogenic mechanisms and that local anesthetics, by its sodium channel blocking properties, has been used as first-line treatment for premature ejaculation, we hypothesized that this compound, by relaxing the smooth muscle and blocking sensory neurons could be potentially useful for both erectile dysfunction and premature ejaculation. The present work was undertaken to check whether the essential oil of C. zehntneri and its major components, estragole, anethole and methyl eugenol would induce relaxations of the corpora cavernosa. In addition, the involvement of nitric oxide from both endothelium and neurons, the role of potassium channels, cyclic nucleotides, soluble guanylate cyclase, protein kinase C and phospholipase A2 and cyclooxygenases products were evaluated. Material and methods Animals Adult male Wistar rats (250–300 g) obtained from the Central Animal House of the Federal University of Ceará were used. The rats were maintained under the conditions of constant temperature (22 ± 2 °C) and relative humidity (55 ± 10%), in 12 h light/dark cycles. The animals were fed standard animal chow (Purina) with free access to drinking water. All animals were randomized into groups. The study protocols were approved by the Institutional Ethics Committee of the Ceara State University, under the protocol number 09231272-01 and are in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States of America National Institutes of Health. Chemicals and reagents Anethole (1-methoxy-4-(1-propenyl)benzene), estragole (1-allyl4-methoxybenzene), methyl eugenol (4-allyl-1,2-dimethoxybenzene), phenylephrine (PE), guanethidine, scopolamine, Nω -Nitro-L-arginine methyl ester hydrochloride (L-NAME), 1H-1,2,4oxadiazole [4,3-a] quinoxalin-1-one (ODQ), acetylcholine chloride, indomethacin, aristolochic acid, forskolin and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemicals Co. (Saint Louis, MO, USA). Sodium pentobarbitone (Hypnol ®) was purchased from -Cristália (São Paulo, Brazil). All the salts and other reagents were of analytical grade and purchased from Vetec (Rio de Janeiro, Brazil). Drugs and constituents preparations and EOCz extraction EOCz and anethole, estragole or methyl eugenol solutions were prepared daily by adding these substances directly to the vehicle (0.1% Tween 80 in water) and then submitted to a vigorous 3–5-min manual agitation or vortexing. Indomethacin was prepared in saline solution plus 3% NaHCO3. The stock solution for ODQ and forskolin was diluted in ethanol and the final concentration of ethanol in the bath during experiments was 0.01%. Aristolochic acid was dissolved in DMSO. The final concentration of DMSO in the bath during experiments was lower that 0.05%. The essential oil of C. zehntneri (EOCz) was extracted from freshly chopped leaves by steam distillation and analyzed chemically as previously described (Oliveira et al., 2001). Briefly, analytical conditions were used as follows: EOCz analyses were performed on a Hewlett Packard 6971 GC/MS. Column: dimethylpolysiloxane DB-1 fused silica capillary column; carrier gas: helium (1 mL/min); injector temperature: 250 °C; detector temperature: 200 °C; column temperature: 35–180 °C at 4 °C/min and then 180–250 °C at 10 °C/min; mass spectra: electronic

