Original Papers
Essential Oil of Croton zehntneri and Its Main Constituent Anethole Block Excitability of Rat Peripheral Nerve
Authors
Kerly Shamyra da Silva-Alves 1, Francisco Walber Ferreira-da-Silva 1, Andrelina Noronha Coelho-de-Souza 1, Aline Alice Cavalcante Albuquerque 1, Otoni Cardoso do Vale 2, José Henrique Leal-Cardoso 1
Affiliations
1 2
Key words " Croton zehntneri l " Euphorbiaceae l " essential oil l " anethole l " extracellular recording l " sciatic nerve l " neuronal excitability l
received revised accepted
June 20, 2014 January 9, 2015 January 13, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1396309 Published online February 25, 2015 Planta Med 2015; 81: 292–297 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence José Henrique Leal-Cardoso, PhD Laboratory of Electrophysiology Superior Institute of Biomedical Sciences State University of Ceará Av. Silas Munguba, 1700 – Campus of Itaperi 60740–903, Fortaleza, CE Brazil Phone: + 55 85 31 01 98 14 Fax: + 55 85 31 01 98 10 [email protected]
Silva-Alves KS et al. Essential Oil of …
Laboratory of Electrophysiology, Superior Institute of Biomedical Sciences, State University of Ceará, Fortaleza, Ceará, Brazil Department of Clinical Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil
Abstract !
Croton zehntneri is an aromatic plant native to Northeast Brazil and employed by local people to treat various diseases. The leaves of this plant have a rich content of essential oil. The essential oil of C. zehntneri samples, with anethole as the major constituent and anethole itself, have been reported to have several pharmacological activities such as antispasmodic, cardiovascular, and gastroprotective effects and inducing the blockade of neuromuscular transmission and antinociception. Since several works have demonstrated that essential oils and their constituents block cell excitability and in view of the multiple effects of C. zehntneri essential oil and anethole on biological tissues, we undertook this investigation aiming to characterize and compare the effects of this essential oil and its major constituent on nerve excitability. Sciatic nerves of Wistar rats were used. They were mounted in a moist chamber, and evoked compound action potentials were recorded. Nerves were exposed in vitro to the essential oil of C. zehntneri and anethole (0.1–1 mg/ mL) up to 180 min, and alterations in excitability (rheobase and chronaxie) and conductibility (peak-to-peak amplitude and conduction velocity) parameters of the compound action potentials were evaluated. The essential oil of C. zehntneri and anethole blocked, in a concentration-dependent manner with similar pharmacological potencies (IC50: 0.32 ± 0.07 and 0.22 ± 0.11 mg/mL,
Introduction !
Croton zehntneri Pax et Hoffm. (Euphorbiaceae) is an aromatic plant native to Northeast Brazil where it is popularly called “canela de cunhã”. The leaves of C. zehntneri are used as a flavoring agent in the preparation of beverages and foods. Teas prepared with the bark and leaves of C.
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respectively), rat sciatic nerve compound action potentials. Strength-duration curves for both agents were shifted upward and to the right compared to the control curve, and the rheobase and chronaxie were increased following essential oil and anethole exposure. The time courses of the essential oil of C. zehntneri and anethole effects on peak-to-peak amplitude of compound action potentials followed an exponential decay and reached a steady state. The essential oil of C. zehntneri and anethole caused a similar reduction in conduction velocities of the compound action potential waves investigated. In conclusion, we demonstrated here that the essential oil of C. zehntneri blocks neuronal excitability and that this effect, which can be predominantly attributable to its major constituent, anethole, is important since these agents have several pharmacological effects likely related to the alteration of excitability. This finding is relevant due to the use of essential oils in aromatherapy and the low acute toxicity of this agent, which exhibits other effects of potential therapeutic usefulness.
Abbreviations !
CAP: EOCz: PPA: TNF-α:
compound action potential essential oil of Croton zehntneri peak-to-peak amplitude tumor necrosis factor α
zehntneri are employed by local people to treat nervous disorders, anxiety, seizures, insomnia, and irritability, to relieve gastrointestinal disturbances [1–3], and as an appetite-stimulating agent [4]. The leaves of this plant have a rich content of essential oil (~ 3 % of leaf dry weight), which has a pleasant odor and comprises several terpenes
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and terpenoids [2]. The EOCz of different regions of Northeast Brazil has shown different chemical compositions [5–7], and it has been reported to exert biological and pharmacological activities such as anthelmintic [8], bactericidal [6], antifungal [9], and larvicidal [10] effects. Furthermore, EOCz samples containing anethole as the major constituent and anethole itself have also been reported to have several pharmacological activities such as antispasmodic [11, 12], cardiovascular [13], and gastroprotective [14] effects. Reported EOCz and anethole effects include the blockade of neuromuscular transmission [15], depressant action on the central nervous system [1, 16], antinociceptive activity [4], and improvement in wound healing [5]. These pharmacological activities of EOCz have been attributed to anethole. Besides these activities, various studies have demonstrated that anethole has potent anti-inflammatory activity, since it was shown to inhibit the cellular response induced by the proinflammatory cytokine TNF-α and to reduce paw edema elicited by substance P, bradykinin, histamine, and TNF-α [17, 18]. Several EOCz and anethole activities are theoretically related to the alteration of cell excitability, for example, antispasmodic, anti-inflammatory, and anti-hypernociceptive activity [11, 17– 19]. Anethole has anti-inflammatory and antinociceptive activities that are highly potent and efficacious (a range of in vivo doses of 3–30 mg/kg for the anti-edematogenic effect and of 30– 100 mg/kg for the antinociceptive effect), making it a candidate therapeutic agent that deserves further study of its effects and mechanisms [18]. Anti-inflammatory and antinociceptive activities may also be related to the alteration of cell excitability, since previous studies have demonstrated that proinflammatory cytokines may specifically interact with receptors and ion channels regulating neuronal excitability [20]. Thus, the alteration of cell excitability appears to be a common mechanism partially or totally underlying the pharmacological effects of the EOCz and anethole. Our research group has demonstrated that various essential oils and their constituents block cell excitability [21–25]. In some cases, this blockade by the constituents, even when being similar to that of the essential oil, could not explain the inhibitory activity of the oil [22]. For all these reasons, we undertook this investigation aiming to characterize and compare the effects of the EOCz and anethole on excitability. Excitability was assessed by extracellular recording of the rat sciatic nerve CAP, and the EOCz and anethole were found to block nerve excitability with similar pharmacological characteristics.
