Erica A Gelfuso

Daiane Suelen L Reis

S R Aguiar

Silmara A Faggion

M M Flávia

Diogo T Gomes

Steve Galan

Ana M Peigneur

S Pereira

Márcia R Mortari

Alexandra O S Cunha

Daiane S R Aguiar

M M Gomes

T Diogo

Galan

Steve Peigneur

Ana M S Pereira

Jan Tytgat
email: jan.tytgat@kuleuven.be

Renê O Beleboni
email: rbeleboni@unaerp.br

New insights in the mode of action of (+

Keywords: Electrophysiology, E. mulungu, Erythrinian alkaloids, GABA, Glutamate, Ion Channels

A c c e p t e d m a n u s c r i p t 1 Introduction

In the last decades, the gradual increase of studies searching for new antiepileptic and anxiolytic drugs has pointed Erythrina mulungu (Mart. ex Benth.) (syn. E. verna Velloso, 1825) as a special source of active compounds [START_REF] Fahmy | Alkaloids of genus Erythrina : An updated review[END_REF]. Flowerings of this plant have been used as raw material for a large set of industrialized herbal preparations in differe nt countries. Several investigations have validated the antidepressant, anxiolytic, anticonvuls a nt and sedative properties popularly acclaimed to E. mulungu [START_REF] Bezerra Carvalho | Regulation of herbal medicines in Brazil[END_REF][START_REF] De Oliveira | Antinociceptive and anti-infl ammatory activity of hydroalcoholic extracts and fractions from Erythrina mulungu[END_REF][START_REF] Durigan | Plantas do cerrado paulista: imagens de uma paisagem ameaçada[END_REF][START_REF] Lorenzi | Plantas medicinais no Brasil : nativas e exóticas[END_REF][START_REF] Rodrigues | Plants with possible anxiolytic and/or hypnotic effects indicated by three brazilian cultures-Indians, Afro-Brazilians, and River-Dwellers[END_REF]Valli et al., 2013).

E. mulungu has been considered as a great source of erythrinian alkaloids. Indeed, the plant flowerings reserve a huge collection of alkaloids with different chemical structures and pharmacological activities, attending as a natural library for a chemical diversity of compounds for which structure-activity relationship deserves strong pharmaceutical attention [START_REF] Feitosa | Aplicação de espectrometria de massas com ionização por elétron na análise de alcaloides do mulungu #[END_REF][START_REF] Majinda | An Update of Erythrinan Alkaloids and Their Pharmacologic a l A c c e p t e d m a n u s c r i p t Activities[END_REF]. Previous studies have demonstrated that (+)-erythravine and (+)-11α-hydroxy-erythravine alkaloids are among those responsible for antiepileptic and anxiolytic properties exhibited by E. mulungu flowerings [START_REF] Faggion | Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11α-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae)[END_REF]Flausino et al., 2007;[START_REF] Gelfuso | Neuroprotective effects and improvement of learning and memory elicited by erythravine and 11α-hydroxy-erythravine against the pilocarpine model of epilepsy[END_REF][START_REF] Rosa | Erysothr ine, an alkaloid extracted from flowers of Erythrina mulungu Mart. ex Benth: Evaluating its anticonvulsant and anxiolytic potential[END_REF]. However, scarce information is presented about the mechanism of action of these alkaloids both at molecular and cellular levels (Flausino et al., 2007;Setti-Perdigão et al., 2013).

