Life Sciences 89 (2011) 540544

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Life Sciences
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Minireview

Essential oils components as a new path to understand ion channel molecular pharmacology
Demetrius Antonio Machado de Arajo a, Christiane Freitas b, Jader Santos Cruz b,
a b

Laboratrio de Tecnologia Farmacutica, Universidade Federal da Paraba, Mail Box 5009, CEP: 58051-970, Joo Pessoa, PB, Brazil Departamento de Bioqumica e Imunologia, Universidade Federal de Minas Gerais, CEP: 31270-901, Belo Horizonte, MG, Brazil

a r t i c l e

i n f o

a b s t r a c t
The discovery and development of new drugs targeting voltage-gated ion channels are important for treating a variety of medical conditions and diseases. Ion channels are molecular nanostructures expressed ubiquitously throughout the whole body, and are involved in many basic physiological processes. Over the years, natural products have proven useful in the pharmacological assessment of ion channel structure and function, while also contributing to the identication of lead molecules for drug development. Essential oils are complex chemical mixtures isolated from plants which may possess a large spectrum of biological activities most of them of clinical interest. Among their bioactive constituents, terpenes are small to mediumsized components and belong to different chemical groups. Various reports have drawn our attention to the fact that terpenes are novel compounds targeting voltage-gated ion channels. The purpose of this review is to provide a focused discussion on the molecular interaction between monoterpenes and phenylpropenes with voltage-gated ion channels in different biological scenarios. 2011 Elsevier Inc. All rights reserved.

Article history: Received 20 December 2010 Accepted 27 April 2011 Keywords: Natural products Ion channels Terpenes Na+ channels Ca2+ channels K+ channels

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Essential oils: new source for new molecules . . . . . . . . . . . . . . . . . Constituents of essential oils that acts in the central and peripheral nervous system Pitfalls and promises in the eld . . . . . . . . . . . . . . . . . . . . . . Conict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540 541 541 543 543 543 543

Introduction Ion channels are integral membrane proteins designed to catalyze ion ux and, as a consequence produce changes in the membrane potential. These molecular nanostructures are present in all types of cells but they have a very important role in excitable cells where they are the main actors responsible for the generation of action potentials. Action potentials are the result of the activity of many different types of ion channels working in concert to carry information from one cell to another in a very controlled way. There is much information available about the function of ion channels (Catterall 2010). Usually, we separate ion channels in two

Corresponding author. Tel.: + 55 31 3409 2668; fax: + 55 31 3409 2613. E-mail addresses: jader.cruz@pq.cnpq.br, jcruz@icb.ufmg.br (J.S. Cruz). 0024-3205/$ see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.04.020

major super-families, voltage-dependent and ligand-dependent ion channels. They are involved in a plethora of distinct physiological processes such as: neurotransmitter release, excitationcontraction coupling, excitationtranscription coupling, control of gene expression, cell development and so on. Therefore, we can argue that the normal ion channels' activity (or function) is crucial for the maintenance of health. Another important point is the realization that ion channel dysfunctions could lead to serious pathological disorders compromising the whole organism. In 2006 the US Drug Administration approved 18 new molecular compounds and two of them had their primary mode of action attributed to ion-channel modulation indicating that ion channels are very attractive and promising drug discovery targets (Dunlop et al., 2008). In this way one would think that these molecular bio-structures are important pharmacological targets for natural-based chemical