Results !
CAP is an electrical signal that results from the summation of axonal action potentials in a nerve bundle, and illustrative traces of rat sciatic nerve CAP under control conditions are shown in " Fig. 1 A to C (left traces). As seen in l " Fig. 1, the CAP showed l two distinct waves, designated here as 1st and 2nd waves (white arrows in figure), as described by Leal-Cardoso et al. The mean values of the control PPA of the CAP was 7.11 ± 0.40 mV (n = 57), and the conduction velocity of its 1st and 2nd waves was 97.05 ± 3.57 and 31.96 ± 1.39 m/s (n = 54), respectively. To ensure that DMSO 0.2 % v/v did not interfere with the CAP parameters, we performed an external control for the activity of the vehicle, DMSO. The sciatic nerve was exposed to DMSO 0.2 % v/v for up to 180 min (the same period of time EOCz and anethole exposure). During this exposure to the vehicle, the PPA and conduction velocity of the 1st and 2nd waves showed a minor monoton-
ic increase and, at the end of this period, these parameters measured 107.90 ± 2.89, 102.98 ± 1.34, and 102.50 ± 1.97 % (n = 6) of the control, respectively, and were not statistically different from the initial control values (statistical comparison done using absolute control values). Concerning EOCz and anethole effects, recordings of the CAP in the control during exposure of the nerve to the EOCz, anethole (0.30 mg/mL), and washout are shown in " Fig. 1 A, B. Both agents blocked PPA and CAP conduction in the l sciatic nerve, and the effects were reversible after the washout. The effects the EOCz and anethole displayed concentration de" Fig. 1 D). Calcupendence in the range of 0.01 to 1.0 mg/mL (l lated IC50 values for the EOCz and anethole on the PPA were 0.32 ± 0.07 and 0.22 ± 0.11 mg/mL (1.48 ± 0.74 mM; R > 0.97 for both agents, n = 4–8), respectively, and there was no statistical difference between the EOCz and anethole (p > 0.05, ANOVA). The blockade induced by lidocaine, a well-known local anes" Fig. 1 C. For comparison, the concentrathetic, is illustrated in l " Fig. 1 D) was 0.02– tion-dependence range for lidocaine (l 0.7 mg/mL (0.1–3 mM) with an IC50 of 0.30 ± 0.16 mg/mL (1.28 mM, n = 4). Concerning the rheobase and chronaxie, two parameters related to nerve excitability [26], the control values were 3.22 ± 0.05 V and 59.75 ± 2.41 µs (n = 12), respectively. Since the IC50 values of the EOCz and anethole were close (0.32 ± 0.07 and 0.22 ± 0.11 mg/mL, respectively), a concentration of 0.30 mg/mL was chosen for both the EOCz and anethole (~ 2 mM) to assess the parameters related to excitability. Sciatic nerve exposure to the EOCz and anethole reduced excitability, and as shown in " Fig. 2 A, the strength-duration curve for both agents was l shifted upward and to the right compared to the control curve. " Fig. 2 B, C). Besides, the rheobase and chronaxie were altered (l The EOCz increased the rheobase to 4.23 ± 0.29 V (n = 6) and chronaxie to 75.17 ± 5.73 µs (n = 6). With anethole, the rheobase and chronaxie increased, respectively, to 4.02 ± 0.15 V (n = 6) and 68.67 ± 4.72 µs (n = 6). These changes were statistically significant for the chronaxie in the presence of the EOCz and for the rheobase in the presence of both agents compared to the control (p < 0.05, ANOVA followed by Dunnettʼs multiple comparison tests). The time course of the EOCz and anethole effects on the PPA of " Fig. 3 A, B. Both agents reduced the PPA in CAP is illustrated in l a concentration-dependent manner, and the reduction in amplitude for higher doses followed an approximately exponential decay, reaching a steady-state level during EOCz and anethole exposure. The time constants for 1.00 mg/mL EOCz and 0.6 mg/ml (~ 4 mM) anethole were 32.15 and 48.78 min (R > 0.97), respectively. The EOCz and anethole (180 min exposure) evoked a decrease in the conduction velocity of the 1st and 2nd waves that was similar for both waves. The EOCz at 0.60 and 1.00 mg/mL and anethole at 0.60 mg/mL blocked all CAP waves, and the conduction velocity could not be determined and was assumed to be zero. Statistical differences were found in CAP velocity with exposure to the EOCz (0.1, 0.3, and 0.45 mg/mL) or anethole (0.30 and 0.45 mg/mL; 2 and 3 mM) compared to the control group (p < 0.05, ANOVA on ranks followed by Dunnʼs comparison test). After 180 min of washout, the PPA of the preparation that was exposed to the EOCz and anethole (0.3 mg/mL for both agents) recovered to 80.34 ± 10.15 (n = 4) and 77.33 ± 14.65 % (n = 5), respectively, of the control.