Around 50 million people worldwide are diagnosed with different types of epilepsy (World Health Organization, 2019). The high prevalence and/or incidence of this neurologic a l disorder negatively impacts on social, health and economic demands from different countries [START_REF] Bell | An unknown quantity -The worldwide prevalence of epilepsy[END_REF]. Similarly, anxiety disorders affect different segments of our modern society and usually appears as a comorbid condition in many epileptic patients (de la [START_REF] De La Loge | PatientsLikeMe® Online Epilepsy Community: Patient characteristics and predictors of poor health-related quality of life[END_REF]. Despite significant differences in the molecular and cellular bases involved on the developme nt A c c e p t e d m a n u s c r i p t of epilepsy and anxiety, the sharing of some pathophysiological mechanisms and neural pathways appears as overlapping explanations for both disorders [START_REF] Hamid | Anxiety symptoms in epilepsy: Salient issues for future research[END_REF]. In this context, an imbalance between GABA and glutamate neurotransmissions and/or between different ion channels in the onset and the development of epilepsy and anxiety has been proposed [START_REF] Hamid | Anxiety symptoms in epilepsy: Salient issues for future research[END_REF][START_REF] Reddy | Experimental models of status epilepticus and neuronal injury for evaluation of therapeutic interventions[END_REF]Zubareva et al., 2018). Moreover, studies with nicotinic receptors demonstrate the relevance of this receptor-channel for both disorders, as well as its relationship with glutamatergic plasticity and cognitive functions such as memory and learning, suggesting an important connection with epilepsy or anxiety and a new avenue for development novel drugs acting on new or diversified pharmacological targets [START_REF] Dani | Nicotinic Acetylcholine Receptors and Nicotinic Choliner gic Mechanisms of the Central Nervous System[END_REF][START_REF] Ghasemi | Pathologic role of neuronal nicotinic acetylcholine receptors in epileptic disorders: Implication for pharmacological interventions[END_REF]Zarrindast and Khakpai, 2019).

Considering this last statement and also the physiopathology underlying both epilepsy and anxiety, it is chiefly appropriate to investigate the potential effects of (+)-erythravine and (+)-11-α-hydroxy-erythravine alkaloids on synaptic events such as of GABA and glutama te binding and uptake as well as on different sodium and potassium channels isoforms or also on different isoforms of cholinergic receptors. This approach may bring a broad body of information useful for a better understanding of different pharmacological aspects related to erythrinian alkaloids and to the ethnopharmacology regarding use of E. mulungu herbal preparations.

Material and Methods

Alkaloids

Flowers of E. mulungu were collected in Rifaina, São Paulo, Brazil (20°07′41,7″; 47°281′54,5″; 1037m; CGEN-Brazil: 02001.005109/2011-10). The alkaloids chromatographic isolation was carried out according to Flausino and colleagues (Flausino et al., 2007). Nuclear A c c e p t e d m a n u s c r i p t magnetic resonance (Varian Inc., USA) was used to confirm the chemical identity and purity degree of (+)-erythravine and (+)-11-α-hydroxy-erythravine.

Ethics statement animal experimentation

The procedures performed with rats were approved by the University of Ribeirão Preto Ethic Committee (06/2008). All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals, (2011) and all efforts were made to minimize animal suffering and to reduce the number of animals used.

The use of the frogs was in accordance with the license number LA1210239 of the Laboratory of Toxicology & Pharmacology, University of Leuven. The use of Xenopus laevis was approved by the Ethical Committee for animal experiments of the University of Leuven (P186/2019). All animal care and experimental procedures agreed with the guidelines of 'European convention for the protection of vertebrate animals used for experimental and other scientific purposes (Strasbourg, 18.III.1986).

