Preliminary screening of six medicinal plants for anticonvulsant activity and further evaluation of Annona muricata for anticonvulsant and related pharmacological effects

Abstract

Annona muricata is a small tree or shrub which can reach 8 m of height found in Africa and other parts of the world. Different parts of the plant are used in traditional medicine for the treatment of diseases such as epilepsy. This study investigated the anticonvulsant, antioxidant, antiglycation, analgesic and safety properties of A. muricata. Plant collection was done in the center region and the plants collected evaluated for their anticonvulsant activity using pentylenetetrazole (PTZ). A. muricata (AnM) was selected to further the work. After qualitative and quantitative phytochemical analysis, two widely used animal models of epilepsy PTZ and picrotoxin (PTX) were tested against the extracts and fractions of AnM. Acetic acid, formalin and the hot plate test were used to investigate its antinociceptive effects and naloxone for the involvement of opioid receptors. 2,2-diphenyl-1- picrylhydrazyl (DPPH) and nitric oxide (NO), were among the antioxidant tests used in this study. Ribose, glucose and glyoxal were used to evaluate the antiglycation activity and the adverse effect evaluated. After anticonvulsant screening of plants with PTZ, A. muricata displayed the highest activity at 200 mg/kg b.w. Consequently, different organs from A. muricata were tested using Pentylenetetrazol (PTZ) and Picrotoxin (PTX) induced seizure in mice. Leaves, roots and twigs in both PTZ and PTX tests were able to delay seizure onset and reduce convulsion duration significantly (p<0.001), while they also significantly increased total protein and albumin values to normal values (p<0.001). The roots which displayed the highest activity were subjected to fractionation. Of all the fractions and the compounds tested, J18 at all the doses tested significantly delayed the appearance of seizure (p<0.001). Qualitative phytochemical screening showed that the different parts of the plant extract contained metabolites as tannins and flavonoids, while the fractions contained alkaloids, saponin, steroid and triterpernes. The quantitative phytochemical analysis showed that the highest content of polyphenols, flavonols and flavonoid, was obtained in leaves, with a value of 230.8 ± 2.386 GAE/g, 6.571 ± 0.048 mg/g (p<0.001) and 9.962 ±0.870 QE/gof extract (p<0.01) respectively. In the DPPH test, the maximum percentage of inhibition was from root extract with a percentage of inhibition of 87.89%; while the scavenging activity of OH was greater in leaves and twigs (69.96%). In the FRAP assay, the root extract presented the highest activity. The percentage of inhibition of NO was highest in leaves, and a slight difference in the activity of the extracts was observed in the ABTS test. In the acetic acid pain model, Annona muricata roots (AnMr) and leaves (AnMl) at 200 mg/kg significantly (p<0.001) reduced mice writhings with respectively 64.65% and 53.84%. Further investigation of AnMr showed that in the 1st phase of the formalin test at 200 mg/kg it significantly (p<0.5) reduced the time for licking or biting the paw by mice (65.44%); but the effects were reduced by co-administration of naloxone. In the 2nd phase of the formalin test, the time of licking or biting the paw by the mice was significantly reduced at 200 mg/kg ofAnMr with a value of 56.80% (p<0.05). On the hot plate test, only the same dose of AnMr significantly (p<0.5) inhibited the time of staying on the plate with a percentage of inhibition of 119.01%. The formation of advanced glycation end product (AGE) was significantly reduced by ribose after 5 days at concentrations ranging from 20 to 95 μg/ml (p<0.001) while the same pattern was observed with glucose at 5, 10, and 95 μg/ml (p<0.001). In the second step using glyoxal, except the concentration of 10 μg/ml, all the tested doses of AnMr extract were able to significantly reduce the formation of AGE (p<0.001). In the acute toxicity test, 30 min after administration the behavior of animals such as locomotion was modified and after an hour some of the animals treated with AnMPf (pulp of fruit) and AnMs (seeds) died, giving LD50 values of 2.31 g/kg and 4.53 g/kg for AnMs and AnMPf respectively. After the 2nd day, the surviving mice were recovering. Using the Macaca mulatta, monkey, rhesus kidney cells (LLCMK2) for the cytotoxicity, the compounds J17 and P106 were safe products with IC50 values higher than the concentrations tested during the 3 days of observation. The developmental toxicity using the FETAX test shows that J17 and J19 at the lowest concentration tested did not empeds the development of embryos, while P106 was responsible for the delay in development observed. The results indicate that the different plants tested, especially A. muricata has anticonvulsant effect in the different models used, exhibiting analgesic and antiglycating effects and antioxidant properties that support its use in traditional medicine. Meanwhile further investigations on characterization of active extracts/fractions and understandings of their mechanisms of action are required.