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Original Papers
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Fig. 1 Illustrative traces of the CAP and concentration-response curve for the EOCz, anethole, and lidocaine. A, B, and C show the CAP for the control (left) and exposure to the EOCz, anethole, and lidocaine [center, 0.3 mg/mL for the EOCz and anethole (2 mM) and ~ 0.25 mg/mL (1 mM) for lidocaine]
and the washout conditions (right). White arrows indicate the 1st and 2nd waves of the CAP and black triangles indicate the stimulus artifact. D shows the EOCz, anethole, and lidocaine concentration-response curves for the PPA of the CAP.
Discussion !
Fig. 2 Effects of the EOCz and anethole on excitability of the sciatic nerve. Strength-duration curves (A) were obtained in the presence of 0.3 mg/mL EOCz (white filled circles) and anethole (black triangles). Rheobase and chronaxie are shown in B and C, respectively. * Indicates a statistically significant difference compared to the control conditions (p < 0.05, ANOVA on ranks followed by Dunnʼs comparison test). # Indicates a statistically significant difference compared to the control curve (p < 0.05, two-way ANOVA followed by the Holm-Sidak test).
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This study demonstrated for the first time that the EOCz and anethole block sciatic nerve conductibility and excitability in a concentration-dependent and reversible manner. Both agents inhibited nerve CAP and increased rheobase and chronaxie, two parameters directly related to excitability. The present investigation adds important information about essential oil and its main constituent on peripheral nerves. The EOCz and anethole effects on excitability have been hypothesized or demonstrated with indirect methods for other tissues including cardiovascular, skeletal, and smooth muscle [11, 13, 15]. Furthermore, the EOCz also has an antinociceptive effect [4] and a depressant effect on the central nervous system [16], which are greatly related to the blockade of excitability. It has been reported that essential oils from Croton nepetaefolius [22], Alpinia zerumbet [24], and rosewood [27] block the CAP of the sciatic nerve in a concentration range similar to that of the EOCz found by us. Usually, the blockade of neuronal activity by essential oils can be attributed to the action of its main constituent, as shown by linalool-rich rosewood oil [27]. However, that is not always the case. The effects of C. nepetaeolius essential oil could not be predominantly explained by the action of its main constituent, 1,8-cineole [22]. In the present study, anethole blocked the CAP with a similar pharmacological potency as the EOCz, and the changes in CAP parameters elicited by a given concentration of anethole and the EOCz were very similar. Together, these findings indicate that the EOCz effects can be attributed to the action of its main constituent, anethole. However, the possible involvement of other minor constituents such as estragole (4.80 %) and 1,8-cineole (2.95 %) cannot be excluded, since these constituents also block the action potential in dorsal root and superior cervical ganglion neurons, respectively [22, 24, 28, 29]. A
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Fig. 3 Time course of the PPA and conduction velocity of the CAP waves after exposure to the EOCz and anethole. Time courses for the EOCz and anethole are shown in A and B, and data were fitted with a single exponential function (solid lines). The conduction velocity of the 1st and 2nd CAP waves
partial contribution of these constituents has been previously reported for the EOCz in the mediation of antispasmodic [11], antinociceptive [4], cardiovascular [13], and skeletal muscle [15] effects. To compare the pharmacological potency of both agents, we performed experiments with lidocaine, a well-known local anesthetic. We found that the EOCz and anethole showed a similar pharmacological potency in reducing the PPA of the CAP compared to lidocaine. Thus, it seems that the EOCz and anethole could act like a local anesthetic in reducing sciatic nerve excitability. We also observed that CAP inhibition by the EOCz and anethole was established in 180 min. This apparently long time course is not exclusive for these agents. Many essential oils, terpenes, and terpenoids show a slow CAP blockade [21–25], and this characteristic can be attributed to the diffusion barrier provided by the various sheaths (peri-, meso-, and endoneurium) from the nerve surface to axon fibers [30]. We investigated the effects of the EOCz and anethole on the rheobase and chronaxie, which are parameters directly related to the measurement of nerve excitability. As demonstrated, both the EOCz and anethole increased the rheobase, meaning that they increased the minimum stimulus level required to generate action potentials [26], reducing the excitability of the sciatic nerve. The mechanism of this decreased excitability, which would lead to the inhibitory action on CAP generation, was not elucidated. However, several terpenes and terpenoids of small molecular weight, such as thymol, eugenol, linalool, carvacrol, and estragole, act on Na+ channels [25, 28, 31–33]. This makes the blockade
is shown in D and C for the EOCz and anethole, respectively. * Indicates a statistically significant difference compared to the control conditions (p < 0.05, ANOVA on ranks followed by Dunnʼs comparison test).
of this ion channel an attractive hypothesis to explain the effect of the EOCz and anethole on nerve excitability. In conclusion, we demonstrated here that the EOCz blocks neuronal excitability and that this effect can be predominantly attributable to its major constituent, anethole. These findings are likely to be relevant to the mechanism of action of various activities of potential therapeutic usefulness of these agents, which deserve further investigation.
Material and Methods !
Plant material, extraction, and chemical analysis Aerial parts of C. zehntneri were collected in September 1998, near the city of Viçosa do Ceará (lat. 3°33′ 48″ S.; long. 41°5′ 41″ W., Ceará, Brazil). C. zenhtneri is a deciduous plant that acquires leaves during the raining season (February to May) and with the small amount of rain that drops during the months of August and September (rain of “caju”). Plant identification was confirmed by Dr. FJ Abreu Matos (Laboratory of Natural Products, Federal University of Ceará, Fortaleza, CE, Brazil). A voucher specimen (No. 277 477) has been deposited in the herbarium Prisco Viana at the Federal University of Ceará. The EOCz was isolated from freshly chopped leaves by steam distillation and analyzed chemically as previously described [4, 5]. Briefly, chromatographic analysis was carried out on a HewlettPackard 6971 system using the following analytical conditions: a dimethylpolysiloxane DB-1 fused silica capillary column (30 m × 0.25 mm; 0.1 µm); helium (1 mL/min) as the carrier gas; 250 °C
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injector temperature; 200 °C detector temperature; a column temperature of 35–180 °C at 4 °C/min and then 180–250 °C at 10 °C/min; and 70 eV electron impact mass spectrometry. The compounds were identified using a mass spectral library search and 13C‑NMR spectroscopy. The chemical composition of the EOCz performed by GC/MS was 85.7 % anethole, 4.8 % estragole, 2.95 % 1,8-cineole, 2.2% β-myrcene, 1.22 % anisaldehyde, 0.9 % trans-caryophyllene, and 2.23 % unidentified.