Neurochemical assays: 3 H-Glutamate and 3 H-GABA uptake and binding

Synaptosomal preparation was performed according to the method described by Gray & Whittaker, Coutinho-Netto et al. and Coutinho-Neto et al. (Gray and Whittaker, 1962;[START_REF] Coutinho-Netto | Is Glutamate a Trigger Factor in Epileptic Hyperactivity[END_REF][START_REF] Coutinho-Neto | The Role of Polar Phytocomplexes on Anticonvulsant effects of Lippia alba (Mill) chemotypes leaf extracts[END_REF]. Cerebral cortices from healthy male Wistar rats (200-250 g) were rapidly removed and homogenized in ice-cold 0.32 M sucrose using Potter-Elhvejen Labo Stirrer LS-50 Yamato-type equipment. The sample was then centrifuged for 10 minutes at 1700 g (4 °C). The supernatant was collected and centrifuged for 20 minutes at 21.200 g (4 °C). The pellet was resuspended in Krebs-phosphate buffer (5 mM, pH 7.4). The synaptosome fraction obtained was used in the glutamate and GABA uptake A c c e p t e d m a n u s c r i p t assays. For the binding assay, the synaptosome fraction was further homogenized in 50 mM Tris-HCl buffer (pH 7.4) and then centrifuged at 3000 g (4 °C) (5 min). The pellet was washed in 50 mM Tris-HCl buffer (pH 7.4) and resuspended in the same buffer. Finally, the obtained synaptic membranes were centrifuged (3000 g/5 min/4 °C) and stored at -20 °C for at least 18 h. Protein content for both synaptosome fraction and the synaptic membrane was determined by the Lowry method modified by Hartree [START_REF] Hartree | Determination of Protein: A Modification of the Lowry Method That Gives a Linear Photometric Response[END_REF]. Uptake assays were started by adding 5 nM of radio-labelled [ 3 H]-GABA (GE Healthcare; 90 Ci/mmol) or 10 nM of radio-labelled [ 3 H]-Glutamate (GE Healthcare; 52 Ci/mmol) to the synaptosome fraction (0.25 mg of protein/ml at final concentration). Binding assays were started by adding of 5 nM of [ 3 H]-GABA or 25 nM of [ 3 H]-Glutamate to the synaptic membranes (1.0 mg of protein/ml at final concentration). Both assays were performed in the presence or absence of increasing final concentrations (0.001-10 µg/mL) of (+)erythravine or (+)-11α-hydroxy-erythravine completed to the final volume with 50 mM Tris-HCl buffer (0.3 ml for binding assays) or Krebs-phosphate buffer (0.5 ml for uptake assays).

Samples were incubated for 3 min at 25 º C or 37ºC respectively for [ 3 H]-GABA and [ 3 H]-Glutamate uptake assays. Incubation was performed for 30 min at temperatures of 25 º C or 37ºC for [ 3 H]-GABA and [ 3 H]-Glutamate binding assays, respectively.

All reactions were carried out in triplicate and stopped by centrifugation (3000 g, 3 min, 4ºC). Pellets were washed twice with ice-cold distilled water and homogenized in absolute methanol for uptake experiments or in 50 mM Tris-HCl buffer for binding assays. Samples of supernatants (uptake experiments) or whole tube content (binding experiments) were transferred to scintillation vials containing 1 ml of the biodegradable scintillation cocktail ScintiVerse (Fisher Scientific, USA) and quantified in a scintillation counter with a counting efficiency of 30-40% for 3 H. Non-specific GABA/Glutamate uptake and GABA/Gluta ma te binding were respectively estimated in parallel probes with non-radiolabelled GABA and

A c c e p t e d m a n u s c r i p t

Glutamate (1 mM, final concentration). These values were subtracted to give the total specific uptake or binding. Results were expressed as % of inhibition of GABA/Glutamate uptake or GABA/Glutamate binding in relation to the control.

Electrophysiological Recordings -Voltage-clamp

Oocytes were obtained through partial ovariectomy from sedated female frogs (Xenopus laevis) by using a solution containing tricaine mesylate and sodium carbonate (1g/L). After collection recovery, oocytes were injected with 20-70 nL of cRNA coding for Nav, Kv channels or nicotinic receptors (nAChR) using a micro-injector (Drummond Scientific, EUA). cRNAs were prepared with mMESSAGE mMACHINE transcription kit T7 or SP6 (Ambion, USA).

Then, the oocytes were incubated in a ND-96 solution (96 NaCl, 2 KCl, 1.8 CaCl2, 2 MgCl2, and 5 HEPES-pH 7.4; in mM) supplemented with gentamicin sulfate (50 mg/L; Rotexmedica, Trittau, Germany). Theophylline (90 mg/L; ABC chemicals, Wauthier Braine, Belgium) was also added in the solution for oocytes injected with cRNA encoding Nav channels. Whole-cell currents were collected under controlled temperature (18-22ºC) using the two-electrode voltage-clamp technique (GeneClamp 500 amplifier; Axon Instruments, Foster City, CA, USA), driven by a pClamp data acquisition system (Molecular Devices, Sunnyvale, CA, USA).