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impact 70 eV. The essential oil of C. zehntneri used in this study was composed, as percentage of the total essential oil weight, by 57% estragole; 28% anethole; 6% methyl eugenol; 2.5% myrcene; 2.5% γ-elemene; 1.5% 1,8-cineole and 0.5% caryophyllene about 2% of the compounds could not be identified. Experimental protocols In vitro measurement of isometric force generation in cavernosal strips Rats (n = 6 per each group except when otherwise stated) were sacrificed by cervical displacement and bleeding under pentobalbital anesthesia. The penis was removed at the level of attachment to the ischium, and immersed in cold Krebs solution (pH 7.4). The composition (mM) of the Krebs solution was: 136.9 mM NaCl; 2.7 mM KCl; 1.8 mM CaCl2; 0.6 mM MgSO4; 11.9 mM NaHCO3; 0.5 mM KH2PO4 and 11.5 mM glucose. The corpora cavernosa tissue was carefully dissected, free from the tunica albuginea, and 10 mm strips were mounted under 0.3 g resting tension in 5 mL organ baths, filled with warmed (37 °C) Krebs solution gassed with a mixture of 95% O2 in 5% CO2. Following an equilibration period of 60 min, tension was induced by the addition of phenylephrine (PE; 10 μM). At the plateau of contraction, relaxation responses to cumulative concentrations of the essential oil of C. zehntneri (1 to 1000 μg/mL) or to its major compounds estragole, anethole or methyl eugenol (10− 9 to 10− 3 M) were recorded by means of a force displacement transducer (FT-60; Narco Bio-System, Houston, TX, USA), coupled to a 4-channel desk model polygraph (DMP-4B, Narco Bio-Systems). The concentration–response curves to anethole and estragole were then obtained, in different set of experiments, in the absence or the presence of L-NAME (500 μM), in order to inhibit both endothelial and neuronal NOS (CelleK et al., 2003); indomethacin (10 μM) to inhibit cyclooxygenases; ODQ (100 μM) to inhibit soluble guanylate cyclase; and aristolochic acid (10 μM) to inhibit phospholipase A2. The concentration–response curves to anethole and estragole were also repeated in tissues precontracted with 60 mM K+ or in tissues precontracted with phenylephrine and incubated with 100 μM barium chloride (BaCl2), a nonspecific potassium channel blocker. In order to evaluate the role of protein kinase C, the relaxant effect of anethole and estragole was probed in a tissue incubated during 30 min with phorbol 12-myristate 13-acetate (PMA;10 μM) and then contracted with phenylephrine. In vitro measurement of relaxation response induced by EFS In another set of experiments, the relaxation induced by transmural electrical field stimulation (EFS) was probed in the absence or presence of 1, 10 or 100 nM of anethole or estragole. The nitrergic relaxation was studied in tissues pretreated with scopolamine (5 μM) and guanethidine (5 μM) for 30 min, and then precontracted with 10 μM phenylephrine as previously described (Cellek and Moncada, 1997; Kasakov et al., 1995). During the plateau phase of the tonic contraction induced by phenylephrine, the tissues were stimulated by EFS (50 V; 0.5 ms; 0.5, 1, 2,4,8,16,32 and 64 Hz). Each train of stimulation lasted 15 s and a 1-minute period was observed between each individual frequency. This EFS-induced relaxation was blocked by TTX, lidocaine or L-NAME (500 μM) and is therefore neurogenic and uses NO as a neurotransmitter. cAMP and cGMP assays In order to measure the intracellular levels of cyclic nucleotides, cavernosal strips (n = 4/each group) were contracted with PE (10 μM) and after the contractile response attained the plateau, the tissues were incubated with either vehicle (DMSO 0.05%), 100 μM anethole, 100 μM estragole, 1 μM forskolin or 1 μM sildenafil. When drugs caused a maximal change in tension, the tissues were immediately frozen in liquid nitrogen. The tissues were homogenized with a buffer enriched with 100 μM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor (IBMX). After centrifugation (3000 ×g, for 15 min, 4 °C), the

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supernatant was extracted with water-saturated diethyl ether, and aliquots of the aqueous phase were dried in a nitrogen atmosphere, and then reconstituted in the buffer of the respective (cAMP or cGMP) kit for analysis. The cyclic nucleotide levels in the solution were measured by the acetylation method with commercially available cAMP and cGMP immunoassay kits (Cayman Chemical Company (Ann Harbor, MI, USA). Protein content was determined using the Bradford method (Bradford, 1976). The cyclic nucleotide levels were expressed in pmol/ mg protein.

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The data were expressed as mean ± standard error of mean (SEM) and compared, when appropriate, by using the unpaired Student's t test or by the one-way ANOVA followed by the Bonferroni test. The concentration sufficient to induce half of maximal relaxation (IC50) was expressed as mean ± 95% confidence interval (CI). The null hypothesis was rejected when the p value was smaller than 0.05.