Solutions and substances Electrophysiology recordings were performed using modified Lockeʼs solution with the following composition (in mM): NaCl 140, KCl 5.6, MgCl2 1.2, CaCl2 2.2, Tris(hydroxymethyl-aminomethane) 10, and glucose 10. The EOCz and anethole were dissolved in DMSO at a final concentration equal to or less than 0.2 % v/v, and stock solutions were prepared daily. Lidocaine (powder) was directly dissolved in Lockeʼs solution to the desired concentrations. The EOCz concentrations used were 0.01, 0.10, 0.30, 0.45, 0.60, and 1.00 mg/mL. For anethole, the concentration range was the same as for the EOCz, excluding 1.00 mg/mL (0.07– 4.0 mM). For lidocaine, the concentration range was 0.02– 0.7 mg/mL (0.1–3.0 mM). Experiments were carried out at room temperature (22 to 26 °C), and all salts, anethole (99 %), and lidocaine (99%) were of analytical grade and purchased from Sigma Chemical or Reagen.
voltage for an active response with a long duration pulse (1000 µs), and chronaxie is the threshold duration for an active response with a stimulus twice the rheobase [26].
Statistical analysis All results are expressed as the mean ± SEM. Statistical significance (p < 0.05) was assessed using one-way analysis of variance (ANOVA) followed by a post hoc comparison test when appropriate. The IC50 of the agents was calculated from the concentrationresponse curves where the experimental points were fitted by a Hill equation.
Acknowledgements !
The authors acknowledge the financial support from Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Universidade Estadual do Ceará (UECE). Also, the authors would like to thank Mr. Pedro Militão de Albuquerque Neto for technical support and for improving the manuscript. Dr. A. Leyva helped with the English editing of the manuscript.
Animals and tissue preparation
Conflict of Interest
Wistar rats (200–300 g) of both sexes were maintained under conditions of constant temperature (22 ± 2 °C), with a 12-h light/ 12-h dark cycle and free access to food and water. All animals were handled in compliance with the Guide for the Care and Use of Laboratory Animals, published by the U. S. National Institutes of Health (NIH Publication 85–23, revised 1996; http://www. nap.edu/readingroom/books/labrats/index.html), and all efforts were made to minimize animal suffering. All procedures described herein were first reviewed and approved by the local animal ethics committee (CEUA/UECE protocol No.: 06 379 067–0, approval date 08/15/2011). The sciatic nerve was dissected from rats sacrificed by asphyxia in a CO2 chamber and was immediately placed in a vessel containing modified Lockeʼs solution. The tissue was used on the same day of dissection as well as the electrophysiological recording.
!
Electrophysiology The sciatic nerve was mounted in a moist chamber and the evoked CAP was recorded as described elsewhere [22, 25]. Sciatic nerve exposure to the EOCz, anethole, and lidocaine was performed only when a stable PPA of the CAP was achieved for at least 30 min, and the agent exposure time was set to 180 min, except for lidocaine in which case drug exposure was set to 30 min. This interval was usually sufficient to allow for the steady-state CAP amplitude to be reached during drug exposure, and this period was followed by a 180-min washout/recovery period. The parameters measured in the evoked CAP were PPA and conduction velocity of the CAP waves. The PPA is the absolute sum of maximum and minimum amplitudes of the CAP, and conduction velocity was determined by the ratio of sciatic nerve length and the time interval between stimulus artifact and the peak of the " Fig. 1 A, left). Besides, strength-dura1st and 2nd CAP waves (l tion curves with voltage square wave stimuli were used to determine the rheobase and chronaxie, which are parameters related to excitability. Rheobase is defined as the threshold stimulus
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There is no conflict of interest.
