Demetrius Antônio Machado de Araújo

Study of anticonvulsant effect of citronellol, a monoterpene alcohol, in rodents

Citronellol is one monoterpene alcohol, which is present in the essential oils of various aromatic plant species. This study evaluated the neuroprotective activity of citronellol on pentylenetetrazol- and picrotoxin-induced convulsions and maximal electroshock-induced seizures in mice. Administration of citronellol significantly reduced the number of animals of convulsion induced by pentylenetetrazol and eliminated the extensor reflex of maximal electroshock-induced seizures test in about 80% of the experimental animals. In addition, administration of citronellol showed protection in the pentylenetetrazol and picrotoxin tests by increasing the latency of clonic seizures. We also investigated the effect of citronellol in the rat isolated nerve using the single sucrose-gap technique. We showed that the amplitude of the compound action potential decreased more than 90% when the monoterpene was incubated for 30 min at 6.4 mM and we did not verify any effect on the repolarization of the compound action potential. Taken together, our results demonstrated an anticonvulsant activity of the citronellol that could be, at least in part, explained by the diminution of the action potential amplitude.

Electrophysiological characterization and molecular identification of the Phoneutria nigriventer peptide toxin PnTx2-61

A cDNA with 403 nucleotides encoding the precursor of the toxin PnTx2‐6 was cloned and sequenced. Subsequent analysis revealed that the precursor begins with a signal peptide and a glutamate‐rich propeptide. The succeeding peptide confirmed the reported sequence of PnTx2‐6. The purified toxin exerted complex effects on Na+ current of frog skeletal muscle. There was a marked decrease of the inactivation kinetics, and a shift to hyperpolarizing potentials of both the Na+ conductance and the steady‐state inactivation voltage dependences, along with a reduction of the current amplitude. The concentration dependence of the modified current suggests a K D of 0.8 μM for the toxin–channel complex.

Evaluation of the sesquiterpene (-)-α-bisabolol as a novel peripheral nervous blocker.

Essential oils are natural, complex and multi-component systems composed mainly of terpenes in addition to some other non-terpenes compounds that are widely used to prevent and treat human diseases. (-)-alpha-Bisabolol is an unsaturated monocyclic sesquiterpene alcohol found as the major constituent of many essential oils, like the German chamomile (Chamomilla recutita (L.) Rauschert), a plant reported to reduce the perception of acute pain and used for centuries for their medicinal properties. Recently, our group demonstrated the antinociceptive-like effect promoted by other terpenes could be associated with the decreased peripheral nerve excitability. Therefore, this study investigated the pharmacological activities of (-)-alpha-bisabolol on mice peripheral nervous system observing the changes on the compound action potential (CAP) characteristics. Using modified single sucrose-gap method in mice sciatic nerves, we acquired CAP recordings in the absence and presence of (-)-alpha-bisabolol (0.5, 1, 5 and 10mM). We observed that this sesquiterpene was able to reduce the neuronal excitability in a concentration-dependent manner, although, such effects were not reversed when the nerve was submitted to wash out. Assessing CAP parameters of depolarization and repolarization, we noticed similarities between (-)-alpha-bisabolol and lidocaine but not with 4-aminopyridine that are considered good blockers for sodium and potassium voltage-gated channels, respectively. Additionally, we also characterized the non-use-dependent profile of (-)-alpha-bisabolol action, in contrast to lidocaine. Thus, we suggested that decreased nervous excitability elicited by (-)-alpha-bisabolol might be caused by an irreversible blockade of voltage-dependent sodium channels.

Anticonvulsant effect of a natural compound α,β-epoxy-carvone and its action on the nerve excitability