The final concentration (10 μM) of each alkaloid was added to the camera containing the oocyte in a bath solution ND-96; in case of action on the channel or receptor tested, a sufficient number of measurements were obtained using additional and serial concentrations to attain statistica l significance. Voltage and current electrodes were filled with 3M KCl, and the resistances of both electrodes were maintained as low as possible (0.5 to 1.5 MΩ). The alkaloids-induced shift in the current-voltage relationship was obtained by averaging the peak amplitude of at least three control responses performed in triplicate. Whole-cell current traces were evoked from a holding potential of -90 mV. For NaV 1.3 and NaV 1.6 channels, whole-cell current traces were A c c e p t e d m a n u s c r i p t evoked every 5s for 100ms to 0mV, which corresponds to the maximal activation of the NaVsubtype in control conditions. The elicited currents were sampled at 20 kHz and filtered at 2 kHz using a four-pole, low-pass Bessel filter. KV1.1, KV1.2, Kv1.4 and KV4.2 currents were evoked by 500ms depolarizations to 0 mV followed by a 500ms pulse to -50 mV, from a holding potential of -90 mV. hERG or KV11.1 peak and tail currents were generated by a 2.5s prepulse from -90 mV, depolarized to +40 mV followed by a 2.5s pulse to -120 mV and then held at -90 mV for 7.4 seconds. For measuring nAChR currents, current traces were evoked from an initial potential of -90 mV to -70 mV for 400 seconds then returned to -90 mV. The receptor was activated by the addition of 300 µM of ACh.

Statistical analysis

Electrophysiological data were analyzed using Origin 7.5 software (Originlab, Northampton, MA, USA) and presented as the result of at least 3 independent experiments (n ≥ 3), the effective dose 50% was calculated through the nonlinear regression with the normalized response. Neurochemical assays were evaluated by one-way ANOVA followed Newman-Keuls using GraphPad Prism 7 (GraphPad Software), considering p <0.05 as statistically significant.

Results

Neurochemical assays: 3 H-Glutamate and 3 H-GABA uptake and binding

We investigated whether the alkaloids (+)-erythravine and (+)-11-α-hydroxyerythravine would act by modifying H 3 -glutamate and H 3 -GABA uptake and/or binding. neither on the uptake nor on the binding of the referred neurotransmitters according to our experimental conditions. The alkaloid concentrations ranged from 0.001 to 10 µg/mL and were broad enough to be pharmacologically relevant and representative.

Electrophysiology

Next, we checked if the alkaloids would act by modulating of sodium and potassium channels and/or nicotinic receptors. Data showed that both alkaloids, (+)-erythravine and (+)-11-α-hydroxy-erythravine, at a final concentration of 10 µM, did not promote signific a nt changes at the activation and/or inactivation of the potassium channels (isoforms KV1.1, KV1.2, Analysis of the potential effects of erythrinian alkaloids at a final concentration of 10 µM in sodium and potassium channels using oocytes of X. laevis performed in triplicate. The data were analyzed using the Originlab and they are presented means ± SEM. However, these alkaloids seem to selectively act on specific isoforms of the nicotinic receptors (Figure 3A and3B). (+)-erythravine and (+)-11-α-hydroxy-erythravine (both at 10 µM) did not promote any significant modulation on ACh-evoked current amplitude mediated by α1β1δɣ and α1β1δε nAChRs. However, differences in the magnitude of action were observed for (+)-erythravine and (+)-11-α-hydroxy-erythravine when tested for others nAChR types.

Indeed, while the (+)-11-α-hydroxy-erythravine (10 µM) presented a lesser and very discrete action for the nAChRs α4β4 (23.52%), α4β2 (10.42%) and α7 (3.56%), on the other hand, (+)erythravine (10 µM) elicited a very significant inhibition of the electric currents mediated by α7 (87.78%), α4β2 (83.65%) and α4β4 (55.73%) nAChR isoforms ( Effects of alkaloids (+)-erythravine and (+)-11-α-hydroxy-erythravine isolated of the plant E. mulungu in nicotinic acetylcholine receptors using oocytes of X. laevis performed in triplica te. The data were analyzed using the Originlab and they are presented means ± SEM.

Considering the higher blocking activity performance of (+)-erythravine for the nicotinic isoforms α7, α4β2, α4β4, the assayed concentrations were extended to 1 and 0.1 µM, allowing to estimate the EC50. The EC50 values (please see Table 3) confirm the high affinity of the alkaloid (+)-erythravine for the α4β2, α4β4 and α7 nAChR isoforms, especially for the case of α4β2 and α7 receptors in which the EC50 values were lower (1.04 and 2.84 µM, respective ly).