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Effects of estragole on the compound action potential of the rat sciatic nerve. Braz J Med Biol Res 2004; 37: 1193–1198 Leal-Cardoso JH, Moreira MH, Pinto-da-Cruz GM, Morais SM, Lahlou S, Coelho-de-Souza AN. Effects of essential oil of Alpinia zerumbet on the compound action potential of the rat sciatic nerve. Phytomedicine 2004; 11: 549–553 Leal-Cardoso JH, Silva-Alves KS, Ferreira-da-Silva FW, Santos-Nascimento T, Joca HC, Macedo FHP, Albuquerque-Neto PM, Magalhães PJC, Lahlou S, Cruz JS, Barbosa R. Linalool blocks excitability in peripheral nerves and voltage-dependent Na+ current in dissociated dorsal root ganglia neurons. Eur J Pharmacol 2010; 645: 86–93 Holsheimer J, Dijkstra EA, Demeulemeester H, Nuttin B. Chronaxie calculated from current-duration and voltage-duration data. J Neurosci Methods 2000; 97: 45–50 Almeida RN, Araújo DAM, Gonçalves JCR, Montenegro FC, Sousa DP, Leite JR, Mattei R, Benedito MAC, Carvalho JGB, Cruz JS, Maia JGS. Rosewood oil induces sedation and inhibits compound action potential in rodents. J Ethnopharmacol 2009; 124: 440–443 Silva-Alves KS, Ferreira-da-Silva FW, Peixoto-Neves D, Viana-Cardoso KV, Moreira-Júnior L, Oquendo MB, Oliveira-Abreu K, Albuquerque AAC, Coelho-de-Souza NA, Leal-Cardoso JH. Estragole blocks neuronal excitability by direct inhibition of Na+ channels. Braz J Med Biol Res 2013; 46: 1056–1063 Ferreira-da-Silva FW, Barbosa R, Moreira-Junior L, dos Santos-Nascimento T, de Oliveira-Martins MD, Coelho-de-Souza AN, Cavalcante FSA, Ceccatto VM, Lemos TLG, Magalhães PJC, Lahlou S, Leal-Cardoso JH. Effects of 1, 8-cineole on electrophysiological parameters of neurons of the rat superior cervical ganglion. Clin Exp Pharmacol Physiol 2009; 36: 1068–1073 Afifi AK, Bergman R. Functional neuroanatomy, 2nd edition. New York: The Mcgraw Hill Companies; 2005 Cho JS, Kim TH, Lim JM, Song JH. Effects of eugenol on Na+ currents in rat dorsal root ganglion neurons. Brain Res 2008; 1243: 53–62 Haeseler G, Maue D, Grosskreutz J, Bufler J, Nentwig B, Piepenbrock S. Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol. Eur J Anaesthesiol 2002; 19: 571– 579 Joca HC, Cruz-Mendes Y, Oliveira-Abreu K, Maia-Joca RP, Barbosa R, Lemos TL, Lacerda Beirão PS, Leal-Cardoso JH. Carvacrol decreases neuronal excitability by inhibition of voltage-gated sodium channels. J Nat Prod 2012; 75: 1511–1517
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Original Papers
Essential Oil of Croton zehntneri and Its Main Constituent Anethole Block Excitability of Rat Peripheral Nerve
Authors
Kerly Shamyra da Silva-Alves 1, Francisco Walber Ferreira-da-Silva 1, Andrelina Noronha Coelho-de-Souza 1, Aline Alice Cavalcante Albuquerque 1, Otoni Cardoso do Vale 2, José Henrique Leal-Cardoso 1
Affiliations
1 2
Key words " Croton zehntneri l " Euphorbiaceae l " essential oil l " anethole l " extracellular recording l " sciatic nerve l " neuronal excitability l
received revised accepted
June 20, 2014 January 9, 2015 January 13, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1396309 Published online February 25, 2015 Planta Med 2015; 81: 292–297 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence José Henrique Leal-Cardoso, PhD Laboratory of Electrophysiology Superior Institute of Biomedical Sciences State University of Ceará Av. Silas Munguba, 1700 – Campus of Itaperi 60740–903, Fortaleza, CE Brazil Phone: + 55 85 31 01 98 14 Fax: + 55 85 31 01 98 10 [email protected]
Silva-Alves KS et al. Essential Oil of …
Laboratory of Electrophysiology, Superior Institute of Biomedical Sciences, State University of Ceará, Fortaleza, Ceará, Brazil Department of Clinical Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil
Abstract !
Croton zehntneri is an aromatic plant native to Northeast Brazil and employed by local people to treat various diseases. The leaves of this plant have a rich content of essential oil. The essential oil of C. zehntneri samples, with anethole as the major constituent and anethole itself, have been reported to have several pharmacological activities such as antispasmodic, cardiovascular, and gastroprotective effects and inducing the blockade of neuromuscular transmission and antinociception. Since several works have demonstrated that essential oils and their constituents block cell excitability and in view of the multiple effects of C. zehntneri essential oil and anethole on biological tissues, we undertook this investigation aiming to characterize and compare the effects of this essential oil and its major constituent on nerve excitability. Sciatic nerves of Wistar rats were used. They were mounted in a moist chamber, and evoked compound action potentials were recorded. Nerves were exposed in vitro to the essential oil of C. zehntneri and anethole (0.1–1 mg/ mL) up to 180 min, and alterations in excitability (rheobase and chronaxie) and conductibility (peak-to-peak amplitude and conduction velocity) parameters of the compound action potentials were evaluated. The essential oil of C. zehntneri and anethole blocked, in a concentration-dependent manner with similar pharmacological potencies (IC50: 0.32 ± 0.07 and 0.22 ± 0.11 mg/mL,
Introduction !
Croton zehntneri Pax et Hoffm. (Euphorbiaceae) is an aromatic plant native to Northeast Brazil where it is popularly called “canela de cunhã”. The leaves of C. zehntneri are used as a flavoring agent in the preparation of beverages and foods. Teas prepared with the bark and leaves of C.
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respectively), rat sciatic nerve compound action potentials. Strength-duration curves for both agents were shifted upward and to the right compared to the control curve, and the rheobase and chronaxie were increased following essential oil and anethole exposure. The time courses of the essential oil of C. zehntneri and anethole effects on peak-to-peak amplitude of compound action potentials followed an exponential decay and reached a steady state. The essential oil of C. zehntneri and anethole caused a similar reduction in conduction velocities of the compound action potential waves investigated. In conclusion, we demonstrated here that the essential oil of C. zehntneri blocks neuronal excitability and that this effect, which can be predominantly attributable to its major constituent, anethole, is important since these agents have several pharmacological effects likely related to the alteration of excitability. This finding is relevant due to the use of essential oils in aromatherapy and the low acute toxicity of this agent, which exhibits other effects of potential therapeutic usefulness.
Abbreviations !