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compounds looking at the development of new therapeutic strategies. In order to validate this saga, highly selective and potent antagonists or even agonists are a prerequisite (Bulaj 2008). Drug discovery efforts posed by the medicinal chemists' community have discovered a rather small number of molecules that modulate ion channels function. Taking all of that in mind, the understanding of how these molecules actually interacts with ion channels are of great interest. Consequently, electrophysiological techniques are extremely useful for the characterization of the biological activity of isolated compounds. Electrophysiological approaches are enormously rich in terms of acquired information and have been considered as the gold-standard assay. Essential oils: new source for new molecules In search for new sources of natural molecules that modulate ion channel behavior, essential oils are both promising and challenging. Plant essential oils are typically composed of volatile aromatic terpenes and phenylpropanoids. These lipophilic substances are classied as monoterpenes and sesquiterpenes based on the number of isoprene units (two and three respectively) besides the phenylpropanoids, which are made up of C6C3 units. These molecules freely cross cellular membranes and may serve various signaling roles inside the cell. In addition, there are some reports indicating that essential oil plant components are active towards ion channels and receptors (Gonalves et al., 2008; de Almeida et al., 2008; Alves et al., 2010). Apart from rational drug design and novel synthetic efforts, natural products are still been investigated for novel chemical structures that may interact with known and unknown pharmacological targets. The pharmacology of ion channels has become a complex research area and as far as we understand the mechanisms controlling ion channel functioning, more potential drug targets are being disclosed. At this point it is worth to remind that over the decades, natural products have undoubtedly contributed to the development of new drugs currently used in clinical practice. More importantly, these remarkable molecules have also been important tools for the discovery of new pharmacological targets such as receptors and/or ion channels (Vriens et al., 2008). One of the most relevant examples is the transient receptor potential (TRP) family of ion channels. This area is very active and there are excellent reviews covering various aspects including the role of natural products in the discovery and pharmacological characterization of TRP channels (for review see Calixto et al., 2005; Vriens et al., 2008) and therefore we will not cover in detail this topic. In this mini-review we will focus on medicinal compounds which modulate ion channels present in different physiological systems. Constituents of essential oils that acts in the central and peripheral nervous system The potential use of essential oils as modulators of ion channels in the treatment of several diseases is indeed exciting. Although most of the published studies quote the popular use of essential oils in the treatment of several nervous system disorders, just a few studies (approximately 3% in a PUBMED search) described the activity and toxic effects of its major components on the nervous system (de Sousa et al., 2006; Goncalves et al., 2010). Only a small fraction of those studies deals with the interaction between terpenes and ion channels. Linalool is a monoterpene that has been the subject of a number of studies and it is one of the major constituents of several essential oils isolated from different plant species. Linalool has been reported to have diverse biological and pharmacological activities (Celik and Ozkaya, 2002; Peana et al., 2002; Bickers et al., 2003; de Almeida et al., 2009). There are reports showing that linalool acts on the central nervous system but through a yet unrevealed mechanism. Linalool

shares high lipid solubility with other lipid soluble odorants that directly affect ion channels activity (Kawai et al., 1997; Kawai, 1999; Kawai and Miyachi, 2000) suggesting that linalool could interact with certain types of ion channels by changing the lipid membrane environment. It was already been described that this monoterpene has sedative effects in vertebrates including humans (Buchbauer et al., 1991; Sugawara et al., 2000), and that the inhalation of linalool can lead to a signicant reduction of motility in mice (Jirovetz et al., 1991). Other possible applications for linalool are as pain modulator, anticonvulsant, hypnotic and hypothermic agent. Elisabetsky et al. (1995) described that linalool inhibits glutamatergic neurons and later Sugawara et al. (2000) observed that it also affected human brain beta waves amplitude. Earlier studies in newt olfactory receptor cells, newt retinal neurons and rat cerebellar Purkinje cells demonstrated that linalool non-selectively but reversibly suppressed the voltagegated currents (Narusuye et al., 2005). In the same report it was shown that linalool reduced KCl-induced intracellular Ca 2+ elevation without affecting the machinery responsible for intracellular Ca 2+ signaling (Narusuye et al., 2005). The pharmacological effects of linalool on somatic sensory neurons have been studied in more detail by Leal-Cardoso's group (LealCardoso et al., 2010). The authors provided a reasonable number of experimental ndings to conclude that inhibition of the voltage-gated Na + channels is probably the major mechanism by which the neuronal excitability is impaired. Taken all together, we may suggest a possible mechanism of action that could be attributed to linalool as a main frame to explain its various effects. As a consequence of a signicant reduction in Ca 2+ inux there is an important neurotransmitter release inhibition in the presynaptic terminals. Similarly, but not independently, linalool could elicit a blockade of voltage-gated Na + channels causing a premature termination of the action potential generation which per se would lead to diminution of neurotransmitter release by exocytosis. Presumably, the body of evidences in the literature support the general mechanism pointed out above but it is clear that further studies are necessary to explore in more detail how linalool is acting. Eugenol, a phenylpropene derivative, is widely used in dentistry as local anesthetic, analgesic, anti-microbial and anti-inammatory agent (Hashimoto et al., 1988; Ohkubo and Kitamura, 1997; Pizzo et al., 2006). In a series of papers from Oh's group it was postulated that the analgesic effects of eugenol in rat dental afferent neurons could be related to its inhibitory effect on voltage-gated Na + channels (Park et al., 2006) and on high voltage-activated Ca 2+ channels (Lee et al., 2005). Surprisingly, both effects did not require TRPV1 activation (Lee et al., 2005; Park et al., 2006). Interestingly, when mammalian central nervous system is acutely exposed to eugenol it causes a general depressant activity leading to sedation, reduction of convulsions induced by electroshock and hypothermia Dallmeier and Carlini (1981).In fact, very little is known about eugenol's mechanism (s) of action especially in the central nervous system. However, it is well known that eugenol blocks rat sciatic nerve compound action potentials probably by acting on the voltagedependent Na + channels. At high concentrations (2 mM) and during brief applications eugenol blocked the action potential without interfering in the resting membrane potential or membrane input resistance. However, at low concentrations (0.6 mM) and longer applications the authors observed a signicant reduction in the input membrane resistance which, as discussed by the authors, raises the possibility of a secondary effect involved in the reduction of neuronal excitability when eugenol was present (Moreira-Lobo et al., 2010). Elucidative studies also showed that eugenol inhibited thermal nociception and capsaicin-induced thermal hyperalgesia in orofacial area indicating that this phenylpropene derivative could also be used for other pathological pain conditions (Park et al., 2009). At this point a note of caution should be presented concerning the activation of TRPV1 channels by eugenol which could evoke excitation of