Table 3 Nicotinic Receptor Concentration for 50% inhibition α4β2

1.04 µM α4β4 9.98 µM α7

2.84 µM Concentration of alkaloid (+)-erythravine for 50% inhibition in different nicotinic acetylcholine receptors considering the higher blocking activity performance electrophysiologic a l experiments previously demonstrated. The data were analyzed using the Originlab.

Discussion

The aim of the present work was to investigate the possible mechanisms of action of the alkaloids (+)-erythravine and (+)-11-α-hydroxy-erythravine. Our results bring forward substantial evidence regarding the mechanisms by which these alkaloids promote their pharmacological effects, especially in the case of (+)-erythravine. In this regard, it is important to notice that erythrinian alkaloids, including (+)-erythravine and (+)-11-α-hydroxyerythravine, have been reported to have anticonvulsant and anxiolytic-like effects in differe nt sets of animal models [START_REF] Faggion | Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11α-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae)[END_REF][START_REF] Gelfuso | Neuroprotective effects and improvement of learning and memory elicited by erythravine and 11α-hydroxy-erythravine against the pilocarpine model of epilepsy[END_REF][START_REF] Rosa | Erysothr ine, an alkaloid extracted from flowers of Erythrina mulungu Mart. ex Benth: Evaluating its anticonvulsant and anxiolytic potential[END_REF].

Epilepsy and anxiety are commonly observed as comorbid disorders [START_REF] Keezer | Comorbidities of epilepsy: Current concepts and future perspectives[END_REF].

Moreover, both diseases shares some neurobiological basis, in particular related to the imbalance between excitatory and inhibitory neurotransmitters [START_REF] Meldrum | Glutamate and Glutamine in the Brain Glutamate as a Neurotransmitter in the Brain: Review of Physiology and Pathology[END_REF]Zarcone and Corbetta, 2017). This is specially reinforced by the well-evidenced use of GABA and/or glutamate-modulating agents for pharmacological treatment of different types of anxiety and epilepsy disorders [START_REF] Averill | Glutamate dysregulation and glutamatergic therapeutics for PTSD: Evidence from human studies[END_REF][START_REF] Mula | Using anxiolytics in epilepsy: Neurobiological, neuropharmacological and clinical aspects[END_REF]. Despite the importance of GABA and glutamate in epilepsy and anxiety, and the fact that the alkaloids inhibit convulsive seizures triggered by GABAergic antagonists and glutamatergic agonists in the acute seizure models A c c e p t e d m a n u s c r i p t [START_REF] Faggion | Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11α-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae)[END_REF], (+)-erythravine and (+)-11-α-hydroxy-erythravine show no signific a nt modifications on uptake and binding of these neurotransmitters.

Although alterations on GABA and glutamate neurotransmitter synaptic events like in case of binding and uptake have been clearly related to the physiopathology and treatment of epilepsy and anxiety, dysfunctions in other structures or synaptic events evolving even GABA and glutamate indirectly or other neurotransmitters also play an important role in the development of these diseases, including the sodium and potassium ionic channels and the nicotinic cholinergic receptors [START_REF] Hamid | Anxiety symptoms in epilepsy: Salient issues for future research[END_REF][START_REF] Mula | Using anxiolytics in epilepsy: Neurobiological, neuropharmacological and clinical aspects[END_REF]Zarrindast and Khakpai, 2019).

The concentration of 10 µM of each alkaloid on voltage-clamp tests is considered relative ly high at the pharmacological point of view and is used in the context of eliminatory prospective assays.

Sodium channels are responsible for the depolarization of the membrane and conduction of action potentials. It is commonly known that the inhibition of sodium channels can explain the antiepileptic and/or anxiolytic action of several drugs, such as carbamazepine, lamotrigine and phenytoin [START_REF] Catterall | Voltage-gated sodium channels at 60: Structure, function and A c c e p t e d m a n u s c r i p t pathophysiology[END_REF]. Among the isoforms of sodium channels related to pharmacological scope/interest of this work, are Nav1.1, Nav1.2, Nav1.3, Nav 1.6, Navβ [START_REF] Dussaule | Psychiatric effects of antiepileptic drugs in adults[END_REF][START_REF] Musto | Recent advances in treatment of epilepsy-rela ted sodium channelopathies[END_REF].