CAP: EOCz: PPA: TNF-α:
compound action potential essential oil of Croton zehntneri peak-to-peak amplitude tumor necrosis factor α
zehntneri are employed by local people to treat nervous disorders, anxiety, seizures, insomnia, and irritability, to relieve gastrointestinal disturbances [1–3], and as an appetite-stimulating agent [4]. The leaves of this plant have a rich content of essential oil (~ 3 % of leaf dry weight), which has a pleasant odor and comprises several terpenes
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and terpenoids [2]. The EOCz of different regions of Northeast Brazil has shown different chemical compositions [5–7], and it has been reported to exert biological and pharmacological activities such as anthelmintic [8], bactericidal [6], antifungal [9], and larvicidal [10] effects. Furthermore, EOCz samples containing anethole as the major constituent and anethole itself have also been reported to have several pharmacological activities such as antispasmodic [11, 12], cardiovascular [13], and gastroprotective [14] effects. Reported EOCz and anethole effects include the blockade of neuromuscular transmission [15], depressant action on the central nervous system [1, 16], antinociceptive activity [4], and improvement in wound healing [5]. These pharmacological activities of EOCz have been attributed to anethole. Besides these activities, various studies have demonstrated that anethole has potent anti-inflammatory activity, since it was shown to inhibit the cellular response induced by the proinflammatory cytokine TNF-α and to reduce paw edema elicited by substance P, bradykinin, histamine, and TNF-α [17, 18]. Several EOCz and anethole activities are theoretically related to the alteration of cell excitability, for example, antispasmodic, anti-inflammatory, and anti-hypernociceptive activity [11, 17– 19]. Anethole has anti-inflammatory and antinociceptive activities that are highly potent and efficacious (a range of in vivo doses of 3–30 mg/kg for the anti-edematogenic effect and of 30– 100 mg/kg for the antinociceptive effect), making it a candidate therapeutic agent that deserves further study of its effects and mechanisms [18]. Anti-inflammatory and antinociceptive activities may also be related to the alteration of cell excitability, since previous studies have demonstrated that proinflammatory cytokines may specifically interact with receptors and ion channels regulating neuronal excitability [20]. Thus, the alteration of cell excitability appears to be a common mechanism partially or totally underlying the pharmacological effects of the EOCz and anethole. Our research group has demonstrated that various essential oils and their constituents block cell excitability [21–25]. In some cases, this blockade by the constituents, even when being similar to that of the essential oil, could not explain the inhibitory activity of the oil [22]. For all these reasons, we undertook this investigation aiming to characterize and compare the effects of the EOCz and anethole on excitability. Excitability was assessed by extracellular recording of the rat sciatic nerve CAP, and the EOCz and anethole were found to block nerve excitability with similar pharmacological characteristics.
Results !
CAP is an electrical signal that results from the summation of axonal action potentials in a nerve bundle, and illustrative traces of rat sciatic nerve CAP under control conditions are shown in " Fig. 1 A to C (left traces). As seen in l " Fig. 1, the CAP showed l two distinct waves, designated here as 1st and 2nd waves (white arrows in figure), as described by Leal-Cardoso et al. The mean values of the control PPA of the CAP was 7.11 ± 0.40 mV (n = 57), and the conduction velocity of its 1st and 2nd waves was 97.05 ± 3.57 and 31.96 ± 1.39 m/s (n = 54), respectively. To ensure that DMSO 0.2 % v/v did not interfere with the CAP parameters, we performed an external control for the activity of the vehicle, DMSO. The sciatic nerve was exposed to DMSO 0.2 % v/v for up to 180 min (the same period of time EOCz and anethole exposure). During this exposure to the vehicle, the PPA and conduction velocity of the 1st and 2nd waves showed a minor monoton-
ic increase and, at the end of this period, these parameters measured 107.90 ± 2.89, 102.98 ± 1.34, and 102.50 ± 1.97 % (n = 6) of the control, respectively, and were not statistically different from the initial control values (statistical comparison done using absolute control values). Concerning EOCz and anethole effects, recordings of the CAP in the control during exposure of the nerve to the EOCz, anethole (0.30 mg/mL), and washout are shown in " Fig. 1 A, B. Both agents blocked PPA and CAP conduction in the l sciatic nerve, and the effects were reversible after the washout. The effects the EOCz and anethole displayed concentration de" Fig. 1 D). Calcupendence in the range of 0.01 to 1.0 mg/mL (l lated IC50 values for the EOCz and anethole on the PPA were 0.32 ± 0.07 and 0.22 ± 0.11 mg/mL (1.48 ± 0.74 mM; R > 0.97 for both agents, n = 4–8), respectively, and there was no statistical difference between the EOCz and anethole (p > 0.05, ANOVA). The blockade induced by lidocaine, a well-known local anes" Fig. 1 C. For comparison, the concentrathetic, is illustrated in l " Fig. 1 D) was 0.02– tion-dependence range for lidocaine (l 0.7 mg/mL (0.1–3 mM) with an IC50 of 0.30 ± 0.16 mg/mL (1.28 mM, n = 4). Concerning the rheobase and chronaxie, two parameters related to nerve excitability [26], the control values were 3.22 ± 0.05 V and 59.75 ± 2.41 µs (n = 12), respectively. Since the IC50 values of the EOCz and anethole were close (0.32 ± 0.07 and 0.22 ± 0.11 mg/mL, respectively), a concentration of 0.30 mg/mL was chosen for both the EOCz and anethole (~ 2 mM) to assess the parameters related to excitability. Sciatic nerve exposure to the EOCz and anethole reduced excitability, and as shown in " Fig. 2 A, the strength-duration curve for both agents was l shifted upward and to the right compared to the control curve. " Fig. 2 B, C). Besides, the rheobase and chronaxie were altered (l The EOCz increased the rheobase to 4.23 ± 0.29 V (n = 6) and chronaxie to 75.17 ± 5.73 µs (n = 6). With anethole, the rheobase and chronaxie increased, respectively, to 4.02 ± 0.15 V (n = 6) and 68.67 ± 4.72 µs (n = 6). These changes were statistically significant for the chronaxie in the presence of the EOCz and for the rheobase in the presence of both agents compared to the control (p < 0.05, ANOVA followed by Dunnettʼs multiple comparison tests). The time course of the EOCz and anethole effects on the PPA of " Fig. 3 A, B. Both agents reduced the PPA in CAP is illustrated in l a concentration-dependent manner, and the reduction in amplitude for higher doses followed an approximately exponential decay, reaching a steady-state level during EOCz and anethole exposure. The time constants for 1.00 mg/mL EOCz and 0.6 mg/ml (~ 4 mM) anethole were 32.15 and 48.78 min (R > 0.97), respectively. The EOCz and anethole (180 min exposure) evoked a decrease in the conduction velocity of the 1st and 2nd waves that was similar for both waves. The EOCz at 0.60 and 1.00 mg/mL and anethole at 0.60 mg/mL blocked all CAP waves, and the conduction velocity could not be determined and was assumed to be zero. Statistical differences were found in CAP velocity with exposure to the EOCz (0.1, 0.3, and 0.45 mg/mL) or anethole (0.30 and 0.45 mg/mL; 2 and 3 mM) compared to the control group (p < 0.05, ANOVA on ranks followed by Dunnʼs comparison test). After 180 min of washout, the PPA of the preparation that was exposed to the EOCz and anethole (0.3 mg/mL for both agents) recovered to 80.34 ± 10.15 (n = 4) and 77.33 ± 14.65 % (n = 5), respectively, of the control.