The anticonvulsant effect of alpha,beta-epoxy-carvone (EC), a monoterpene monocyclic, was investigated in three animal models. EC at 300 or 400 mg/kg promoted protection of 75% and 87.5%, respectively, against convulsions induced chemically by pentylenetetrazole (PTZ) and it was efficient in prevents the tonic convulsions induced by maximal electroshock (MES) in doses of 200, 300 or 400 mg/kg, resulting in 25%, 25% and 100% of protection, respectively. This monoterpene was also capable to promote an increase of latency for development of convulsions induced by picrotoxin (PIC) at 300 or 400 mg/kg and presented a significant protection against convulsions at doses of 200, 300 or 400 mg/kg, resulting in 12.5%, 12.5% and 100% of protection, respectively. On the other hand, the anticonvulsant effect of EC, was not affected by pretreatment with flumazenil (FLU), a selective antagonist of benzodiazepine site of GABA(A) receptor. Additionally was observed that EC treatment reduced the levels of in vitro lipoperoxidation and decreased (21.2%) the amplitude of compound action potential after 30 min of incubation. The present results clearly indicate the ability of EC to modulate the anticonvulsant and antioxidant effects. However, our data suggests that the action mechanisms are not due a direct activation of the GABA(A) benzodiazepine receptors, but could be associated with the reduction of isolated nerve excitability, possibly involving a voltage-gated Na(+) channels blockade.

Essential oils components as a new path to understand ion channel molecular pharmacology.

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 identification 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 medium-sized 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.

Distinct effects of carvone analogues on the isolated nerve of rats

Carvone (p-mentha-6,8-dien-2-one) is a monoterpene ketone found as the main active component of various essential oils. It is obtained by distillation and occurs naturally as the enantiomers (+)- and (-)-carvone. Our group have shown that the in vivo antinociceptive activity of (-)-carvone is impaired with decreased nerve excitability. To better characterize the neuropharmacology of such a monoterpene, we investigated the profile of several carvone analogues to establish a structure-function relationship related to the compound action potential (CAP) inhibitory effect. We performed ex vivo assays to evaluate the effects of (+)- and (-)-carvone, carvacrol, (-)-carveol, and limonene on CAP characteristics using a modified single sucrose-gap method. Our results demonstrated that (-)-carvone was less potent (IC(50)=10.7+/-0.07 mM) in reducing nerve excitability than its enantiomer, (+)-carvone (IC(50)=8.7+/-0.1mM), although they shared a similar mode of action, since their effects were partially extinguished by nerve washing and also by reduction of depolarization velocity, probably as a result of voltage-gated sodium channel blockades. In a structure-activity relationship study, we demonstrated that hydroxyl groups in the (-)-carveol and carvacrol molecules enhanced the CAP blocking-effect, while the absence of oxygen moiety in (+)-limonene resulted in the effect being almost abolished. Therefore, inhibition of CAP conduction in peripheral nerves by monoterpenes could expand our understanding concerning the pharmacology of such natural bioactive compounds. Moreover, activation or inhibition of nerve excitability with these tested monoterpenes can be achieved by altering their chemical structures, and this can lead to further implications for target-directed drug design.

Antiproliferative effects of lectins from Canavalia ensiformis and Canavalia brasiliensis in human leukemia cell lines

The antiproliferative activity of lectins Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr) were studied using human leukemia MOLT-4 and HL-60 cell lines. It was revealed that both ConA and ConBr were markedly cytotoxic to cells using MTT and NAC assays. The IC(50) values were approximately 3 and 20 μg/mL for ConA and ConBr, respectively, for both MOLT-4 and HL-60 cells. However, in normal human peripheral blood lymphocytes, the lectins were not cytotoxic, even when tested at concentrations as high as 200 μg/ml. Using comet assay, the lectins produced a rate of DNA damage exceeding 80% in MOLT-4 and HL-60 cells. Fluorescence analysis revealed the morphology characteristic of apoptosis, with low concentrations of apoptotic bodies and fragmented DNA (5 μg/ml). Flow cytometric analysis demonstrated an accumulation of cells in the sub-G1 cell cycle that is characteristic of DNA fragmentation, and a decrease in membrane integrity at high concentrations. Lastly, we evaluated the alterations in mitochondrial potential that reduced after treatment with lectins. Our results indicate that ConA and ConBr inhibited cell proliferation selectively in tumor cells and that apoptosis was the main death mechanism. Therefore, lectins can be considered a class of molecules with a high antitumor activity potential.