Different from sodium channels, an increase in the conductance of K + ions results in neuronal hyperpolarization, exerting an inhibitory effect on the neuronal pathway functio n [START_REF] Barrese | Neuronal potassium channel openers in the management of epilepsy: Role and potential of retigabine[END_REF]. Thus, studies with isoforms of potassium channels are of great value, especially those in which mutations in the genes responsible for their expression are already described in the literature being related to epilepsy, for example, Kv 1.1 (gene KCNA1/ gene

LGI1), Kv 1.2 (KCNA2) e Kv 4.2 (gene KCND2) [START_REF] Barrese | Neuronal potassium channel openers in the management of epilepsy: Role and potential of retigabine[END_REF][START_REF] Errington | Voltage Gated ion Channels: Targets for Anticonvulsant Drugs[END_REF][START_REF] Meldrum | Molecular Targets for Antiepileptic Drug Development[END_REF]. However and despite the absence of some important sodium channels in our representative experimental panel (such as Nav1.1 and Navβ), (+)-erythravine A c c e p t e d m a n u s c r i p t and (+)-11-α-hydroxy-erythravine did not work through a negative or positive modulation of sodium and/or potassium channels, respectively, at least not for the isoforms and alkaloids at the concentrations here studied.

Finally, we investigated the participation of nicotinic receptors on the mode of action of both alkaloids. These receptors are functionally linked to different ion channels and GABAergic and glutamatergic neurotransmission pathways, thus being importantly related to the pathophysiology and treatment of different neurological disorders, including epilepsy and anxiety [START_REF] Mula | Using anxiolytics in epilepsy: Neurobiological, neuropharmacological and clinical aspects[END_REF]Zarrindast and Khakpai, 2019). Indeed, the nicotinic cholinergic receptors can modulate the release of neurotransmitters, being activated via Ca 2+ influx and are described as important structures in several physiological processes such as learning and memory and motor control [START_REF] Dineley | Nicotinic ACh receptors as therapeutic targets in CNS disorders[END_REF]Zarrindast and Khakpai, 2019). The modulation of α4β2 is suggested to play an important role in genetic epileptic syndromes as well as in the autosomal dominant nocturnal frontal lobe epilepsy [START_REF] Garibotto | Nicotinic receptor abnormalities as a biomarker in idiopathic A c c e p t e d m a n u s c r i p t generalized epilepsy[END_REF]. On the other hand, the α7 isoform is a major nicotinic cholinergic receptor that acts presynaptically by modulating the release of neurotransmitters, such as glutamate and GABA [START_REF] Dineley | Nicotinic ACh receptors as therapeutic targets in CNS disorders[END_REF]Zarrindast and Khakpai, 2019). It is demonstrated that the increase in the glutamate clearance elicited by nicotine action on α7 has a neuroprotective effect against neuroinflammation and neurodegenerative disorders [START_REF] Dineley | Nicotinic ACh receptors as therapeutic targets in CNS disorders[END_REF].

