Bioactivity Guided Fractionation of Methanol Leaf Extract of Phyllanthus Muellerianus for its Anticonvulsant and Anxiolytic Activities in Mice

Abstract

Introduction Phyllanthus muellerianus (Kuntze) Exell. (Phyllanthaceae) is an evergreen monoecious shrub widely distributed in tropical and subtropical regions of the world. In South-eastern Nigeria, preparations from the leaf is used in treating pain, epilepsy, convulsion, and other central nervous system disorders. This study was aimed to evaluate the anticonvulsant and anxiolytic properties of leaf extract of Phyllanthus muellerianus (PM) in mice. Materials and methods The methanol leaf extract (ME) of PM was fractionated successively with solvents of increasing polarities to obtain hexane (HF), ethyl acetate (EF) and methanol (MF) fractions. The extract and fractions (100, 200 and 400 mg/kg p.o) were evaluated for anticonvulsant activity using pentylenetetrazol (PTZ)-induced seizure model in mice as activity-guide. EF being the most active fraction was further fractionated with column chromatography and afforded six sub-fractions (F1-F6). The sub-fractions (200 and 400 mg/kg p.o) were evaluated for anticonvulsant activity using PTZ-induced seizure model. ME, EF and F5 which gave the most potent anticonvulsant activity were evaluated for anxiolytic and neurotoxic activities using open field and motor coordination tests respectively at the doses of 200 and 400 mg/kg p.o. Acute toxicity of ME was carried out using Lorke's method. Results were analysed using ANOVA followed by LSD post hoc test. Differences between means were considered significant at p<0.05. Results ME at 200 and 400 mg/kg offered 20% and 40% protection to the mice in PTZ-induced seizures respectively. EF and its F5 sub-fraction completely abolished seizures in mice offering 100% protection and 0% mortality each better than the phenobarbitone standard group (83.3% protection). In the open field test, ME at 400 mg/kg significantly (p<0.05) decreased both the number of line crossing and the number of assisted rearing and was similar to that produced by diazepam. At 200 mg/kg ME significantly (p<0.05) increased the exploratory activities. EF and F5 elicited significant (p<0.05) increase in line crossing. EF significantly (p<0.05) increased both the locomotor and exploratory activities in the mice. ME, EF and F5 significantly (p<0.05) decreased the duration of grooming and number of urination streaks. The total duration of freezes in the ME, EF and F5 were significantly (p<0.05) increased. ME at 400 mg/kg significantly (p<0.05) reduced the time of fall of the mice from the rotarod in the same way as diazepam while EF and F5 (at a lower dose of 200 mg/kg) evoked significant (p<0.05) increase in the time of fall from the rotarod. In acute toxicity study, no mortality was recorded after both phases of the toxicity study. Therefore the LD50 was found to be greater than 5 g/kg. Conclusion The ME possesses anticonvulsant, sedative, and anxiolytic activities while EF and its sub-fraction, F5 possess anticonvulsant and anxiolytic activities devoid of sedative and cognitive impairment. Therefore, F5 may provide a lead for the development of non-sedating anticonvulsant and anxiolytic formulations.