AIM OF THE STUDY: Our study focuses on previously unreported anti-depressant activity of E. variegata bark ethanolic extract (EBE) and determination of its mechanism of action possibly through regulation of monoamine oxidase activity in mouse brain homogenates.
MATERIALS AND METHODS: EBE was characterized using standard protocols for phytochemical analysis, followed by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) analysis. Anti-depressant activity of EBE (50, 100, 200 and 500 mg/kg) was evaluated in Swiss white albino mice using acute and chronic forced swim test (FST) models. Furthermore, the potential use of the extract as an adjunct to selective serotonin reuptake inhibitor (SSRI), escitalopram, was evaluated using the chronic unpredictable mild stress test model wherein inhibitory effects on monoamine oxidase (MAO) A and B were assessed by spectrophotometric-chemical analysis in mouse whole brain homogenates.
RESULTS: The extract showed significant reduction in immobility time periods in both acute (200 mg/kg) and chronic (100, 200 and 500 mg/kg) FST models. When used as an adjunct with escitalopram (15 mg/kg), the extract (100, 200 and 500 mg/kg) showed significantly greater inhibition of MAO-A and B activities when compared to escitalopram alone (30 mg/kg). Phytochemical analysis of EBE revealed presence of sugars, steroids, glycosides, alkaloids and tannins. LC-MS and GC-MS analysis identified components such as 2-amino-3-methyl-1-butanol, phenylethylamine, eriodictyol, daidzein and pomiferin, N-ethyl arachidonoyl amine, inosine diphosphate, trimipramine, granisetron, 3,4-dihydroxymandelic acid, ethyl ester, tri-TMS and dodecane, previously reported for their anti-depressant activity.
CONCLUSIONS: The study thus demonstrated potential for use of the E. variegata bark ethanolic extract as an adjunct to currently available SSRI treatment. The study also identified components present in E. variegata bark ethanolic extract that may be responsible for its anti-depressant activity. Furthermore, the study thus confirms the traditional use of E. variegata barks in improving CNS function through its anti-depressant like activity.
METHODS: The rPvAMA1 protein was heterologous expressed using a tag-free Profinity eXact(TM) system and codon optimized BL21-Codon Plus (DE3)-RIL Escherichia coli strain and further refolded by dialysis for renaturation. Binding peptides toward refolded rPvAMA1 were panned using a Ph.D.-12 random phage display library.
RESULTS: The rPvAMA1 was successfully expressed and refolded with three phage-displayed dodecapeptides designated as PdV1 (DLTFTVNPLSKA), PdV2 (WHWSWWNPNQLT), and PdV3 (TSVSYINNRHNL) with affinity towards rPvAMA1 identified. All of them exhibited positive binding signal to rPvAMA1 in both direct phage assays, i.e., phage ELISA binding assay and Western blot binding assay.
DISCUSSION: Phage display technology enables the mapping of protein-protein interactions based on a simple principle that a library of phage particles displaying peptides is used and the phage clones that bind to the target protein are selected and identified. The binding sites of each selected peptides toward PvAMA1 (Protein Data Bank, PDB ID: 1W8K) were in silico predicted using CABS-dock web server. In this case, the binding peptides provide a valuable starting point for the development of peptidomimetic as antimalarial antagonists directed at PvAMA1.