SOME NEUROPHARMACOLOGICAL STUDIES ON THE STEM BARK OF RANDIA NILOTICA STAPF (RUBIACEAE)

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Department of Medicine

ABSTRACT
The commonest practical use of behavioural tests is probably in the search for new drugs. Medicinal plants have been used in the development of new drugs and continue to play an invaluable role in the drug discovery process. These plants/herbs are relatively cheap and available and their use depends on ancestral experience. The majority of the population in developing countries remain dependent on them for healthcare. Randia nilotica stapf is a lowland shrub or tree widespread in the Sudan and reported from lowland habitats in Central and East Africa as well as Cameroon and Nigeria. In ethnomedicine, a decoction is used orally for treatment of mental breakdown and for convulsions. It is also used against epilepsy and madness. The main objective of this project is to establish scientific basis for the use of Randia nilotica in traditional medicine for the treatment of neurodegenerative and neuropsychiatric illnesses. It also seeks to determine active chemical constituents of Randia nilotica as well as its acute toxicity profile. The test systems employed for primary screening of Randia nilotica included Lorke method (1983) of acute toxicity determination, diazepam induced sleep, hole board test, pentobarbitone induced sleep, performance on treadmills (rota rod), mouse beam walk assay and amphetamine induced stereotype behaviour test. Others were electroshock induced convulsion test, pentylenetetrazole induced convulsion test, strychnine induced convulsion test as well as forced swim test in rats and tail suspension test in mice. In addition, electroencephalographic studies in rats was conducted. In these experiments, rats (180-250g), Swiss albino mice (18 –25g) and black ranger cockerels (1 day old) were used where appropriate. The diazepam induced sleep method was used to elucidate the more potent central nervous system active morphological parts of Randia nilotica (leaf, stem bark, root bark) as well as its fractions (saponins, flavonoids, aqueous butanolic residue) and sub-fractions (SF1-SF5). Data vii generated was analysed using t-test and significant difference determined using one-way analysis of variance (ANOVA) followed by Dunnet?s and Scheffe?s post hoc tests. Results were expressed as mean ?standard error of the mean (?SEM). Values that are <0.05 were considered significant. Results of studies carried out revealed high potency for stem bark extract compared to leaf or root bark extracts. This is evident in the increase in duration of sleep as observed in diazepam sleep test which was significant (p < 0.001). It increased from 38.2?5.78 min. (Normal Saline) to 165.8?22.9 min. (stem bark), 113.5?12.8min. (leaf) and 64?13.0min. (root bark). Moreover, the activity of the stem bark was observed at 20mg/kg dose compared to 200mg/kg (10 times higher) for the leaf extract. Similarly, the saponin fraction exhibited higher potency compared to flavonoid or aq. butanolic residue of the stem bark extract. Sleeping time was increased significantly (p < 0.05) compared with normal saline i.e from 38.2?6min to 117?20.8min. The flavonoid and water fractions also showed significant increases (p < 0.005) in sleeping time to 68.6?11min and 77.0?10.0min. respectively compared to normal saline. These increases in sleep duration were observed with the extracts at 20mg/kg (flavonoid, water fraction) and 2mg/kg (saponin fraction). This indicated that the saponin fraction is ten times more potent than either flavonoid or aqueous fractions. SF1 showed least decrease in onset and highest increase in sleep duration followed by SF4 and then SF2. The LD50 values determined by intraperitoneal administration in mice for stem bark, root bark and leaf extracts were 282.2mg/kg, 282.2mg/kg and 2154.1mg/kg respectively. Those of the saponin, flavonoid, aqueous fraction were 11.1mg/kg, 471.2mg/kg and 471.2mg/kg respectively. viii In the hole board experiment, the hydroalcoholic stem bark extract showed a dose dependent decrease in number of head dips which was significant (p < 0.05) at 20mg/kg body weight from 7.7?2.5 mean number of head dips (normal saline) to 1.3?0.8. Similarly, the saponin fraction showed a significant (p < 0.05) dose dependent decrease in mean number of head dips in this experiment from 7.7?2.5 (normal saline) to 