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nociceptors even knowing that this phenomenon is rather weak when compared to other TRPV1 agonists (for example, capsaicin). Nav1.7, Nav1.8 and Nav1.9 are Na +channel subtypes that can be found predominantly in nociceptive DRG neurons (Akopian et al., 1996). There are a number of studies suggesting that their expression and functional properties are modied following inammation or nerve injury (Dib-Hajj et al., 1998; Cummins et al., 2007). Great effort has been made in order to a better understanding of how eugenol actually exerts its analgesic effect. As a consequence several mechanisms have been proposed to explain the eugenol analgesia. Recently, a well designed study elucidating the involvement of voltage-gated Na + channels in the eugenol-elicited analgesia came up. To that purpose, Cho et al. (2008) pursued a detailed kinetic analysis to investigate the effects of eugenol on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na + currents in acutely dissociated rat dorsal root ganglion neurons. Their ndings clearly indicate that eugenol inhibits voltage-gated Na + currents through its interaction with both resting and inactivated Na + channels. The authors also evaluated the recovery from inactivation for both Na + currents. Eugenol slowed the recovery from inactivation. One possible implication that one would expect is a decrease in neuronal excitability leading to nerve conduction blockade. To complete their investigation Cho and colleagues demonstrated that the eugenol inhibition of TTX-R and TTX-S Na + currents did not show any stimulation frequency dependence which is a behavior distinct from other typical anesthetics (Hille,2001). As we indicated above eugenol presents various pathways to provoke analgesia. There are substantial experimental evidences that argues in favor of the participation of voltage-gated Ca 2+ channels as key elements in the transmission of pain signals (McGivern and McDonough, 2004; Lee et al., 2005; Chung et al., 2008; Zamponi et al., 2009; Perret and Luo, 2009). Knowing that eugenol is related to capsaicin in terms of chemical structure and that capsaicin has been demonstrated to inhibit high voltage-gated Ca 2+ channels (Petersen et al., 1989; Bleakman et al., 1990), these information combined have motivated studies to investigate whether eugenol would interact with high voltage-gated Ca 2+ channels. Two studies (Lee et al., 2005; Chung et al., 2008) addressed the question of whether eugenol would cause an inhibitory effect on Ca 2+ channels. Lee et al. (2005) used rat dental primary afferent neurons and C2D7 cells stably expressing the human N-type Ca 2+ channel. Altogether, they found that eugenol did elicit an inhibitory effect on high voltage-gated Ca 2+ current in all neurons tested and importantly these effects were not exclusive to capsaicin-sensitive primary afferent neurons. What would be the implication for that? These ndings suggest that the mechanisms by which eugenol and capsaicin induced Ca 2+ current inhibition might be somehow different. Further, the authors conrmed this contention by using C2D7 cells (heterologously expressing Cav 2.2 the molecular counterpart for the N-type Ca 2+ channels) that do not contain endogenous TRPV1 receptor. In this better controlled system eugenol caused the same effects as previously described. In 2008, Chung and colleagues using a similar experimental strategy reported that eugenol inhibited Cav 2.3 channels (R-type Ca2+ current), and conrming the earlier results the inhibitory effect was not related to the activation of TRPV1 receptor. The authors, based on their interesting ndings, proposed that due to the complete lack of TRPV1 involvement in eugenol's inhibitory effects, an analog of eugenol might be developed to substitute for capsaicin as a potential analgesic drug without exhibiting irritant actions (Chung et al., 2008). In excitable cells such as neurons, voltage-gated K + channels serve to repolarize or hyperpolarize the membrane. Therefore, pharmacological activation of K + channels in excitable cells reduces excitability. On the other hand, K + channels inhibition would cause an increase in excitability. Overall, K + channels constitute potential drug targets for the treatment of diverse diseases from cancer to cardiovascular