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Results Effect of EOCz, methyl eugenol, anethole and estragole on the corpora cavernosa strips relaxation in rats In the initial set of experiments, designed to compare both the potency and efficacy of the compounds on the relaxation of phenylephrine pre-contracted corpora cavernosa, we probed the essential oil (1 ng to 1 mg/ml), anethole, estragole and methyl eugenol at concentrations ranging from 10−9 to 10−3 M. The maximal relaxation and potency of EOCz, anethole, estragole and methyl eugenol are shown in Table 1. The averaged concentration–response curves are shown in Fig. 1. The amplitude of the contractions induced by 10 μM phenylephrine was similar to the contractions obtained with 60 mM K+. The average maximal amplitude of phenylephrine induced contractions were 0.38 ± 0.02 g and 0.41 ± 0.02 g for anethole and estragole studies, respectively. Similarly, the maximal amplitude of 60 mM K+-induced contraction was 0.36 ± 0.05 g and 0.46 ± 0.01 g, respectively. On the other hand, the concentration–response curve to estragole was displaced to the left and the maximal relaxation increased in tissues precontracted with 60 mM K+ and compared to the response obtained in phenylephrine precontracted tissues (Table 1 and Fig. 2a). The relaxant response to anethole was slightly increased, but without statistical significance, when tissues were precontracted with K+ 60 mM (Table 1 and Fig. 2b). Thereafter, tissues were pretreated with 100 μM barium chloride for 30 min before performing the concentration response curve to estragole (Table 1 and Fig. 3a). Keeping the same trend, barium chloride did not affect the maximal relaxation induced by anethole (Table 1 and Fig. 3b). The relaxation induced by estragole was attenuated in tissues pre’treated for 30 min with L-NAME (500 μM), a nonspecific nitric oxide synthase inhibitor. The maximal relaxation induced by estragole was blunted by 37.5% (IC50 = 1.47 μM [CI = 0.56 to 3.83 μM]) (p b 0.05, n = 5) (Fig. 4a). On the other hand, the relaxation induced by anethole was insensitive to L-NAME (IC50 = 0.45 μM [CI = 0.087 to

C

D

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Fig. 1. Panel A shows the concentration–response curves to the essential oil of Croton zehntneri (EOCz; 0.001 to 1000 μg/mL) or its major components (estragole, anethole or methyl eugenol) at concentrations ranging from 10−9 M to 10−3 M on corpora cavernosa strips precontracted with 10 μM phenylephrine (PE). The data are expressed as mean ± SEM (n = 7). *p b 0.05, ANOVA followed by Bonferroni vs. methyl eugenol. #p b 0.05, ANOVA followed by Bonferroni vs. EOCz. Panels B, C, D and E are original traces depicting the effect of EOCz, estragole, anethole and methyl eugenol, respectively.

Table 1 Relative potency and efficacy of EOCz and its major components. Drugs

pEC50 [95% CI]—μM

Emax (%)

Estragole (PE) Anethole (PE) EOCz (PE) Methyl eugenol (PE) Estragole (K+) Anethole (K+) Estragole (Ba2+) Anethole (Ba2+)

0.6 [0.62 to 6.69] 0.96 [0.26 to 3.4] – 1.7 [0.5 to 5.7] 0.084 [0.032–22.3] 0.37 [0.06–2.15] 1.14 [0.092–14] 0.78 [0.08–7.8]

76.6 66.7 62.8 45.8 91.9 77.0 63.7 58.4

PE = phenylephrine, K+ = KCl 60 mM, Ba2+ = barium chloride.

± ± ± ± ± ± ± ±

9.0 7.9 6.9 3.8 3.7 9.5 4.7 5.0

2.39 μM]) (Fig. 4b). Similarly, a 30-minute incubation period of tissues with 30 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) decreased the maximal response to estragole by 20.5% (IC50 = 0.84 μM [CI = 0.27 to 2.58 μM]), while the maximal relaxant response to anethole was not affected by such pretreatment (Fig. 5a and b). Remarkably, a 30-min period pretreatment with indomethacin (10 μM) blunted the maximal relaxant response to estragole by 68.2% (IC50 = 1.30 μM [CI = 0.74 to 2.29 μM]) (Fig. 6a) and to anethole by 46.6%.(IC50 = 1.66 μM [CI = 0.23 to 11.5 μM]) (Fig. 6b).