2.0?0.6 (5mg/kg), 1.2?0.4 (1.0mg/kg), 0.3?0.0 (2.0mg/kg) and 2.2?1.7 (Diazepam, 1mg/kg). The stem bark extract did not produce any significant observable effect on motor coordination as determined in the rota rod test. Similarly, its saponin fraction did not produce any observable effect on motor coordination using the rota rod test. In the Beam walk assay, significant difference (p<0.001) was observed in mean number of foot slips between stem bark extracts of Randia nilotica (5, 10, 20mg/kg body weight), normal saline and Diazepam (control, 1.5mg/kg). The least number of foot slips was observed with normal saline (0.4?0.2) which subsequently increased in diazepam treated mice (10.8?1.9). Diazepam also showed a significant (p <0.001) increase in number of foot slips compared to normal saline and extracts at 0.5, 1.0 and 2.0mg/kg (1.4?0.6, 1.8?0.7, 1.4?0.7 respectively). No difference was however observed in time to reach goal box between controls and extracts at all doses tested. A biphasic effect was observed with stem bark extracts of Randia nilotica (5, 10, 20mg/kg) on amphetamine-induced stereotyped behaviour in mice. There was a significant (p<0.001) reduction in jumping/climbing episodes produced by the extract from 937.0?22.3 (normal saline) to 103.2?31.2 (5mg/kg), 33.0?11.7 (10mg/kg), 69.8?13.8 (20mg/kg) and 13.0?5.0 (Chlorpromazine, 2mg/kg). Reduction in sniffing was also observed with extract at 5mg/kg (83.4?18.6), which is significant (p<0.05) compared with normal saline (1308.8?13.2). Higher doses paradoxically produced increases rather than decreases in sniffing. The extract (20mg/kg) attenuated mean count in limb licking from 201.8?15.5 (Normal Saline) to 55.6?19.5. Lower doses produced increases in limb licking. The results of the anticunvulsant tests were also mixed. In the maximal electroshock test. The stem bark extract of Randia nilotica protected chicks against hind limb tonic extension (HLTE) by 90% at 20mg/kg comparable to phenobarbitone (PBT) with 90% protection as well. Both 5mg/kg and 10mg/kg produced a 50% inhibition. The saponin fraction on the other hand, protected mice against HLTE by 50% at 2mg/kg body weight. 0.5 and 1.0mg/kg of the saponins showed only a 20% inhibition compared with PBT (20mg/kg) which sowed 80% protection. On the other hand, the stem bark and saponin extracts did not show any effect on pentylenetetrazole (sc-PTZ) induced and strychnine induced seizure tests in mice. However, the mean number of myoclonic body twitches was significantly (p<0.05) reduced by the stem bark extract in the sc-PTZ induced seizure test. 5mg/kg produced a 161% reduction while 10 and 20mg/kg produced 250% and 265% reduction respectively. In the tail suspension test in mice, the HA stem bark extract of Randia nilotica showed no effect on duration of immobility time in mice at the doses tested. But some dose dependent decrease in immobility time was observed in the forced swim test in rats. The reduction was however not significant when compared to control. The saponins on the other hand showed a significant (p<0.05) decrease in immobility time at 2.5mg/kg (82.4 ?7.9) and 10mg/kg (80.0?20.7) compared with normal saline (108.6?24.2). Desynchronisation in the rat electroencephalograph (EEG) of the hyperstriatum (HS), optic tectum (OT) and pontine reticular formation (RF) was observed with the stem bark extract at 20mg/kg and 40mg/kg. A slight increase in muscle activity was also observed. At 80mg/kg however, there was synchronisation of HS, RF and OT while a reduction in electromyographic (EMG) activity was observed. The saponin fraction of Randia nilotica (2.5mg/kg) slightly synchronized EEG of the HS, OT and RF but with weak activation in EMG of the rat. At 5mg/kg and 10mg/kg body weight, synchronization of the HS, RF and OT was observed as well as a decrease in EMG activity throughout the period of observation. These results suggested significant sedative effects of the stem bark extract of Randia nilotica, its saponin fraction and subfraction (SF1). This may lend some scientific support for the uses of the plant in traditional medicinal practice in the treatment of neuropsychiatric and neurological diseases. It may also be suggested that the extracts are relatively safe at the dose levels tested. It can be concluded therefore that Randia nilotica merits further attention in the search for a new compound against neurodegenerative and neuropsychiatric disorders.