disorders (Wulff et al., 2009). Eugenol has both excitatory and inhibitory effects (Lee et al., 2005; Park et al., 2006; Li et al., 2007). The excitatory effect attributed to eugenol comes to its activation of TRPV1 channels that evokes an inward current capable to provoke a membrane depolarization. This effect would possibly be the one responsible for the irritant actions of eugenol. However, it was reported that eugenol inhibits voltage-gated K + currents causing a prolongation of the action potential (Li et al., 2007). Interestingly, eugenol only increased the action potential duration in a small subpopulation of trigeminal ganglion neurons (about 13%). The authors raised an important issue when comparing the concentration ranges where eugenol blocked Na + and Ca 2+ currents indicating that eugenol is less potent towards Ca 2+ channels than Na + or K + channels. In summary, the inhibition demonstrated by Li and colleagues is likely to contribute to the pungent effects of eugenol. At this point we have a clear picture of how eugenol may affect neuronal electrical activity. One important question that may be relevant in the context of this review is whether eugenol (or any other constituent of essential oils) carries the potential for anti-convulsant effects. A careful examination over the literature revealed only a few studies pointing out the possible effects of eugenol in central nervous system (Dallmeier and Carlini, 1981; Wie et al., 1997; Masago et al., 2000; Irie et al., 2004). Throughout this review we showed the complex pharmacological prole of eugenol in blocking different ion channels. Interesting to note is the fact that other anticonvulsant substances seems to exert its effects via multiple mechanisms Araujo et al. (2003). From a functional viewpoint, the simultaneous action of eugenol over distinct ion channels may prevent the ability of neuronal cells to trigger action potentials and therefore makes it very attractive as an anticonvulsant agent. In a very elegant study, Mller et al. (2006) made use of different but complementary biological assays to explore the neurophysiologic properties of the action of eugenol. In order to do that the authors investigated eugenol effects on 1) epileptiform eld potentials elicited by removal of extracellular Mg 2+, 2) spreading depression induced by focal KCl microinjections, 3) electrically evoked eld potentials, and 4) long term potentiation in rat neocortical and hippocampal tissues. From their analyses we can conclude that eugenol can suppress epileptiform eld potentials and spreading depression, probably through inhibition of synaptic plasticity (Mller et al., 2006). These very promising results indicate that further studies are needed to clarify in more detail the putative benecial effect of eugenol in the treatment of epileptic patients. Another constituent of essential oils that has been studied in the last 10 years is menthol. It was rst described as a cooling compound and it had a prominent role in the elucidation of the so called cold sensors (McKemy et al., 2002). Menthol is a primary activator of the cold and menthol-sensitive TRPM8 channels (McKemy et al., 2002). After its binding to TRPM8 channels expressed in sensory neurons there is a large increase in intracellular Ca 2+ level which indirectly facilitates glutamate release. This process is very important in the modulatory effects of peripheral nociception provoked by menthol. Until recently there was no evidence for distinct roles of menthol in the nervous system. Mounting evidence, however, points to the fact that menthol could have effects in the central nervous system. Umezu et al. (2001) have shown that menthol when administered intravenously elicited profound effects on rodent behavior. The authors reasoned that menthol caused its effects by acting on the central nervous system. One would probably ask the question: How menthol reaches central nervous system? Menthol is absorbed and then by crossing the blood-brain barrier (due to its lipophilic nature) produces its effects on the neurons. Zhang et al. (2008) explored the effect of menthol in central neurons and they clearly demonstrated the central actions of menthol on hippocampal neurons. More interestingly, they showed a specic function of menthol in arresting the excitation of hippocampal neuronal cells by selectively augmenting tonic GABA inhibition. These ndings are very relevant for the eld

