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Nantenine alkaloid presents anticonvulsant effect on two classical animal models.

Summary

The present study investigated the anticonvulsant and convulsant profiles of nantenine, an aporphine alkaloid found in several vegetal species. At lower doses (20-50 mg/kg, i.p) the alkaloid proved to be effective in inhibiting pentylenotetrazol- (PTZ 100 mg/kg, s.c) and maximal electroshock-induced seizures (80 mA, 50 pulses/s, 0.2 s), suggesting its potential as an anticonvulsant drug. However, at higher doses ([greater than or equal to] 75 mg/kg, i.p) a convulsant activity was observed. Comparing the present in vivo nantenine effects on seizures with previous in vitro biphasic action on [Na.sup.+], [K.sup.+]-ATPase activity, the convulsant effect appears to be related to inhibition of these phosphatase at high doses whereas anticonvulsant effect, observed at low doses, seems attributable to its stimulation and the resultant decrease of [Ca.sup.2+]-influx into the cell.

Key words: Nantenine, Aporphine, Alkaloid, Anticonvulsant activity, Convulsant activity

* Introduction

The aporphine alkaloids are derivable from the benzyltetrahydroisoquinolines by the abstraction of two hydrogens in a such a manner that the two benzene nuclei form part of a 9,10-dihydrophenanthrene. The aporphine occurs most abundantly in the Papaveraceae, but they are also widespread in Anonaceae, Lauraceae and Moniminaceae families (Manske, 1954).

Derivatives of the aporphine molecule comprise pharmacologically active compounds such as apomorphine, an agonist of dopamine receptors, and alkaloids with antidopaminergic actions such as the bulbocapnine, boldine and glaucine (Zetler, 1988).

Besides their recognized dopaminergic effects, the aporphines still presents antagonistic activities on [alpha]1-adrenergic (Ivorra, 1993a; Baldessarini et al., 1994) and 5-H[T.sub.2]-serotonergic receptors (Alzueta et al., 1992; Catret et al., 1992), and blocking activity on voltage-dependent channels of calcium (Anselmi et al., 1991; Ivorra et al., 1992, 1993a, b). Other compounds structurally related to the aporphines, as the benzyltetrahydroisoquinolines (cularine, isocrasifoline and laudanosine) and the bisbenzyltetrahydroisoquinolines (antioquine, tetrandine) are also described as inhibitors of the C[a.sup.+2] influx through voltage-operated calcium channels (Ivorra et al., 1992, 1993a). The blocking activity of calcium channels increases furthermore the therapeutic potentiality of the aporphine alkaloid group.

Nantenine is an aporphine alkaloid found in several vegetal species. It was first isolated by Takase and Ohashi (1926) from fruit of Nandina domestica Thunberg, which was used to treat asthma, whooping cough, pharynx tumor and uterine bleeding in Japan for many years. Pharmacological studies showed that it shares some of the activities of the other aporphines, as well as of calcium channels blocking agents. In vitro assays demonstrated that the alkaloid, in small doses, antagonizes the contractions induced by [alpha]1-adrenergic an 5H[T.sub.2]-serotonergic agonists (Shoji et al., 1984; Alzueta et al., 1992) and that, as it happens with the calcium channel blockers, causes bradicardia and negative inotropism (Takase and Ohashi, 1926).

As regards nantenine effects on the central nervous system, contrasting actions according to the dose have been reported. Nantenine exerts a sedative effect at low doses whereas induces ataxia and convulsions at high doses (Takase and Ohashi, 1926).

Considering the anticonvulsant potential of calcium channel blockers (Meyer et al., 1986; Czuczwar et al., 1990a, b; Kamal et al., 1990) and the importance of natural products as sources of new drugs (Phillipson, 1979), the present experiments were designed to investigate the effect of nantenine on two classical models for anticonvulsant activity. The convulsant activity previously observed at high doses was also investigated.