Rosewood oil induces sedation and inhibits compound action potential in rodents

AIM OF THE STUDY Aniba rosaeodora is an aromatic plant which has been used in Brazil folk medicine due to its sedative effect. Therefore, the purpose of the present study was to evaluate the sedative effect of linalool-rich rosewood oil in mice. In addition we sought to investigate the linalool-rich oil effects on the isolated nerve using the single sucrose-gap technique. MATERIALS AND METHODS Sedative effect was determined by measuring the potentiation of the pentobarbital-induced sleeping time. The compound action potential amplitude was evaluated as a way to detect changes in excitability of the isolated nerve. RESULTS The results showed that administration of rosewood oil at the doses of 200 and 300 mg/kg significantly decreased latency and increased the duration of sleeping time. On the other hand, the dose of 100 mg/kg potentiated significantly the pentobarbital action decreasing pentobarbital latency time and increasing pentobarbital sleeping time. In addition, the effect of linalool-rich rosewood oil on the isolated nerve of the rat was also investigated through the single sucrose-gap technique. The amplitude of the action potential decreased almost 100% when it was incubated for 30 min at 100 microg/ml. CONCLUSIONS From this study, it is suggested a sedative effect of linalool-rich rosewood oil that could, at least in part, be explained by the reduction in action potential amplitude that provokes a decrease in neuronal excitability.

Rotundifolone-Induced Relaxation is Mediated by BKCa Channel Activation and Cav Channel Inactivation

Rotundifolone is the major constituent of the essential oil of Mentha x villosa Hudson. In preliminary studies, rotundifolone induced significant hypotensive, bradycardic and vasorelaxant effects in rats. Thus, to gain more insight into the pharmacology of rotundifolone, the aim of this study was to characterize the molecular mechanism of action involved in relaxation produced by rotundifolone. The relaxant effect was investigated in rat superior mesenteric arteries by using isometric tension measurements and whole-cell patch-clamp techniques. Rotundifolone relaxed phenylephrine-induced contractions in a concentration-dependent manner. Pre-treatment with KCl (20 mM), charybdotoxin (10(-7) M) or tetraethylammonium (TEA 10(-3) or 3 × 10(-3) M) significantly attenuated the relaxation effect induced by rotundifolone. Additionally, whole-cell patch-clamp recordings were made in mesenteric smooth muscle cells and showed that rotundifolone significantly increased K(+) currents, and this effect was abolished by TEA (10(-3)  M), suggesting the participation of BK(Ca) channels. Furthermore, rotundifolone inhibited the vasoconstriction induced by CaCl(2) in depolarizing nominally Ca(2+) -free medium and antagonized the contractions elicited by an L-type Ca(2+) channel agonist, S(-)-Bay K 8644 (2 × 10(-7)  M), indicating that the vasodilatation involved inhibition of Ca(2+) influx through L-type voltage-dependent calcium channels (Ca(v) type-L). Additionally, rotundifolone inhibited L-type Ca(2+) currents (I(Ca) L), affecting the voltage-dependent activation of I(Ca) L and steady-state inactivation. Our findings suggest that rotundifolone induces vasodilatation through two distinct but complementary mechanisms that clearly depend on the concentration range used. Rotundifolone elicits an increase in the current density of BK(Ca) channels and causes a shift in the steady-state inactivation relationship for Ca(v) type-L towards more hyperpolarized membrane potentials.

N-salicyloyltryptamine, a new anticonvulsant drug, acts on voltage-dependent Na+, Ca2+, and K+ ion channels.

The aim of this work was to study the effects of N‐salicyloyltryptamine (STP), a novel anticonvulsant agent, on voltage‐gated ion channels in GH3 cells. In this study, we show that STP at 17 μM inhibited up to 59.2±10.4% of the Ito and 73.1±8.56% of the IKD K+ currents in GH3 cells. Moreover, the inhibitory activity of the drug STP on K+ currents was dose‐dependent (IC50=34.6±8.14 μM for Ito) and partially reversible after washing off. Repeated stimulation at 1 Hz (STP at 17 μM) led to the total disappearance of Ito current, and an enhancement of IKD. In the cell‐attached configuration, application of STP to the bath increased the open probability of large‐conductance Ca2+‐activated K+ channels. STP at 17 μM inhibited the L‐type Ca2+ current by 54.9±7.50% without any significant changes in the voltage dependence. STP at 170 μM inhibited the TTX‐sensitive Na+ current by 22.1±2.41%. At a lower concentration (17 μM), no effect on INa was observed. The pharmacological profile described here might contribute to the neuroprotective effect exerted by this compound in experimental ‘in vivo’ models.