The most important result presented on this work is the selective inhibitory action of (+)-erythravine on the nicotinic receptors α4β4 and especially on α4β2 and α7 isoforms to the detriment of others evaluated in this study. The action of (+)-erythravine in nicotinic receptors, can at least partially, explain its anxiolytic and anticonvulsant properties as previous ly demonstrated [START_REF] Faggion | Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11α-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae)[END_REF][START_REF] Rosa | Erysothr ine, an alkaloid extracted from flowers of Erythrina mulungu Mart. ex Benth: Evaluating its anticonvulsant and anxiolytic potential[END_REF]Setti-Perdigão et al., 2013). The results from this study are added up and are in accordance to Setti-Perdigão and colleagues (2013) data, which showed an important selectivity of (+)-erythravine for nicotinic receptors A c c e p t e d m a n u s c r i p t specifically expressed in the CNS (α4β2 e α7) (Setti-Perdigão et al., 2013) to the detriment of others studied isoforms expressed in skeletal neuromuscular junctions (α1β1δε), ganglio nic nicotinic receptors (α7, α3β4) [START_REF] Fagerlund | Propofol and AZD3043 inhibit adult muscle and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes[END_REF][START_REF] Lebbe | Structure-function elucidatio n of a new α-Conotoxin, Lo1a, from conus longurionis[END_REF]. This reinforces the probable participation of the inhibitory mechanism of specific nicotinic receptors (particula r ly α4β2 e α7) in the anxiolytic and anticonvulsant actions of (+)-erythravine. However, it is important to note that (+)-11-α-hydroxy-erythravine showed an almost null or very discrete effect on the same type of receptors. Thus, since (+)-11-α-hydroxy-erythravine and erythravine modulated α4β2, α4β2 and α7 nicotinic receptors at different extents and they have a very similar pharmacological performance as anxiolytic and anticonvulsant as previously demonstrated [START_REF] Faggion | Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11α-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae)[END_REF][START_REF] Flausino | Anxiolytic effects of erythrinian alkaloids from Erythrina mulungu[END_REF], it is possible to assume that other neuronal and synaptic structures or neurotransmission pathways are involved on the mode of action of the erythrinian alkaloids besides the nicotinic receptors. Moreover, it cannot be excluded that small differences between the chemical structure of these alkaloids can affect the selectivity and affinity of target-ligand interactions on nicotinic receptors composed by the α4, α7, β2, β3 and β4 subunits. Thus, the erythrinian alkaloids represent a phytochemical class for which the study of structure-activity relationship along their chemical diversity deserves a strong pharmaceutic a l attention, important for the rational development of new drugs with action on the CNS, including those useful against anxiety and epilepsy.

Conclusion

In this work we have described the mechanism of action of alkaloids, unlike most of the commercially available anxiolytic and anticonvulsant drugs, does not involve modulation of the synaptic events related to GABA and glutamate binding or uptake, neither involves sodium or potassium channels. Particularly in the case of (+)-erythravine, an inhibitory modulation on

Figures

  Figures 1A and 1B present data obtained from H 3 -GABA and H 3 -Glutamate synaptosoma l

Figure 1

 1 Figure 1 Analysis of the potential effects of erythrinian alkaloids on binding and reuptake of [ 3 H]GABA and L-[ 3 H]Glutamate

Figure 2

 2 Figure 2 Current-Voltage relation for the potassium and sodium channels

Figure

  Figure 3A Current-Voltage relation for the nAChR

Figure

  Figure 3B Current-Voltage relation for the nAChR A c c e p t e d m a n u s c r i p t

Table 1

 1

	Isoform	Control	(+)-erythravine 10µM (+)-α-OH-erythravine 10µM
	KV1.1	0.598 ± 0.076	0.599 ± 0.099	0.659 ± 0.085
	KV1.2	0.073 ± 0.067	0.093 ± 0.068	0.066 ± 0.066
	KV 1.4	1.966 ± 1.908	1.989 ± 1.904	2.001 ± 1.895
	KV 4.2	0.413 ± 0.030	0.417 ± 0.067	0.415 ± 0.104
	KV11.1	-1.063 ± 0.247	-0.981 ± 0.150	-1.038 ± 0.242
	NaV1.3	-0.585 ± 0.037	-0.575 ± 0.033	-0.535 ± 0.035
	NaV1.6	-0.546 ± 0.085	-0.546 ± 0.075	-0.547 ± 0.063

Table 2

 2

).

Table 2 Isoform % inhibition (+)- erythravine 10µM % inhibition (+)-α-OH-

 2

	erythravine 10µM

nicotinic receptors with a selective action for the isoforms of α4β4, α4β2 and α7 was observed, which may explain at least partially its mode of action.

Funding

This work was supported by FAPESP (São Paulo Research Foundation, scholarship process nr: 04/14151-1; E.A.G.), CAPES (scholarship process nr: 6243/15-0; E.A.G.) and CNPq (309874/2017-3). Jan Tytgat was funded by GOC2319N and GOA4919N (F.W.O. Vlaanderen) and CELSA/17/047 (BOF, 447 KU Leuven). Steve Peigneur is a Postdoctoral fellow supported by KU Leuven funding (PDM/19/164).

Setti-Perdigão, P., Serrano, M.A.R., Flausino, O.A., Bolzani, V.S., Guimarães, M.Z.P., Castro, N.G., 2013. Erythrina