* Materials and Methods

Animals

Male albino mice weighing 30-35 g were used. They were housed under controlled conditions of light (7:00-19:00 h), temperature (22 [+ or -] 2 [degrees]C) and humidity (60%) with food and water ad libitum. Animals used in this study were maintained in accordance with the guidelines of the Committee on Care an Use of Laboratory Animals Resources, National Research Council, USA.

Drugs and Chemical

Sodium chloride (Merck S.A. Industrias Quimicas), valproic acid (Abbot Laboratorios do Brasil), baclofen (Biogalenica Quimica e Farmaceutica Ltda--CibaGeigy), diazepam (Roche Produtos Quimicos e Farmaceuticos), phenobarbital (Rhodia Produtos Quimicos e Farmaceuticos), pentylenetetrazol (Sigma Chemical Co., USA) and diphenylhydantoin (Sarsa Laboratorio Silva Araujo-Roussel) were used. Nantenine was kindly provided by Professors Otto R. Gottlieb and Massayoshi Yoshida, Instituto de Quimica da Universidade de Sao Paulo, Brazil.

Nantenine dilution was made in a HCl 0.01N solution and the pH was adjusted to 5.0-5.5 with NaOH 0.1N.

Anticonvulsant activity

All tests were carried out in a quiet room between 9:00 and 17:00 h. Thirty minutes before the tests, groups of 10 mice were treated with nantenine 10, 25, 40, 50 mg/kg, body w. (i.p), diphenylhydantoin 25 mg/kg, body w. (i.p.) or saline (NaCl 0.9%, i.p., 0.1 ml/10 g, v/w). For induction of maximal electroshock seizures (MES), a 80 Hz variable current (mA), 50 pulses/s frequency and 0.2 s duration (previously determined as 99.9% effective), was applied auricularly. Flexion time of front limbs, extension time of hind limbs and the number of deaths were registered and used to calculate the ratio extensor:flexor time and mortality rate, respectively (Swinyard, 1972). The effect of nantenine 10, 25, 40, 50 mg/kg body w., (i.p) or saline (NaCl 0.9%, i.p., 0.1 ml/10 g, v/w) on chemically induced seizures was investigated by injecting pentylenetetrazol (100 mg/kg, body w., s.c) in pretreated mice (Chen et al., 1954; Swinyard and Kupferberg, 1985). The animals were placed, individually, in glass beakers and their behavioral responses were recorded during thirty minutes. Clonus (at least 3s of continuous limb movement), tonus (complete hind limb extension) and mortality were recorded in terms of seizure percentage and latency.

Convulsant activity

Groups of 10 mice were pretreated with valproic acid 375 mg/kg body w. (i.p), diazepam 2.5 mg/kg body w. (i.p) phenobarbital 30 mg/kg body w. (i.p), baclofen 20 mg/kg (i.p) or saline (NaCl 0.9%, i.p, 0.1ml/10g, v/w) thirty minutes before receiving nantenine 105 mg/kg body w. (i.p) (a dose previously determined as 100% convulsant). The animals were placed, individually, in glass beakers and their behavioral responses were recorded during thirty minutes. Clonus (at least 3s of continuous limb movement), tonus (complete hind limb extension) and mortality were recorded in terms of seizure percentage and latency.

Statistical Analysis

Extensor:flexor ratio and latency to convulsive episode results were submitted to Kruskal-Wallis non-parametric variance analysis followed by Mann-Whitney U test. Fisher's exact test was performed to determine significance of other anticonvulsant and convulsant properties results. Significance was set at p < 0.05.

* Results

Anticonvulsant activity

Electroshock Maximal Seizures

Except for the dose of 10 mg/kg, body w., nantenine (20, 40 and 50 mg/kg, body w.) pretreatment significantly reduced extensor:flexor ratio and mortality in animals submitted to electroconvulsive shock (Fig. 1). At higher doses, 40 and 50 mg/kg, the percentage of death in tonic was reduced to zero. On these experimental conditions, diphenylhydantoin 25 mg/kg, body w. totally inhibited the electroshock-induced seizure (data not shown).