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In order to evaluate whether anethole or estragole (1, 10 or 100 nM) would affect the relaxation induced by nitrergic nerve activation; we studied their effects on a frequency–response relaxation evoked by transmural electrical field stimulation in phenylephryne pre-contracted strips. Anethole increased (about 2-fold) the relaxation induced by EFS at low frequencies (1, 2 and 4 Hz) at both 10 and 100 nM. The relaxations obtained at higher frequencies (16, 32 and 64 Hz) were also increased, usually in the range of 50–70% (p b 0.01)(Fig. 9b). Estragole induced an even higher increased relaxation to EFS at lower frequencies (0.5, 1, 2 and 4 Hz), from 5 to 9 fold increase and 94 to 172% at higher frequencies (16, 32 and 64 Hz) (Fig. 9a). Measure of the levels of cyclic nucleotides The incubation of corpora cavernosa strips, with anethole, did not change the tissue levels of cGMP but estragole did increase cGMP by 78% over the control levels (Fig. 10a). On the other hand, the tissue levels of cAMP were increased 2-fold by anethol and 3-fold by estragole (Fig. 10b). Discussion Concentration–response curves for the essential oil of C. zenthneri (EOCz), estragole, anethole and eugenol showed that anethole and estragole had higher efficacy than the crude essential oil and the structurally related compound, eugenol. The methoxy function at the paraESTRAGOLE(PE) ESTRAGOLE (K+)

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ANETHOLE LOG (M) Fig. 3. Effect of relaxation caused by estragole in tissues pre-incubated for 30 min with barium chloride (BaCl2; 100 μM). Values are expressed as mean ± SEM of the percentage of relaxation of tissues contracted with phenylephrine. No statistical difference was observed (p N 0.05). (ANOVA followed by Bonferroni).

position in anethole and estragole, while at the meta-position in eugenol, is probably determinant for such difference. Both anethole and estragole induced a strong relaxation with a relative high potency, when compared to their effects on other tissues. In a previous study, it was shown that both anethole and estragole are also able to relax rat aortic rings precontracted with phenylephrine (Soares

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The inhibition of PLA2 enzymes with aristolochic acid did not blunt the relaxation induced by anethole (Fig. 7a and b). Similarly, a 30-min period incubation with phorbol myristate (PMA; 10 μM), did not blunt the relaxation curve for anethole or estragole (Fig. 8b).

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ANETHOLE LOG (M) Fig. 2. Effect on relaxation caused by estragole (A) or anethole (B) in corpus cavernosum strips pre-contracted with 60 mM KCl. Values are expressed as mean ± SEM of the percentage of relaxation of tissues contracted with 60 mM KCl or 10 μM phenylephrine. * p b 0.05 ANOVA followed by Bonferroni.

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ANETHOLE LOG (M) Fig. 4. Effect of treatment with N-L-nitro arginine methyl ester (L-NAME) on strips of corpus cavernosum relaxed by estragole (A) or anethole (B). Values are expressed as mean ± SEM of percentage of relaxation of 10 μM phenylephrine precontracted tissue. *p b 0.05, ANOVA followed by Bonferroni.

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et al., 2007). The concentration necessary to relax aortic rings is about 700–900 times higher than to relax the rat corpora cavernosa. Similarly these compounds were shown to relax intestinal smooth muscle and the IC50 for this effect was 7500 times higher than to relax the corpora cavernosa (Coelho-de-Souza et al., 1997).

Fig. 7. Effect on relaxation caused by estragole (A) or anethole (B) in strips of cavernous bodies incubated during 30 min with PLA2 inhibitor, aristolochic acid. Values are expressed as mean ± SEM the percentage of relaxation of tissues contracted with 10 μM phenylephrine. No statistical difference was observed (p N 0.05). (ANOVA followed by Bonferroni).

The same pattern of very low potency was shown for anethole and estragole regarding skeletal muscle and neuronal preparations. For instance, at 3 mM the twitch of the rat phrenic-diafragm was reduced by anethole (but not blocked), while estragole actually enhanced

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Fig. 5. Effect of treatment with ODQ in relaxation evoked by estragole (A) or anethole (B) in the rat corpus cavernosum. Values are expressed as mean ± SEM of the percentage of relaxation of 10 μM phenylephrine precontracted tissue. *p b 0.05 ANOVA followed by Bonferroni.

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ANETHOLE LOG (M) Fig. 6. Effect of treatment with indomethacin on relaxation induced by estragole in rat corpora cavernosa. Values are expressed as mean ± SEM of the percentage of relaxation of relaxation of 10 μM phenylephrine precontracted tissue. *p b 0.05 ANOVA followed by Bonferroni.

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ANETHOLE LOG (M) Fig. 8. Effect on relaxation caused by estragole (A) or anethole (B) in strips of cavernous bodies incubated during 30 min with an activator of protein kinase C, phorbol myristate. Values are expressed as mean ± e.p.m. the percentage of relaxation of tissues contracted with PE 10 μM. *p b 0.05, ANOVA followed by Bonferroni.