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because menthol could be used as an antiepileptic drug. In the same study, Zhang and colleagues used two different strategies to elicit epileptic activity and they provided compelling evidence that menthol does exert anticonvulsant effect and it is totally consistent with the enhanced tonic GABAergic inhibition which prevents epileptiform neuronal hyperexcitability. Watt et al. (2008) using Xenopus oocyte heterologous expression system decided to investigate a possible modulation of recombinant human GABAA receptors by menthol. This study was carried out to determine whether menthol shares or not sites of action with other most common sedative and anesthetic drugs. Firstly, they tested a number of menthol analogs looking for an enhancement of GABA currents. Secondly, they described structurefunction relationships centered on receptor modulation. Thirdly, they nally explored whether menthol can act as a general anesthetic using an established biological assay. The authors then concluded that menthol and its chemical analogs share general anesthetic action with propofol (a well known anesthetic), probably through similar binding sites on the GABAA receptor. It has been well known the antibacterial and antimycotic activity of thymol (Botelho et al., 2007; Bakkali et al., 2008). However, some authors have unequivocally demonstrated that this monoterpene showed effects on voltage-gated ion channels. One of such studies performed in isolated canine and human ventricular cardiomyocytes demonstrated that thymol acted on cardiac ion channels in a concentration-dependent manner provoking inhibitory effects on Ltype Ca 2+ currents and on various types of K + currents such as, transient outward and delayed rectier (Magyar et al., 2002). The authors point to the fact that the mechanism of action may be different when thymol blocks L-type Ca 2+ channels and K + channels. In a follow up study Magyar et al. (2004) investigated in more detail the effects of terpenoid phenol derivatives (such as carvacrol, thymol and eugenol) on L-type Ca 2+ current in isolated cardiac myocytes. The authors then concluded that the blocking effect on cardiac Ca 2+ currents may be related to the chemical nature of the substituent in the benzene ring. In a very interesting study Haeseler et al. (2002) compared thymol and menthol in their capacity to evoke a blockade in voltage-gated Na + currents. They discovered that both, thymol and menthol inhibited voltage-gated neuronal and skeletal muscle Na + channels in resting and inactivated states which strongly indicates a voltage-dependent blockade which shares great similarity to the local anesthetic lidocaine. Excitationcontraction coupling is a very important physiological process that maintain under control muscle contraction. It has been reported that thymol had major effects on excitationcontraction coupling in skeletal muscle of rodents (Szentesi et al., 2004). In a series of well designed experiments Szentesi et al. (2004) demonstrated that thymol increases sarcoplasmatic reticulum (SR) Ca 2+ release by acting directly on the intracellular ryanodine receptor Ca 2+ channel. Thymol also had effects on intracellular Ca 2+ handling in canine and guinea pig cardiac preparations (Szentandrassy et al., 2004). In a more recent study it was reported that thymol increased intracellular Ca 2+ concentration in pituitary GH3 cells (Shen et al., 2009). The authors provided evidence to state that thymol depletion of intracellular Ca 2+ stores is related to thapsigargin-sensitive and -insensitive reservoirs. Pitfalls and promises in the eld While many important ndings related to natural products have already been uncovered, we are still far from a through comprehension of this very important topic of research. Many questions and challenges remain to be solved. The main bottleneck for properly exploring the molecular diversity of plant derived natural products is an efcient purication platform that allows isolation in enough quantities that can be screened against ion channels to look for new pharmacological proles.

Another important point to be discussed is related to the limitations in identifying new compounds with highly desirable biological activity. This limitation is worsened by seasonal or environmental variations that would certainly have a direct impact in the biochemical composition of living organisms causing real problems in the earlier purication steps. Finally, we would like to make a reminder as to what was pointed out by Vries and colleagues in a recent review it is obviousthat naturally occurring substances especially those derived from higher plants, will continue to be essential tools in the discovery of therapeutic targets necessary for the development of new innovative drugs.
Conict of interest statement The authors declare that there are no conicts of interest.

Acknowledgments This work has been supported by research grants to Dr. D.A.M. Arajo and Dr. J.S. Cruz (CNPq and FAPEMIG). Christiane Freitas held an undergraduate scholarship from CNPq-RENORBIO. References
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