[FIGURE 1 OMITTED]

Pentylenetetrazol induced seizures

None of the tested doses (10, 20, 40 and 50 mg/kg, body w.) successfully inhibited the pentylenetetrazol-induced seizures. However, an inhibition of 30, 60 and 90% on tonic phase occurrence was observed with nantenine 20, 40 and 50 mg/kg, respectively. A significant reduction in the percentage of death in tonic (60, 90 and 100%, respectively) was also observed. (Fig. 2).

[FIGURE 2 OMITTED]

Convulsant activity

Nantenine showed a dose-dependent convulsant activity at doses ranging from 75 to 105 mg/kg. At the highest dose seizure in 100% of treated animals (latency mean time = 5.4 [+ or -] 0.5 min) was observed, followed by death on 30% of them (Fig. 3).

[FIGURE 3 OMITTED]

The previous treatment with phenobarbital 30 mg/kg and diazepam 2.5 mg/kg totally inhibited the nantenine-induced seizure. For both drugs, the inhibition was followed by 10 or more hours of sleep. It is important to emphasize that the doses of phenobarbital and diazepam used were not hypnotic by itself. After valproic acid 375 mg/kg, seizure was observed in 20% of treated animals, half of which (10%) died during the period of observation. Pretreatment with baclofen 20 mg/kg, inhibited the occurrence of seizure in 60% of animals followed by a significant increase in latency for convulsive episodes (16.75 [ + or -] 4.51 minutes) and a death percentage of 10%.

Diphenylhydantoin 25 mg/kg, (100% anticonvulsant on electroshock-induced seizures) was ineffective on inhibiting nantenine-induced seizures. On the contrary, a significant decrease in the latency for convulsive episodes (3.10 [+ or -] 0.23 minutes) was observed as well as an increase from 30 to 70 in the mortality rate (Fig. 4).

[FIGURE 4 OMITTED]

* Discussion

Nantenine proved to be effective in inhibiting tonic component of pentylenetetrazol-induced and maximal electroshock seizures. On the other hand, at higher doses (75, 87.5, 100 and 105 mg/kg, body w.) nantenine produced convulsive episodes, an effect that should be compared to that observed with diphenylhydantoin. Diphenylhydantoin, which was expected to inhibit nantenine-induced seizures, on the contrary, significantly reduced seizure latency and increased the mortality of animals from 30% to 70%, suggesting a synergism between the two drugs. Aside from the apparent loss of benefit when both drugs were jointly administered, nantenine and diphenylhydantoin had very similar profiles as anticonvulsant agents. At lower doses, diphenylhydantoin was effective in controlling convulsions, while at higher doses induces a toxic state characterized by irritability, ataxia and opisthotonic convulsions (Gruber et al., 1940). Much of the anticonvulsant, excitatory and toxic effects of phenytoin can be explained by its inhibitory activity on [Na.sup.+] influx, [Ca.sup.2+] uptake and action on [Na.sup.+]-[K.sup.+] active transport across excitatory tissue or epithelial cells membranes. In excitatory tissue, the greatest effect was apparently the reduction of passive [Na.sup.+] influx and/or a reduction of the [Na.sup.+] currents through voltage-dependent channels, with a consequent reduction in neuronal activity. Phenytoin may also influence the transport of [Na.sup.+] and [K.sup.+] through synaptic membranes, mainly when it is damaged, as occurs in the epileptogenic center (focus) (Escueta et al., 1971, 1974). In this case, phenytoin appears to stimulate the [Na.sup.+]-[K.sup.+] active transport, a process that similar to the reduction of [Na.sup.+] influx, increases the gradient of [Na.sup.+] across the cellular membrane, diminishes the [Ca.sup.2+] influx (Woodbury, 1980), alters the membrane potential and reduces the release of neurotransmitters (Albers et al., 1994). Its efficacy in reducing the synaptosomal release of excitatory neurotransmitters would partially explain phenytoin's anticonvulsant effects. On the other side, phenytoin may also block the release in inhibitory synapses, in such a manner that the final result will be the sum of its effects on these systems. As such, the final effect in its interaction with other drugs will also depend on the sum of effects. Similar to what occurred in the interaction with nantenine, phenytoin heightens the actions of convulsant agents, such as bicuculline and picrotoxin, which possess mechanisms involving the reduction of the inhibitory gabaergic transmission (Stone and Javid, 1982).