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FREQUENCY (Hz) Fig. 9. Effect of estragole (A) or anethole (B) at doses of 1, 10 and 100 nM in the EFS-induced relaxation on corpora cavernosa strips (EFS-50 V; 5 ms, 0.5–64 Hz). *p b 0.05, ANOVA followed by Bonferroni.

twitches following direct electrical stimulation (Albuquerque et al., 1995). These substances were also shown to block the compound action potential of the sciatic nerve or dorsal root ganglia cells studied in vitro, but this only occurs when the drugs are incubated for long periods of time (tens of minutes) and at concentrations ≥ 2 mM (Silva-Alves et al., 2013; Leal-Cardoso et al., 2004). The maximal relaxant response to estragole is greatly blunted in tissues pretreated with L-NAME, a non-selective inhibitor of the synthesis of nitric oxide (Rees et al., 1990). Therefore, it seems that the relaxation induced by estragole has a nitric oxide-dependent component. This conclusion has been reinforced by the fact that ODQ, a highly selective, irreversible, heme-site inhibitor of soluble guanylyl cyclase (Garthwaite et al., 1995) also greatly decreases the concentrationdependent relaxation induced by estragole. Another result of our study that supports this argument is the finding that estragole increases cGMP in strips of corpora cavernosa while anethole does not. In addition, neither L-NAME nor ODQ affected anethole-induced relaxation what excludes a NO-cGMP dependent mechanism for this compound. This is in agreement with previous findings from Soares et al. (2007) which

found that anethole-induced relaxation in rat aortic rings was not sensitive to L-NAME. Remarkably, anethole increased the amplitude of the relaxationinduced by transmural electrical field stimulation (EFS) at all frequencies studied. Estragole also increased EFS-induced relaxations in all frequencies but only in the higher concentration used, i.e., 100 μM. It is worth to mention that 10 μM estragole increased the EFS-induced relaxation at low frequencies. The mechanisms by which anethole would promote increased nitrergic relaxations were not investigated. One possible explanation is that anethole would decrease nitric oxide scavenging by superoxide. For instance, some reports have been published showing antioxidant properties of anethole derivatives (Lado et al., 2004; Tissie et al., 1990; Mansuy et al., 1986). The concentration needed to induce neuronal blockade is very high, i.e., above 2 mM (Silva-Alves et al., 2013; Leal-Cardoso et al., 2004) and those compounds probably do not affect neuronal excitability of autonomic fibers at low concentrations. The relaxation induced by anethole and estragole is probably not greatly dependent on the activation of potassium channels leading to

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A

*#

cGMP (pmol/mg of protein)

1200

1000

800

600

*

400

200

0 DMSO

Anethole

Estragole

Sildenafil

B 3500

*§

cAMP (pmol/mg of protein)

3000 2500 2000 1500

*#

1000

* 500

0 DMSO

Anethole

Estragole

Forskolin

Fig. 10. Effect of anethole (100 μ M) and estragole (100 μ M) in cGMP (A) or cAMP (B) levels in homogenates of corpora cavernosa strips pre-contracted with phenylephrine. Values are expressed as mean ± SEM. The measurements were made in duplicate from four strips per group. *p b .05, unpaired Student's t test, vs. DMSO. # p b 0.05, unpaired Student's t test, versus Anethole. §p b 0.05, “unpaired Student's t test”, versus Estragole.

hyperpolarization. For instance, such relaxations are not blunted in the presence of high potassium (60 mM) and are only marginally affected in the presence of barium chloride, an unspecific potassium channel blocker. Both procedures greatly reduce the relaxation induced by potassium channel openers (Hille, 2001; Knot et al., 1996). Anethole, at high concentrations, was shown to promote contraction of aortic rings by opening of voltage dependent calcium channels-VDCC (Soares et al., 2007) but this effect was not seen in our study. Actually, estragole may decrease calcium entry through VDCC, since the estragole-induced relaxation is higher in tissues precontracted by high potassium; such electromechanical coupling is highly dependent on calcium entry through VDCC. This higher relaxation was not due to a higher tonus achieved with the high potassium solution since the contractions were similar to that achieved with 10 μM phenylephrine. On the other hand, in the rat corpora cavernosa, both the activity and density of membrane PKCα and PKC are increased during sustained phenylephrine contractions (Husain et al., 2004). In addition, the association of phenylephrine with phorbol myristate greatly potentiates the corpus cavernosum contraction. It is reasonable to hypothesize that a compound that inhibits PKC activity would have erectogenic activity, since the basal detumescence is maintained by the sympathetic

