Anticonvulsant, Antidepressant and Anxiolytic Effects of Mallotus Oppositifolius (Geiseler) Müll. Arg. (Euphorbiaceae)

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Mallotus oppositifolius is used in Ghana for CNS disorders but very little scientific evidence exists to support its use. Thus central effects of 70% v/v hydroalcoholic extract of the leaves of Mallotus oppositifolius (MOE) was assessed. Anticonvulsant effects of the extract in acute and chronic seizure models were evaluated. The study also investigated the effect of the extract on animal models of depression and anxiety. In a preliminary screening of the central effects of the extract, oral dose of MOE induced sedation (1000 – 3000 mg kg-1); caused neuromuscular deficits in the rotarod test (300 – 3000 mg kg-1); reduced spontaneous locomotor activity in the activity cage; exhibited anticonvulsant effect (30 – 3000 mg kg-1) and central analgesic effect in the tail immersion test (100 – 3000 mg kg-1). The LD50 was approximately 6000 mg kg-1 in mice. M. oppositifolius (10 - 100 mg kg-1, p.o.) exhibited anticonvulsant effect in the picrotoxin and strychnine induced seizure tests. The extract significantly delayed onset of myoclonic jerks and clonic convulsions; decreased the frequency and duration of clonic convulsions in these models. In the MES test, the extract caused a significant and dose dependent decrease in the duration of tonic limb extensions. In the pilocarpine induced status epilepticus, MOE delayed the onset of clonic convulsions and decreased the duration of these seizures. Furthermore, the extract protected mice against mortality induced by 4-aminopyridine and delayed the onset of both clonic and tonic convulsions. Flumazenil, a GABAA/benzodiazepine antagonist, reversed the anticonvulsant effect of the extract in the PTZ-induced seizure test suggesting enhancement of GABAA neurotransmission is involved in the anticonvulsant effect of the extract. Isobolographic analysis of the combination of diazepam and extract showed a synergistic effect but the mode of action of this effect may not be dependent on enhancement of GABAA neurotransmission since flumazenil failed to reverse their anticonvulsant effect. Oral doses of MOE (10 - 100 mg kg-1), fluoxetine (3 - 30 mg kg-1) and imipramine (3 - 30 mg kg-1) decreased the frequency of immobility and immobility periods of mice in both the FST and TST when compared to control group, indicating significant antidepressant activity. In the open space swim test, a chronic depression model, MOE demonstrated antidepressant-like effect on the first day of treatment and sustained it throughout the period of drug treatment. MOE decreased immobility time while increasing the distanced travelled by the mice. The depression induced in this model induced significant impairment in spatial learning and memory in the Morris water maze—this was reversed by the extract and fluoxetine but not imipramine. Extract, fluoxetine and imipramine treatments did not have significant effects on weight variation. A day after the 14th day of drug treatment, the antidepressant effect was still significant. A 3-day subcutaneous pretreatment with 200 mg kg-1 para-chlorophenylalanine (pCPA), reversed the antidepressant effect of MOE and fluoxetine but not imipramine, suggesting that serotoninergic enhancement may be involved in the behavioural effect of the extract. This was confirmed by the ability of the extract to potentiate the head twitch responses induced by 5-hydroxytryptophan in mice, a model sensitive to 5-HT2A receptor activation. Pretreatment with α-methyldopa (400 mg kg-1) however, failed to reverse the behavioural effect of the extract and fluoxetine treatments in the forced swim test. The same result as above was observed for extract and fluoxetine treatments when mice were pretreated with reserpine (1 mg kg-1) or a combination of α-methyldopa (200 mg kg-1) and reserpine (1 mg kg-1). This suggests that the antidepressant effect of the extract may not be dependent on central noradrenergic mechanisms. Administration of D-serine (600 mg kg-1), a full agonist on the glycine site of the NMDA receptors, reversed the antidepressant effect of the extract, fluoxetine and desipramine in both the TST and FST. D-cycloserine (2.5 mg kg-1), a partial agonist potentiated this behavioural effect in both extract and fluoxetine treated mice but not desipramine in both the TST and FST. This suggests possible involvement of glycine/NMDA receptor or pathway antagonism in the antidepressant effect of the extract. MOE slightly increased curling score in the tail suspension test and this was significantly potentiated by D-cycloserine, suggestive of possible opioidergic activity. MOE (10 - 100 mg kg-1, p.o.) showed anxiolytic effect in the three anxiety models used namely; elevated plus maze, light-dark box and open field tests. M. oppositifolius treatment significantly increased the percentage of centre entries and the percentage time spent in the centre of the open field. M. oppositifolius also increased the time spent in the lit area and the latency to leave the lit area in light/dark box. In the EPM, it significantly increased open arm activities by increasing percentage open arm entries and duration. MOE also decreased risk assessment behaviours such as the head dips, stretch-attend postures and rearing. Acute and subacute toxicity in rats did show deaths after 14 day treatment with the extract (30 – 3000 mg kg-1). Extract treatment did not affect weight of rats or the relative organ weights. Haematological or serum biochemical parameters were not affected except increases in serum bilirubin (300 and 3000 mg kg-1), urea and creatinine (30 and 100 mg kg-1). Histopathological examination did not reveal toxic effect on the stomach, heart, liver and spleen. There were however some morphological changes of the kidney at 30 mg kg-1. These results suggest that the extract has anticonvulsant effect possibly through enhancement of GABAergic, glycinergic and potassium channel activation or increased potassium conductance. Possible inhibition of muscarinic and glutamatergic transmission may also be involved. The antidepressant-like effects of the extract may be due to the interplay of serotoninergic, glycine/NMDA and opioidergic pathways. The extract also demonstrated anxiolytic-like effects possibly by the involvement of GABAergic and serotoninergic mechanisms.
A thesis submitted to the School of Graduate Studies, Kwame Nkrumah University of Science and Technology in partial fulfilment of the requirement for the award of Doctor of Philosophy Degree, August-2012