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Fig. 1 Illustrative traces of the CAP and concentration-response curve for the EOCz, anethole, and lidocaine. A, B, and C show the CAP for the control (left) and exposure to the EOCz, anethole, and lidocaine [center, 0.3 mg/mL for the EOCz and anethole (2 mM) and ~ 0.25 mg/mL (1 mM) for lidocaine]
and the washout conditions (right). White arrows indicate the 1st and 2nd waves of the CAP and black triangles indicate the stimulus artifact. D shows the EOCz, anethole, and lidocaine concentration-response curves for the PPA of the CAP.
Discussion !
Fig. 2 Effects of the EOCz and anethole on excitability of the sciatic nerve. Strength-duration curves (A) were obtained in the presence of 0.3 mg/mL EOCz (white filled circles) and anethole (black triangles). Rheobase and chronaxie are shown in B and C, respectively. * Indicates a statistically significant difference compared to the control conditions (p < 0.05, ANOVA on ranks followed by Dunnʼs comparison test). # Indicates a statistically significant difference compared to the control curve (p < 0.05, two-way ANOVA followed by the Holm-Sidak test).
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This study demonstrated for the first time that the EOCz and anethole block sciatic nerve conductibility and excitability in a concentration-dependent and reversible manner. Both agents inhibited nerve CAP and increased rheobase and chronaxie, two parameters directly related to excitability. The present investigation adds important information about essential oil and its main constituent on peripheral nerves. The EOCz and anethole effects on excitability have been hypothesized or demonstrated with indirect methods for other tissues including cardiovascular, skeletal, and smooth muscle [11, 13, 15]. Furthermore, the EOCz also has an antinociceptive effect [4] and a depressant effect on the central nervous system [16], which are greatly related to the blockade of excitability. It has been reported that essential oils from Croton nepetaefolius [22], Alpinia zerumbet [24], and rosewood [27] block the CAP of the sciatic nerve in a concentration range similar to that of the EOCz found by us. Usually, the blockade of neuronal activity by essential oils can be attributed to the action of its main constituent, as shown by linalool-rich rosewood oil [27]. However, that is not always the case. The effects of C. nepetaeolius essential oil could not be predominantly explained by the action of its main constituent, 1,8-cineole [22]. In the present study, anethole blocked the CAP with a similar pharmacological potency as the EOCz, and the changes in CAP parameters elicited by a given concentration of anethole and the EOCz were very similar. Together, these findings indicate that the EOCz effects can be attributed to the action of its main constituent, anethole. However, the possible involvement of other minor constituents such as estragole (4.80 %) and 1,8-cineole (2.95 %) cannot be excluded, since these constituents also block the action potential in dorsal root and superior cervical ganglion neurons, respectively [22, 24, 28, 29]. A
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Fig. 3 Time course of the PPA and conduction velocity of the CAP waves after exposure to the EOCz and anethole. Time courses for the EOCz and anethole are shown in A and B, and data were fitted with a single exponential function (solid lines). The conduction velocity of the 1st and 2nd CAP waves
partial contribution of these constituents has been previously reported for the EOCz in the mediation of antispasmodic [11], antinociceptive [4], cardiovascular [13], and skeletal muscle [15] effects. To compare the pharmacological potency of both agents, we performed experiments with lidocaine, a well-known local anesthetic. We found that the EOCz and anethole showed a similar pharmacological potency in reducing the PPA of the CAP compared to lidocaine. Thus, it seems that the EOCz and anethole could act like a local anesthetic in reducing sciatic nerve excitability. We also observed that CAP inhibition by the EOCz and anethole was established in 180 min. This apparently long time course is not exclusive for these agents. Many essential oils, terpenes, and terpenoids show a slow CAP blockade [21–25], and this characteristic can be attributed to the diffusion barrier provided by the various sheaths (peri-, meso-, and endoneurium) from the nerve surface to axon fibers [30]. We investigated the effects of the EOCz and anethole on the rheobase and chronaxie, which are parameters directly related to the measurement of nerve excitability. As demonstrated, both the EOCz and anethole increased the rheobase, meaning that they increased the minimum stimulus level required to generate action potentials [26], reducing the excitability of the sciatic nerve. The mechanism of this decreased excitability, which would lead to the inhibitory action on CAP generation, was not elucidated. However, several terpenes and terpenoids of small molecular weight, such as thymol, eugenol, linalool, carvacrol, and estragole, act on Na+ channels [25, 28, 31–33]. This makes the blockade
is shown in D and C for the EOCz and anethole, respectively. * Indicates a statistically significant difference compared to the control conditions (p < 0.05, ANOVA on ranks followed by Dunnʼs comparison test).
of this ion channel an attractive hypothesis to explain the effect of the EOCz and anethole on nerve excitability. In conclusion, we demonstrated here that the EOCz blocks neuronal excitability and that this effect can be predominantly attributable to its major constituent, anethole. These findings are likely to be relevant to the mechanism of action of various activities of potential therapeutic usefulness of these agents, which deserve further investigation.