activation of the α1-adrenergic receptor. Nevertheless, the relaxant effect of anethole and estragole, was not different in corpora cavernosa pre-incubated with phorbol 12-myristate 13-acetate. This rules out a significative role for PKC inhibition, as a mechanism for anethole and estragole-induced smooth muscle relaxation. However, the relaxant response to both anethole and estragole was greatly blunted by indomethacin, especially regarding estragole. The release of relaxant prostanoids seems to have a predominant role in the relaxation of corpora cavernosa induced by both anethole and estragole. The main source of such compounds is the endothelium of the cavernous sinusoids and the smooth muscle of the corpora cavernosa (Kun et al., 2008; Angulo et al., 2002; Jeremy et al., 1986). A point that should be noted is that both compounds increased cAMP production, which is the main second messenger for relaxant prostanois such as PGE2 acting through EP2 receptors. The major prostanoid synthesized in the corpora cavernosa is PGE2 and the expression of both EP2 and EP4 receptors were demonstrated (Moreland et al., 2001). Furthermore, the inhibition of the prostaglandin G/H synthase by indomethacin is able to decrease basal cAMP levels in the corpora cavernosa by a half, showing that these prostanoids have a role in the regulation of corpora cavernosa smooth muscle tonus. Basal release of relaxant prostanoids has been demonstrated in equine and human penile resistance arteries (Prieto et al., 1998; Angulo et al., 2002). In our study, both anethole and estragole increased cAMP levels, in the corpora cavernosa strips. Anethole increased 2-fold and estragole increased 3-fold, the intracellular levels of cAMP and therefore its possible that these compounds would relax corpora cavernosa smooth muscle, by a mechanism dependent on the synthesis and release of relaxant prostanoids, which would then activate EP2/EP4 receptors leading to increased cAMP formation. The cAMP mediated relaxation primarily involves PKA activation and subsequent decreases in the intracellular calcium concentration and/or the sensitivity of the contractile apparatus to calcium (Murray, 1990). A cross-talk between prostanoids and the NO-cGMP-PKG pathway cannot be excluded. Endogenous prostanoids are able to increase the release of neuronal NO (Ferrer et al., 2004). Furthermore, it was shown that a single intracavernosal injection of PGE1 in Sprague-Dawley rats results in a dose-related release of NO in the corpora cavernosa (Escrig et al., 1999). This is reinforced by the fact that the relaxation induced by either estragole or anethole is blunted to the same extent by the association of indomethacin and L-NAME, when compared to the effect of indomethacin alone. Nevertheless, the reason why estragole does activate this cAMP pathway more effectively than anethole is not clear and was not investigated in the present study. Nevertheless, this relaxant activity is not sensitive to the blockade of phospholipase A2 enzymes by a sufficient concentration of aristolochic acid (Lima et al., 2008) and hence, a nonspecific effect due to direct damage to the corpora cavernosa tissues by these compounds was ruled out. Since the relaxation induced by estragole and anethole was blunted after nitric oxide synthase or prostaglandin G/H synthase it is reasonable to suggest that these compounds might be inducing nonspecific release of autacoids through activation of TRP channels. For instance, estragole and anethole have chemical similarities with capsaicin and the TRPV1 antagonist capsazepine is able to block the hypotension and bradycardia associated with the intravenous injection of anethole or estragole (de Siqueira et al., 2006b). A previous study showed that capsaicin does induce dose-dependent, non-tachyphylactic, NOindependent relaxations of the rabbit isolated corpus cavernosum (Teixeira et al, 1998). Nevertheless, this hypothesis was not tested herein and deserves further investigation. The higher potency of these compounds to relax corpora cavernosa smooth muscle rather than other smooth muscle and its slight local anesthetic activity may form the pharmacological basis for the use of such substances as leading compounds in the search of alternative treatments of erectile dysfunction.