Recent data indicates nantenine has a dose-dependent inverse effect on the [Na.sup.+], [K.sup.+]-ATPase of synaptosomal membranes (Ribeiro and Rodriguez de Lores Arnaiz, 2000, 2001). At low concentrations, the alkaloid stimulates, whereas at high concentrations it inhibits these phosphatase. Considering the biphasic action of nantenine in seizure, the inhibition of [Na.sup.+], [K.sup.+]-ATPase could explain the convulsive effect brought on by high doses of nantenine, as such inhibition seemingly acts in countless neurotoxic processes directly or indirectly involved in convulsive activity (Lees, 1991). Conversely, the alkaloid anticonvulsant effect could be, at least in part, attributable to the stimulation of these phosphatase activity and the resultant decrease of [Ca.sup.2+]-influx into the cell (as described above to phenytoin).

Moreover, in light of the potential of aporphines and structurally related alkaloids as calcium channel blockers, one could speculate that, at lower doses, a direct inhibitory effect of nantenine on the [Ca.sup.2+] influx could be also related to the anticonvulsant effects, as well as heightening the hypnotic-sedative effects of phenobarbital and diazepam. Classic calcium channel blockers such as verapamil and nifedipine act as anticonvulsants (Czuczwar et al., 1990a, b; Kamal et al., 1990; Meyer et al., 1986) and heighten the anesthetic effects of barbiturates and benzodiazepines (Dolin and Little, 1986; Dolin et al., 1991. In addition, phenobarbital (Blaustein and Ector, 1975) and chlordiazepoxide (Leslie et al., 1980) alone also are effective in reducing the entry of voltage-dependent calcium in synaptosomes. At higher doses, the blockage of calcium influx in inhibitory synapses could contribute to the appearance of a toxic convulsive situation, which is aggravated by the interaction with diphenylhydantoin. Nevertheless, the role of changes in other regulatory mechanisms of neuronal excitability in the manifestation of convulsions should not be discarded, considering that anticonvulsants such as baclofen (which increases [K.sup.+] conductance by activation of GABAB receptors) and valproic acid (which prolongs the inactivation of the voltage-dependent [Na.sup.+] channel, increases GABA transmission through mechanisms that involve stimulation of GAD and/or inhibition of GABA-T activity) (Meldrum, 1994) reduced the percentages of convulsion and death associated with nantenine.

Acknowledgements

This research was supported by fellowship from Conselho Nacional de Pesquisa (CNPq). The authors would like to thank Mrs Teotila R. R. Amaral for capable technical assistance.

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* Address

R. A. Ribeiro, Universidade Federal de Sao Paulo, Escola Paulista de Medicina, Departamento de Farmacologia, Rua Botucatu 862, Edificio Jose Leal Prado, 04023-970, Sao Paulo, SP, Brazil.

Fax: ++55-11-3064-0179; e-mail:rosanari@terra.com.br

R. A. Ribeiro (1) and J. R. Leite (2)

(1) Departamento de Farmacologia

(2) Departamento de Psicobiologia, Universidade Federal de Sao Paulo, Escola Paulista de Medicina, Rua Botucatu 862, Edificio Jose Leal Prado, Sao Paulo, Brazil
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Author:Riberio, R.A.; Leite, J.R.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:3BRAZ
Date:Jul 1, 2003
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