Material and Methods !
Plant material, extraction, and chemical analysis Aerial parts of C. zehntneri were collected in September 1998, near the city of Viçosa do Ceará (lat. 3°33′ 48″ S.; long. 41°5′ 41″ W., Ceará, Brazil). C. zenhtneri is a deciduous plant that acquires leaves during the raining season (February to May) and with the small amount of rain that drops during the months of August and September (rain of “caju”). Plant identification was confirmed by Dr. FJ Abreu Matos (Laboratory of Natural Products, Federal University of Ceará, Fortaleza, CE, Brazil). A voucher specimen (No. 277 477) has been deposited in the herbarium Prisco Viana at the Federal University of Ceará. The EOCz was isolated from freshly chopped leaves by steam distillation and analyzed chemically as previously described [4, 5]. Briefly, chromatographic analysis was carried out on a HewlettPackard 6971 system using the following analytical conditions: a dimethylpolysiloxane DB-1 fused silica capillary column (30 m × 0.25 mm; 0.1 µm); helium (1 mL/min) as the carrier gas; 250 °C
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injector temperature; 200 °C detector temperature; a column temperature of 35–180 °C at 4 °C/min and then 180–250 °C at 10 °C/min; and 70 eV electron impact mass spectrometry. The compounds were identified using a mass spectral library search and 13C‑NMR spectroscopy. The chemical composition of the EOCz performed by GC/MS was 85.7 % anethole, 4.8 % estragole, 2.95 % 1,8-cineole, 2.2% β-myrcene, 1.22 % anisaldehyde, 0.9 % trans-caryophyllene, and 2.23 % unidentified.
Solutions and substances Electrophysiology recordings were performed using modified Lockeʼs solution with the following composition (in mM): NaCl 140, KCl 5.6, MgCl2 1.2, CaCl2 2.2, Tris(hydroxymethyl-aminomethane) 10, and glucose 10. The EOCz and anethole were dissolved in DMSO at a final concentration equal to or less than 0.2 % v/v, and stock solutions were prepared daily. Lidocaine (powder) was directly dissolved in Lockeʼs solution to the desired concentrations. The EOCz concentrations used were 0.01, 0.10, 0.30, 0.45, 0.60, and 1.00 mg/mL. For anethole, the concentration range was the same as for the EOCz, excluding 1.00 mg/mL (0.07– 4.0 mM). For lidocaine, the concentration range was 0.02– 0.7 mg/mL (0.1–3.0 mM). Experiments were carried out at room temperature (22 to 26 °C), and all salts, anethole (99 %), and lidocaine (99%) were of analytical grade and purchased from Sigma Chemical or Reagen.
voltage for an active response with a long duration pulse (1000 µs), and chronaxie is the threshold duration for an active response with a stimulus twice the rheobase [26].
Statistical analysis All results are expressed as the mean ± SEM. Statistical significance (p < 0.05) was assessed using one-way analysis of variance (ANOVA) followed by a post hoc comparison test when appropriate. The IC50 of the agents was calculated from the concentrationresponse curves where the experimental points were fitted by a Hill equation.
Acknowledgements !
The authors acknowledge the financial support from Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Universidade Estadual do Ceará (UECE). Also, the authors would like to thank Mr. Pedro Militão de Albuquerque Neto for technical support and for improving the manuscript. Dr. A. Leyva helped with the English editing of the manuscript.
Animals and tissue preparation
Conflict of Interest
Wistar rats (200–300 g) of both sexes were maintained under conditions of constant temperature (22 ± 2 °C), with a 12-h light/ 12-h dark cycle and free access to food and water. All animals were handled in compliance with the Guide for the Care and Use of Laboratory Animals, published by the U. S. National Institutes of Health (NIH Publication 85–23, revised 1996; http://www. nap.edu/readingroom/books/labrats/index.html), and all efforts were made to minimize animal suffering. All procedures described herein were first reviewed and approved by the local animal ethics committee (CEUA/UECE protocol No.: 06 379 067–0, approval date 08/15/2011). The sciatic nerve was dissected from rats sacrificed by asphyxia in a CO2 chamber and was immediately placed in a vessel containing modified Lockeʼs solution. The tissue was used on the same day of dissection as well as the electrophysiological recording.
!
Electrophysiology The sciatic nerve was mounted in a moist chamber and the evoked CAP was recorded as described elsewhere [22, 25]. Sciatic nerve exposure to the EOCz, anethole, and lidocaine was performed only when a stable PPA of the CAP was achieved for at least 30 min, and the agent exposure time was set to 180 min, except for lidocaine in which case drug exposure was set to 30 min. This interval was usually sufficient to allow for the steady-state CAP amplitude to be reached during drug exposure, and this period was followed by a 180-min washout/recovery period. The parameters measured in the evoked CAP were PPA and conduction velocity of the CAP waves. The PPA is the absolute sum of maximum and minimum amplitudes of the CAP, and conduction velocity was determined by the ratio of sciatic nerve length and the time interval between stimulus artifact and the peak of the " Fig. 1 A, left). Besides, strength-dura1st and 2nd CAP waves (l tion curves with voltage square wave stimuli were used to determine the rheobase and chronaxie, which are parameters related to excitability. Rheobase is defined as the threshold stimulus
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There is no conflict of interest.
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