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Conclusion In conclusion, anethole and estragole promote relaxation of the rat corpora cavernosa with a mechanism predominantly dependent of release of autacoids. The main autacoids involved are NO and prostanoids, with consequent increase of intracellular levels of cyclic nucleotides. Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgments This research was funded by CNPq—National Reseach Council, Brazil (481318/2012-7). References Albuquerque AAC, Sorenson AL, Leal-Cardoso JH. Effects of essential oil of Croton zehntneri, and anethole and estragole on skeletal muscles. J Ethnopharmacol 1995; 49:41–9. Andersson KE. Mechanism of erection and basis for pharmacological treatment of erectile dysfunction. Pharmacol Rev 2011;63(4):811–59. Andersson KE, Wagner G. Physiology of penile erection. Physiol Rev 1995;75:191–236. Angulo J, Cuevas P, La Fuente JM, Pomerol JM, Ruiz-Castañé E, Puigvert A, et al. Regulation of human penile smooth muscle tone by prostanoid receptors. Br J Pharmacol 2002; 136(1):23–30. Batatinha MJM, de Souza-Spinosa H, Bernadi MM. Croton zehntneri: possible central nervous system effects of the essential oil in rodentes. J Ethnopharmacol 1995;45:53–7. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248–54. Cavalcanti JM, Leal-Cardoso JH, Diniz IR, Portella VG, Costa CO, Linard CF, et al. The essential oil of Croton zehntneri and trans-anethole improves cutaneous wound healing. J Ethnopharmacol 2012;144:240–7. Cellek S, Moncada S. Nitrergic control of peripheral sympathetic responses in the human corpus cavernosum: a comparison with other species. Proc Natl Acad Sci U S A 1997; 94(15):8226–31. CelleK S, Foxwell NA, Moncada S. Two phases of nitrergic neuropathy in streptozotocininduced diabetic rats. Diabetes 2003;52(9):2353–62. Coelho-de-Souza AN, Barata EL, Magalhães PJC, Lima CC, Leal-Cardoso JH. Effects of the essential oil of Croton zehntneri, and its constituent estragole on intestinal smooth muscle. Phytother Res 1997;11:299–304. Coelho-de-Souza AN, Criddle DN, Leal-Cardoso JH. Selective and modulatory effects of the essential oil of Croton zehntneri on isolated smooth muscle preparations of the guinea pig. Phytother Res 1998;12:189–94. Craveiro AA, Andrade CHS, Matos FJA, Alencar JW. Anise-like flavor of Croton aff zehntneri Pax. Et Hoffm. J Agric Food Chem 1978;26:772–3. de Siqueira RJ, Leal-Cardoso JH, Couture R, Lahlou S. Role of capsaicin-sensitive sensory nerves in mediation of the cardiovascular effects of the essential oil of Croton zehntneri leaves in anaesthetized rats. Clin Exp Pharmacol Physiol 2006a;33(3):238–47. de Siqueira RJ, Magalhães PJC, Leal-Cardoso JH, Duarte GP, Lahlou S. Cardiovascular effects of the essential oil of Croton zehntneri leaves and its main constituents, anethole and estragole, in normotensive conscious rats. Life Sci 2006b;78(20):2365–72. Escrig A, Marin R, Mas M. Repeated PGE1 treatment enhances nitric oxide and erection responses to nerve stimulation in the rat penis by upregulating constitutive NOS isoforms. J Urol 1999;162(6):2205–10. Ferrer M, Salaices M, Balfagón G. Endogenous prostacyclin increases neuronal nitric oxide release in mesenteric artery from spontaneously hypertensive rats. Eur J Pharmacol 2004;506(2):151–6.

81

Garthwaite J, Southam E, Boulton CE, Nielsen EB, Schmidt K, Mayer B. Potent and selective inhibition of nitric oxide- sensitive guanylil cyclase by 1 h-[1,2,4]oxadiolo[4,3-a] quinoxalin-1-one. Mol Pharmacol 1995;48:184–8. Hille B. Ion channels of excitable medicine and biology. 3th ed. Sunderland MA: Sinauer; 2001. Husain S, Young D, Wingard CJ. Role of PKCα and PKCi in phenylephrine-induced contraction of rat corpora cavernosa. Int J Impot Res 2004;16:325–33. Jeremy JY, Morgan RJ, Mikhailidis DP, Dandona P. Prostacyclin synthesis by the corpora cavernosa of the human penis: evidence for muscarinic control and pathological implications. Prostaglandins Leukot Med 1986;23:211–6. Kasakov L, Cellek S, Moncada S. Characterization of nitrergic neurotransmission during short- and long-term electrical stimulation of the rabbit anococcygeus muscle. Br J Pharmacol 1995;115:1149–54. Knot HJ, Brayden JE, Nelson MT. Calcium and potassium channels. In: BÁRÁNY M, editor. Biochemistry of smooth muscle contraction. San Diego: Academic Press; 1996. p. 203–19. Kun A, Kiraly I, Pataricza J, Marton Z, Krassoi I, Varro A, et al. C-type natriuretic peptide hyperpolarizes and relaxes human penile resistance arteries. J Sex Med 2008;5: 1114–25. Lado C, Then M, Varga I, Szoke E, Szentmihályl K. Antioxidant property of volatile oils determined by the ferric reducing ability. Z Naturforsch C 2004;59:354–8. Leal-Cardoso JH, Fonteles MC. Pharmacological effects of essential oils of plants of the Northeast of Brazil. An Acad Bras Cienc 1999;71:207–13. Leal-Cardoso JH, Matos-Brito BG, Gonçalves-Lopes-Jr JE, Viana-Cardoso KV, Sampaio Freitas AB, Brasil RO, et al. Effects of estragole on the compound action potencial of the rat sciatic nerve. Braz J Med Biol Res 2004;37:1193–8. Lima AA, Nascimento NR, Fanq GD, Yotseff P, Toyama MH, Guerrant RL, et al. Role of phospholipase A2 and tyrosine kinase in Clostridium difficile toxin A- induced disruption of epithelial integrity, histologic inflamation damage and intestinal secretion. J Appl Toxicol 2008;28:849–57. Mansuy D, Sassi A, Dansette PM, Plat M. A new potent inhibition of lipid peroxidation in vitro and in vivo, the hepatoprotective drug anisyldithiolthione. Biochem Biophys Res Commun 1986;135(3):1015–21. Moreland RB, Albadawi H, Bratton C, Patton G, Goldstein I, Traish A, et al. O2- dependent prostanoid synthesis activates functional PGE receptors on corpus cavernosum smooth muscle. Am J Physiol Heart Circ Physiol 2001;281(2):552–8. Murray KJ. Cyclic AMP, and mechanisms of vasodilation. Pharmacol Ther 1990;47(3): 329–45. Oliveira AC, Leal-Cardoso JH, Santos CF, Morais SM, Coelho-de-Souza AN. Antinociceptive effects of the essential oil of Croton zehntneri in mice. Braz J Med Biol Res 2001; 34(11):1471–4. Ponte EL, Sousa PL, Rocha MV, Soares PM, Coelho-De-Sousa AN, Leal-Cardoso JH, et al. Comparative study of the anti-edematogenic effects of anethole and estragole. Pharmacol Rep 2012;64(4):984–90. Prieto D, Simonsen U, Hernández M, García-Sacristán A. Contribution of K+ channels and ouabain-sensitive mechanisms to the endothelium-dependent relaxations of horse penile small arteries. Br J Pharmacol 1998;123(8):1609–20. Raubenheimer O. Anethol vs oil of anise. J Am Pharm Assoc 1912;1:337–42. Rees DD, Palmer RM, Schulz R, Hodson HF, Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 1990;101(3): 746–52. Silva-Alves KS, Ferreira-da-Silva FW, Peixoto-Neves D, Viana-Cardoso KV, Moreira-Júnior L, Oquendo MB, et al. Estragole blocks neuronal excitability by direct inhibition of Na+ channels. Braz J Med Biol Res 2013;46(12):1056–63. Soares POG, Lima RF, Pires AF, Souza EP, Assreuy AMS, Criddle DN. Effects of anethole and structural analogues on the contractility of rat isolated aorta: involvement of voltagedependent Ca 2+—channels. Life Sci 2007;81(13):1085–93. Teixeira CE, Bento AC, Lopes-Martins RA, Teixeira SA, von Eickestedt V, Muscará MN, et al. Effect of Tityus serrulatus scorpion venom on the rabbit isolated corpus cavernosum and the involvement of NANC nitrergic nerve fibres. Br J Pharmacol 1998;123(3): 435–42. Tissie G, Latour E, Coquelet C, Bonne C. Singlet oxygen-induced damage to rat lenses in vitro: protection by anisydithione. Adv Exp Med Biol 1990;264:529–32.